Tecnai on-line help manual -- Options - UT Southwestern

<strong>Tecnai</strong> on-line help Options 5Options Software version 21 TEM Low Dose and Spotscan (FP5407/00, FP5408/00)1.1 Introduction to Low Dose imagingMany materials damage in the electron microscope. In some cases the damage is tolerable as it is toosmall to affect the validity of the information extracted from the electron microscopy. In other cases it iscrucial to limit the amount of damage because the damage itself is the ultimate limitation to theinformation sought (and thus not microscope-related parameters such as contrast or resolution). Sincethe electron dose needed for the exposure is relatively fixed (because the dose determines the noise inthe electron images), the strategy of Low Dose imaging is simply to reduce the electron dose prior toexposure to an absolute minimum (preferably none but that is not achievable). For that purpose the LowDose software provides a number of dedicated tools.Low Dose is designed such that it allows Low Dose recording on late as well as CCD camera.1.1.1 Beam blankerIn order to prevent accidental exposure of the specimen by electrons the Low Dose software uses abeam blanker. This blanker deflects the beam in the electron gun by a gun tilt (by an amount that can becalibrated by the user). The beam blanker is used to blank the beam during state transitions (see below)and as the actual exposure shutter for the Low Dose exposure. It is also under user control, so the usercan blank the beam for as long as needed.1.1.2 Low Dose statesLow Dose contains three states that have a certain degree of optical independence:• Search• Focus• ExposureThe general idea is to use the Search state to search the specimen under very Low Dose and highcontrastconditions for suitable areas (commonly followed strategies are to use one of the lower SA or Mimagnifications with a relatively large defocus or use defocussed diffraction). Once a suitable area hasbeen identified (alternatively suitable areas can be stored and the stage driven from one stored positionto the next), Low Dose is switched to the Focus state. Typically the Focus state is set to a magnificationthat is well-suited to focussing and astigmatism correction. In order to prevent damage to the area of thespecimen that is intended for recording, the Focus state uses an off-axis image shift (with acorresponding beam shift). The Focus image shift is controlled by distance and rotation. By changing therotation only the off-axis shift can be used circle around the exposure area. Two separate Focus off-axisshifts can be set (useful e.g. when one of the shifts is on a grid bar or for focussing a tilted specimens. Inthe latter case set the image shift to either side of the exposure area).

<strong>Tecnai</strong> on-line help Options 7Options Software version 2d) Switch to Exposure, Focus and Search again and check the conditions (especially centering ofthe beam).e) If necessary, recenter the beam and repeat the procedure.2. Operationa) Switch to the Search state and locate a number of good specimen areas. Store these using theStage control panel function.(If the microscope is equipped with an off-axis TV that must be used in the Search state, usethe TV toggle (Detector configuration) to switch to the off-axis detector position.)b) Press the Blank button (blank the beam).c) Move to the first good area (or the best) and wait a short time to allow the goniometer to settle.d) Press the Blank button again (unblank the beam).e) If necessary, center the area accurately with the goniometer.f) Switch to Focus and focus the image.(If the microscope is equipped with an on-axis CCD that must be used in the Focus state, usethe TV toggle (Detector configuration) to switch to the on-axis detector position.)g) If necessary, press the Blank button (blank the beam).h) Press the Expose button (or the Exposure button on the <strong>Tecnai</strong> Control Pad) to make theexposure.(If the microscope is equipped with an off-axis TV that must be used in the Search state, thedetector shift is set to on-axis automatically before the exposure and reset to its originalposition afterwards.)i) After the exposure is finished, go to the Exposure state and check the conditions (unlessanother low-dose exposure must be made at the same position).1.1.4 An overview of Search, Focus and Exposure opticsThe settings of Search, Focus and Exposure achieve a certain amount of decoupling between the states.In general the Search state is almost totally independent of Focus and Exposure, while some settings ofthe Focus state are coupled to the Exposure state.Spotsize The spotsize (C1 lens) is independent for all three states.Intensity The intensity (C2 lens) is independent for all three states.Illumination modes The illumination modes are independent between Search on the one hand andFocus and Exposure on the other (Focus and Exposure are thus the same by definition). This meanse.g. that Search may be in Microprobe and Focus/Exposure in Nanoprobe.Imaging modes The imaging modes are independent between Search on the one hand and Focus andExposure on the other (Focus and Exposure are thus the same by definition). This means e.g. thatSearch may be in diffraction or LM and Focus/Exposure in HM imaging (Microprobe).Focus The focus settings are independent between Search on the one hand and Focus and Exposureon the other (Focus and Exposure are thus the same by definition). Separate settings are stored by thesoftware for:• HM Image focus• LM Image focus• D (HM) diffraction• LAD (LM) diffractionFocus differences between magnifications used for the Focus and Exposure states can be eithercompensated (properly) by using the Parfocal Magnification series alignment. As an alternative, thefocus difference can be measured and entered as a single-exposure series and series switched on. Thesoftware will the automatically apply the focus change (but only during the actual recording of theexposure, not when the state is changed from Focus to Exposure).Beam shift The Search and Focus states react to beam shifts in Exposure states and in their ownstates. The beam shift is built up as follows. First of all any 'User' beam shift at the start of using Low

<strong>Tecnai</strong> on-line help Options 9Options Software version 2These buttons and knobs are assigned in the Options tab of the Low Dose flap-out. Low Dose keepstrack of any other assignment to these buttons and knobs (e.g. on alignment or stigmation). If a button orknob is given another function, its will no longer affect Low Dose (so pressing L1 when the L1 functionhas been changed doesn't switch to Search anymore). As soon as the button or knob has beenreleased, it gets back its Low Dose functionality.When Low Dose is stopped (by pressing the Low Dose button or exiting the <strong>Tecnai</strong> User Interface), thebuttons and knobs get back their original setting.1.1.7 User levels in Low DoseThe Low Dose software supports two user levels, 'user' and 'supervisor'. Supervisor settings (both LowDose settings and calibrations) are stored separately from those of the user. When a new user startsLow Dose for the first time, the settings and calibrations used are those defined by the supervisor.Thereafter the user has set his/her own settings and these will be retrieved when the software is started.The only way thereafter to go back to the supervisor settings is to have the supervisor save settings andcalibrations files and load these files as a user. Low Dose settings and calibrations saved by Factory orService are equivalent to Supervisor settings.1.1.8 SpotscanSpotscan (FP5408/00) is an option for the Low Dose software. It relies on hardware installed in themicroscope PC and microscope and will not function without the proper hardware.The Low Dose version with spotscan supports two modes of exposure:• The normal 'flood-beam' exposure wherein the whole exposure area is illuminated by a broad beamand exposed together.• The spotscan exposure wherein a smaller beam is stepped across the area to be exposed and eachspot is exposed individually.Spotscan settings are defined in the Spotscan tab of the Low Dose Control Panel flap-out.

<strong>Tecnai</strong> on-line help Options 10Options Software version 21.2 Low Dose Control PanelThe Low Dose Control Panel contains the functions used for LowDose electron microscopy. A more extensive description of LowDose and its use is given on the Low Dose page.Note: The magnifications (or camera lengths) listed for the threeLow Dose states are the so-called reference values (the valueson the plate camera, that is, the ones seen with the screen up).These fixed values are used to avoid confusion (which couldoccur when Low Dose would continuously adjust values whenthe screen goes up or down). The values with the screen downare roughly 10% lower than with the screen up.Low Dose buttonThe Low Dose button activates or deactivates Low Dose. The status can be seen from the color of thebutton (gray is off, yellow is on). When Low Dose is activated, it saves the currently active settings of themicroscope and changes them to Low Dose settings (Search at start-up, any other state when Low Dosehas been active previously). When Low Dose is switched off, it restores the microscope to the status ithad before Low Dose was switched on.Blank buttonThe Blank button toggles the beam blanker on and off. The beam is blanked (that is, no electrons comedown the column) when the button is yellow. The beam blanker uses an offset on the gun tilt which iscalibrated (by supervisor or user).Peek buttonThe Peek button toggles the diffraction peek function on and off. The button is visible only when thefunction is enabled in the Options tab of the Low Dose flap-out. It is enabled only when:• The Exposure state is active.• The microscope is in diffraction.• The function itself is enabled (Options tab).StatusThe status line displays the current Low Dose status as well as any error messages..

<strong>Tecnai</strong> on-line help Options 11Options Software version 2Search buttonThe Search button switches Low Dose to the Search state.Search settingsThe settings used for the Search state (microscope mode, magnification or camera length, spot size,intensity and image shifts) are displayed underneath the Search button.Search start buttonThe Start button starts image acquisition on the CCD camera in the Search state. It is enabled only whena CCD has been identified, selected for operation in Search and Low Dose is in the Search state.Focus buttonPressing the Focus button switches the Low Dose function to the Focus state.Focus substatesThe Focus state has two substates, 1 and 2, that can have different settings for the off-axis beam andimage shifts. Switching between the two substates is done by selecting the required radio button on theLow Dose window or pressing the <strong>Tecnai</strong> User Button selected when the Focus state is already active.Focus settingsThe settings used for the Focus state (magnification or camera length, spot size, intensity and imageshifts) are displayed underneath the Focus button. The microscope mode is not displayed because it is,by definition, the same as for the Exposure state.Focus start buttonThe Start button starts image acquisition on the CCD camera in the Focus state. It is enabled only whena CCD has been identified, selected for operation in Focus and Low Dose is in the Focus state.Exposure buttonPressing the Exposure button switches the Low Dose function to the Exposure state. This is not thesame as making the plate-camera exposure! The Exposure state activated by the Exposure button ismeant for setting up the conditions under which the Low Dose exposures will be made.Exposure settingsThe settings used for the Exposure state (microscope mode, magnification or camera length, spot size,and intensity; in addition the exposure time is shown at the bottom) are displayed underneath theExposure button.Flap out buttonPressing the flap-out button leads to the Low Dose flap-out, containing the Spotscan (if present),Settings, Calibrate, Options and TV /CCD tabs.

<strong>Tecnai</strong> on-line help Options 12Options Software version 21.2.1 Expose panelExpose buttonWhen the Expose button (or the <strong>Tecnai</strong> Exposure button on the left-hand Control Pad) is pressed, theactual Low Dose exposure is started. The progress of the exposure is displayed in the status as well asby the status of the state and blank buttons. The medium for exposure (plate camera or CCD) dependson the setting chosen.SeriesThe Series check box enables the automatic exposure of a series. The settings for the series are definedin the Series panel. The Series check box is only enabled when at least one setting has been defined forseries.DoubleThe Double check box enables double exposure (multiple exposures on a single plate). The exposuretimes for the double exposures are defined in the Double panel. The Double check box is only enabledwhen at least one setting has been defined for double. Double and Series or Use spotscan cannot beused together.Dim screenIn order to prevent light from the monitor from striking the plate (which is lying open in the projectionchamber during the exposure; it is therefore always important to switch room lights off and cover thewindows of the projection chamber), the monitor screen can be blanked during the exposure.Note: Should the screen dimmer fail to be removed after the exposure, then it can always be removed bypressing Shift+Esc on the keyboard. Do not use Alt+Tab to switch to another window because that will(in most cases) not work.Use spotscanOnly on systems where spotscan is installed :When the Use spotscan option is enabled the exposure(s) will be taken as spotscans, not flood-beamexposures. The Exposure time will change to spot dwell time (exposure time per spot). The spotscan asexecuted is defined in the Spotscan tab of the flap-out. Spotscan and pre-exposure or Double cannot becombined.Exposure timeBecause the conditions during operation (search and focus) are not suitable for measuring the exposuretime to be used during the actual Low Dose exposure (due to differing magnification/camera length, spotsize and intensity settings), the exposure time must be set by the operator. Switch to the Exposure stateinitially and determine the exposure time as indicated by the plate camera of the <strong>Tecnai</strong> microscope. If

<strong>Tecnai</strong> on-line help Options 13Options Software version 2necessary, adjust the exposure settings (spot size, intensity) and then enter the desired exposure timeon the Plate camera panel (type or enter by pressing the up or down spin buttons). If the CCD is used forexposure, the proper exposure time must be entered in the camera setup (DigitalMicrograph or <strong>Tecnai</strong>Control Panel for MSC cameras). Low Dose itself does not define CCD settings.While Low Dose is active, the manual exposure of the plate camera is set to the same setting as theexposure time for Low Dose plate exposures. When Low Dose is switched off, the plate camera manualexposure time setting that was active before Low Dose was switched on is restored.If spotscan is used :The dwell time (in milliseconds) determines how long each individual spot will be exposed. The scanboard that drives the beam itself allows dwell times down to 1 microsecond. The beam deflection coils ofthe microscope cannot support such high scan rates (the scan will not consist of individually steppedspots but a blurred line) and advised dwell times are 10 milliseconds and higher.To estimate the dwell time, select the required spotscan settings, go to the Exposure state and insert thesmall viewing screen. Read off the auto exposure time in the Plate camera control panel of themicroscope. Estimate the ratio of the spot diameter and the small screen diameter. Divide the measuredexposure time by the square of the ratio (it goes by area). This will be the dwell time.If the spotscan spot is much smaller than the small screen, increase the magnification so the spot makesup about 1/3 to 1/2 of the small screen. Estimate the ratio as described above but modify it further bydividing it by the square of the ratio of the measurement magnification and the true exposuremagnification (once again it goes by area).For good results, you may want to bracket initial attempts (take one exposure at a 3x shorter dwell time,one at the dwell determined and another at a 3x longer dwell time) and thereby calibrate the requireddwell times.If Double is active : The exposure time as indicated is not used. Instead the exposure times as definedfor double exposure are used.Wait timeIn order to allow the microscope to stabilize after the mechanical 'shocks' from the loading of the plate awaiting time is set before the exposure is taken. For high resolution allow at least 5 seconds waiting time.At lower magnification or lower resolution a shorter waiting time can be set.Pre-exposureThe Low Dose mode allows pre-exposing the specimen (the beam is put on the specimen but theexposure shutter of the microscope stays closed so this exposure has no effect on the plate) to allowstabilization (in case of charging). When this function is enabled, it is possible to choose a pre-exposuretime.Note: When pre-exposure is active, the resolution of the exposure may not be as good as ultimatelyachievable in Low Dose due to drift of the shutter (below the specimen) during the actual exposure,especially if the waiting time after the pre-exposure is short (1 second or less). During Low Doseexposure without pre-exposure the shutter is opened several seconds before the beam is unblanked forthe Low Dose exposure.Wait time after pre-exposureUnless a waiting time is set after the pre-exposure, the real Low Dose exposure takes place as quicklyas possible after the pre-exposure. When the waiting time is enabled (through the checkbox), the waitingtime can be set.

<strong>Tecnai</strong> on-line help Options 14Options Software version 21.2.2 Focus panelThe off-axis shift for the Focus state is controlled by distance and(rotation) angle. Thereby it is possible to circle around an area ofinterest by simply changing the rotation angle, without any dangerto accidentally moving towards the real area of interest. Circlingmay be necessary, e.g. in the case where the off-axis area lies ona grid bar. Once calibrated the 0 and 180° angles will lie along thetilt axis. Focussing with a tilted specimen can be done either at 0 or180° (where the focus will be the same as on the area of interestprovided the specimen is flat) or at 90 and 270° with an equal offaxisdistance and taking the focus setting halfway in between.DistanceThe off-axis shift distance can be set with the distance trackbar and the Multifunction X knob. Thedistance should be set such that the off-axis shift is sufficient to ensure that the Focus-state beam doesnot overlap onto the Exposure area, while as the same time keeping it as small as possible. The trackbaris active only in the Focus state.AngleThe off-axis shift rotation angle can be set with the angle trackbar and the Multifunction Y knob. Thetrackbar is active only in the Focus state.1.2.3 Series panelThe series panel contains settings used for automated throughfocusseries. The through-focus series is totally flexible. It simplywill execute as many exposures as there are entries in the list,changing the focus by the amount in the list for the particularsetting. Multiple entries with the same value, or the value 0 areallowed. It is also allowed to enter a single non-zero value In thatcase each Low Dose exposure will be made with the focusbetween Focus state and Exposure state offset by the specifiedamount (making it e.g. possible to focus in the Focus state tominimum contrast - close to zero or Gaussian focus - and haveeach Low Dose image recorded at -1000 nm).Note: The focus settings are not absolute but are values relative to the focus set when the exposureseries is started.Focus settingThe Focus settings list is filled by entering values for the focus (underfocus is taken as negative) in theFocus setting edit control in nanometers and pressing the Add button.Focus settings listThe Focus settings list contains the list of values used for the through-focus series.Add buttonThe Add button allows insertion of a new setting into the focus settings list. The value for the new focusmust be inserted into the Focus setting edit control. The maximum number of values is 20.

<strong>Tecnai</strong> on-line help Options 15Options Software version 2Delete buttonThe Delete button allows removal of settings from the list. Select a setting and press Delete.Move upThe through-focus exposures are recorded in the sequence as they occur in the list. To change thesequence, select a setting and press Move up to move it up in the list.Move downThe through-focus exposures are recorded in the sequence as they occur in the list. To change thesequence, select a setting and press Move down to move it down in the list.1.2.4 Double panelThe double panel contains settings used for recording doubleexposure (multiple exposures on a single plate). The doubleexposure setup is totally flexible. It simply will execute as manyexposures as there are entries in the list, changing the exposuretimes as indicated in the list for the particular setting.The double exposure sequence is similar to that for a normal low-dose exposure except that the plate isnot removed until the last double exposure has been done (or the exposure is cancelled). After oneexposure has been recorded, further execution pauses until the operator presses the Expose button (onthe Low Dose Control Panel) or the Exposure button (on the left-hand <strong>Tecnai</strong> Control Pad) again.Pressing either of these buttons while an exposure is taking place (the Expose button is yellow) cancelsfurther exposure, otherwise it starts the next exposure in the sequence. When dim screen is active, thedimmed screen will display a message alerting the operator to press Exposure to start the nextexposure.Between individual exposures of a double exposure sequence, it is possible to lower the screen and lookat the image. Be aware, however, that the Low Dose state remains in Exposure. The Peek and BeamBlanker functions become accessible when the screen is lowered.When Double exposure is selected in the Expose tab, the Low Dose exposure time as listed becomesmeaningless, and the double exposure times in the list are used instead.Exposure timeThe exposure times list is filled by entering values for the exposure time (in seconds) in the exposuretime edit control and pressing the Add button.Exposure times listThe exposure times list contains the list of values used for the double exposure times. The maximumnumber of values is 20.Add buttonThe Add button allows insertion of a new setting into the exposure times list. The value for the newexposure time must be inserted into the exposure time edit control.

<strong>Tecnai</strong> on-line help Options 16Options Software version 2Delete buttonThe Delete button allows removal of settings from the list. Select a setting and press Delete.Move upThe double exposures are recorded in the sequence as they occur in the list. To change the sequence,select a setting and press Move up to move it up in the list.Move downThe double exposures are recorded in the sequence as they occur in the list. To change the sequence,select a setting and press Move down to move it down in the list.1.3 Spotscan tabOnly if spotscan is installed: The Spotscan tab of the Low DoseControl Panel contains the controls for defining and inspectingthe spotscan.PatternThe spotscan can be executed in either a hexagonal pattern of a square pattern. In the case of ahexagonal pattern, the closest packing of the spots is achieved, with each second row of spots displaced1/2 spot (as a result each spot has six nearest neighbors, arranged in a hexagon around it). In thesquare pattern, the spots lie at the corners of squares (and each spot has four neighbors: above, left,right and below).OrientationThe spotscan pattern can be rotated through 360 degrees. When calibrated, the 0 and 180° directionshave the x direction of the spotscan coincide with the a tilt axis of the CompuStage.

<strong>Tecnai</strong> on-line help Options 17Options Software version 2Focus correctionAn automatic focus correction can be applied to spotscan exposures for tilted specimens. The focuscorrection required is autmatically derived from the CompuStage alpha tilt angle. If you use pre-tiltedspecimens or pre-tilted cartridges, specify an offset angle to compensate for the difference between theCompuStage alpha tilt value and the real tilt angle of the specimen. For focus correction to workproperly, it is very important that the orientation angle is set to 0 and the following calibrations have beendone:• Image/beam shift for the active mode (found in the Alignments Control Panel).• Spotscan Pivot points• Spotscan Beam shift• If the size of the spots is affected by the objective-lens current (especially smaller spots and innanoprobe), Spotscan Focus-Intensity.SetupThe spotscan setup contains a number of functions to set the scan area and distance between spots.The total number of spots is indicated.Note: The TEM magnification to be used for the spotscan should be set before switching any of thespotscan setup functions on, because the optimum beam deflection range is not reset when themagnification is changed while spotscan setup functions are active (in this way the scan can beobserved, for example, at lower magnification).NoneWhen None is checked, the spotscan setup is disabled and spotscan wobbles or beam shifts areswitched off. This is necessary after a setup, otherwise the microscope is not usable.OutlineTo set the total area scanned by the beam, you can either use the Outline function or set X and Yseparately. In the Outline function the beam is moved continuously to the four corners of the scan range(for a 'normal' setting, this usually implies that the spots are just off the viewing screen and thus invisible- you can lower the magnification to check). Adjust the X and Y scrollers to set the required scan frame(should be at least the size of a negative). The scan area size is indicated above the X and Y sliders (inarbitrary units when not calibrated, otherwise in micrometers).Static XTo set the total area scanned by the beam, you can either use the Outline function or set X and Yseparately. With the Static X function the beam is moved to the extreme X position of the scan range.Adjust the X scroller to set the required scan frame (should be at least the size of a negative). The scanarea size is indicated above the X and Y sliders (in arbitrary units when not calibrated, otherwise inmicrometers).Static YTo set the total area scanned by the beam, you can either use the Outline function or set X and Yseparately. With the Static Y function the beam is moved to the extreme Y position of the scan range.Adjust the Y scroller to set the required scan frame (should be at least the size of a negative). The scanarea size is indicated above the X and Y sliders (in arbitrary units when not calibrated, otherwise inmicrometers).X, Y slidersThe X and Y sliders become activated when Outline, Static X (X slider only) or Static Y (Y slider only) areon. They control the size of the scan range for the X and Y directions.

<strong>Tecnai</strong> on-line help Options 18Options Software version 2Spot wobbleTo determine what the distances between the spot should be, click on Spot wobble. The spot will now bewobbled back and forth and the scroller can be adjusted until the spot just touch each other. The spotdistance is indicated above the spot slider (in arbitrary units when not calibrated, otherwise inmicrometers). The number of spots in the total scan is also indicated.Note: The optimum beam deflection range (reset when spotscan setup functions are switched on)affects the spot distance. Therefore always check the spot distance after setting the scan range.PreviewThe result of the spotscan setup can be viewed on the viewing screen to check that everything is alrightfor the exposure. This function is available only in the Low Dose Exposure state (press the Exposurebutton). The progress of the scan is indicated by the progress bar in the top of the panel (blueincreasingly fills the bar).ViewPressing the View button starts and stops the spot scan.SlowerWhen the Slower button is pressed, the spot dwell time is increased by a factor 2.FasterWhen the Faster button is pressed, the spot dwell time is reduced by a factor 2.To 0,0When the button To 0,0 is pressed, the scan is stopped and the beam is moved to the 0,0 position(center of the scan frame). If the beam is not centered on the screen, it should be shifted to the centerwith the normal beam shift (or Align beam shift alignment). The control of the AC beam deflection coils(used by spotscan) remains on.Shift awayWhen the Shift away button is pressed, the beam will be moved to an extreme X,Y position. It should notbe visible on the screen now (unless the magnification has been lowered from the exposuremagnification used during the setup). The control of the AC beam deflection coils (used by spotscan)remains on.AC offTo switch the AC coils (used by spotscan) control off after the preview, press the AC off button.

<strong>Tecnai</strong> on-line help Options 19Options Software version 21.4 Settings tabThe Settings tab of the Low Dose Control Panel contains controlsto are related to the Low Dose settings.Search ResetPressing the Reset button resets the currently active Searchsettings to zero. What gets reset depends on the Mode only / Allchoice. The button is only active in the Search state.Search Mode only / AllWhen Mode only is selected, only those settings of the mode(HM, LM, or Nanoprobe; Imaging or Diffraction) currently activeon the microscope are reset to zero. When All is selected, allsettings are reset.Search MediumThe choice between (Viewing) Screen, TV and CCD for theSearch state is made by selecting one of the items in the dropdownlist. (See picture on the right).Focus ResetPressing the Reset button resets the currently active Focussettings to zero. What gets reset depends on the Mode only / Allchoice. The button is only active in the Focus state.

<strong>Tecnai</strong> on-line help Options 20Options Software version 2Focus Mode only / AllWhen Mode only is selected, only those settings of the mode (HM, LM, or Nanoprobe; Imaging orDiffraction) currently active on the microscope are reset to zero. When All is selected, all settings arereset.Focus MediumThe choice between (Viewing) Screen, TV and CCD for the Focus state is made by selecting one of theitems in the drop-down list.Exposure ResetPressing the Reset button resets the currently active Exposure settings to zero. What gets resetdepends on the Mode only / All choice. The button is only active in the Exposure state.Exposure Mode only / AllWhen Mode only is selected, only those settings of the mode (HM, LM, or Nanoprobe; Imaging orDiffraction) currently active on the microscope are reset to zero. When All is selected, all settings arereset.Exposure MediumThe choice between Plate (Camera) and CCD exposure is made by selecting one of the items in thedrop-down list.FilenameSettings can be loaded from and saved to file. When a filename has been defined (through a Load orSave), the name will be listed. When a file is loaded, the entry also lists which file version was loaded. Ifthe same settings are used all the time (for any particular user), there is no need to save and loadsettings since the currently active Low Dose settings are always saved upon exiting and restored uponopening of the program (for each user individually).Load buttonOpens a standard Open File dialog that allows selection of a file with Low Dose settings. This file will beread and settings in the file will be made active. The file name must have the extension lds.Save buttonIf no file name for Low Dose settings has been defined, this opens a standard Save File dialog thatallows entering a file name under which the currently active Low Dose settings will be stored. The filename must have the extension lds (the extension will be added automatically by the software).Save As buttonOpens a standard Save File dialog that allows entering a file name under which the currently active LowDose settings will be stored. The file name must have the extension lds (the extension will be addedautomatically by the software).

<strong>Tecnai</strong> on-line help Options 21Options Software version 21.5 Calibrate tabThe Calibrate tab of the Low Dose Control Panel contains thefunctions necessary for the Low Dose calibrations.Blanker buttonThe Blanker button starts the beam blanker calibration procedure. The beam blanker relies on a usercalibratedoffset of the gun tilt. Once the gun tilt has been aligned properly the beam blanker calibrationcan be done. The user then turns the Multifunction X knob until the electron beams is no longer visible.For safety with the beam blanker it is advisable to turn the gun tilt a bit further than the point where thelight disappear (safety margin).Image / Tilt buttonThe Image / Tilt button starts the Image shift / Alpha tilt calibration procedure. This calibration ensuresthat the Low Dose Focus substate off-axis shift angles 0 and 180 will be along the Alpha tilt axis of theCompuStage.The procedure (for the HM and LM modes) consists of:a) Preparation, wherein the program sets the microscope to a specific mode, magnification and spotsize. The user must center a recognizable image feature.b) The stage will move the feature along the X axis of the stage (which is parallel to the Alpha tilt axis)by approximately 90% of the fluorescent screen. The user must now recenter the feature using theimage shift.Peek buttonThe Peek button starts the peek calibration procedure. The peek function uses a user-calibrated offset ofthe gun tilt. Once the gun tilt has been aligned properly the peek calibration can be done. The user then

<strong>Tecnai</strong> on-line help Options 22Options Software version 2turns the Multifunction X knob until the electron beams is about 10% of the initial setting (almost no morevisible).Spotscan pivot points buttonWhen Spotscan is installed only: The Spotscan pivot points button starts the pivot points alignmentprocedure (there is no such alignment procedure in the standard <strong>Tecnai</strong> software). For proper operationof the spotscan, the beam shift pivot points (in this case for the AC - or STEM - deflection coils) must bealigned for HM, LM and Nanoprobe (the latter not on a <strong>Tecnai</strong> 10). The pivot point aligned is similar tothose in the normal <strong>Tecnai</strong> alignment procedures. This procedure is accessible only from the Low DoseExposure state (press the Exposure button).Note: This is an alignment and not a Low Dose calibration. The results are stored in the operator'salignments.Spotscan beam shift buttonWhen Spotscan is installed only: The Spotscan beam shift button starts the beam shift calibrationprocedure (there is no such alignment procedure in the standard <strong>Tecnai</strong> software). The spotscan beamshift calibration procedure determines the beam shifts for HM, LM and Nanoprobe (the latter not on a<strong>Tecnai</strong> 10). Because the spotscan uses different coils (AC) than the normal beam shifts (DC), thesecalibrations must be done separately. This procedure is accessible only from the Low Dose Exposurestate (press the Exposure button).The procedure (for the three modes) simply consists of:a) Preparation, wherein the program sets the microscope to a specific mode, magnification and spotsize.b) The user must focus the beam and center it accurately.c) Move the beam to the edge of the screen (the point where the phosphor stops) with the scroller atthe bottom. If the edge cannot be reached, move the scroller to its end point. The magnification willbe changed for another attempt (until the edge can be reached). If too little of the range of thedeflection is used (the scroller is at less than half of the range), a setting in the microscope will bechanged and the calibration step is redone to get the proper definition.d) The program recenters the beam.e) Repeat with for Y.From the known screen size and magnification the program calculates a calibration factor that convertsthe beam shift values to micrometers.Note: Part of this adjustment is an alignment. Those results are stored in the operator's alignments.Spotscan Focus-Intensity buttonWhen Spotscan is installed only: The size of the spots can be affected when the objective-lens current ischanged (to some extent in normal Microprobe HM but especially in Nanoprobe) which can result indisturbing effects when Spotscan focus correction is applied for tilted specimens as the the spots usedfor recording will get progressively smaller along the scan. When the Focus-Intensity calibration hasbeen done, Low Dose will automatically compensate the spot size change with the Intensity. Theprocedure consists of focusing the spot for HM and Nanoprobe for two objective-lens settings (controlledby Low Dose).

<strong>Tecnai</strong> on-line help Options 23Options Software version 2Calibration instructionsInstructions during the calibration procedures will be displayed here.OK buttonPressing the OK button (or the L1 user button on the <strong>Tecnai</strong> Control Pads) proceeds with the calibrationprocedure to the next step.Cancel buttonPressing the Cancel button cancels the calibration procedure. All intermediate settings determined so farwill be removed. The microscope returns to its starting position.FilenameCalibrations can be loaded from and saved to file. When a filename has been defined (through a Load orSave), the name will be listed. If the same calibrations are used all the time (for any particular user),there is no need to save and load calibrations since the currently active Low Dose calibrations arealways saved upon exiting and restored upon opening of the program (for each user individually).Load buttonOpens a standard Open File dialog that allows selection of a file with Low Dose calibrations. This file willbe read and calibrations in the file will be made active. The file name must have the extension cal.Save buttonIf no file name for Low Dose calibrations has been defined, this opens a standard Save File dialog thatallows entering a file name under which the currently active Low Dose calibrations will be stored. The filename must have the extension cal (the extension will be added automatically by the software).Save As buttonOpens a standard Save File dialog that allows entering a file name under which the currently active LowDose calibrations will be stored. The file name must have the extension cal (the extension will be addedautomatically by the software).

<strong>Tecnai</strong> on-line help Options 24Options Software version 21.6 Options tabThe Options tab of the Low Dose Control Panel contains a rangeof optional settings.Enable peekIf this option is checked, the Peek button becomes visible and, when appropriate, enabled.Use TV / CCDWhen a TV or CD camera is present on the microscope, it can be used during working with Low Dose.First define what camera's are located where in the TV / CCD tab and then switch on Use TV / CCD.After that define in the Settings tab for which Low Dose states a camera should be used.Multifunction knobs to Search shiftWhen this option is selected the Multifunction knobs of the <strong>Tecnai</strong> Control Pads will be switchedautomatically to the Search image shift (in image mode). Note that there is no other way of setting theimage shift in Low Dose (the Image shift alignment has the same result, though it uses a differentparameter to achieve the effect).Multifunction knobs to Focus shiftWhen this option is selected the Multifunction knobs of the <strong>Tecnai</strong> Control Pads will be switchedautomatically to the Focus image shift (in image mode). Otherwise the way to set the Focus image shiftis though the track bar sliders in the Focus panel.NormalizationsMagnetic lenses suffer from hysteresis - an effect that makes the magnetic field difficult to setreproducibly because the effective field depends on the direction of change (up or down). The magnetic

<strong>Tecnai</strong> on-line help Options 25Options Software version 2field of a lens can be made reproducible by normalization, a procedure wherein the lens is cycled updown-upand then back to the desired setting. Hysteresis can affect the position of the beam (due tochanges in C1 - spot size and C2 - intensity) as well as the image or diffraction pattern (mostly theprojector system consisting of the diffraction, intermediate and projector 1 and 2 lenses). In order toachieve reproducible switching between the Low Dose states it is necessary to normalize lenses. Inorder to allow full flexibility in this, the user can select which lenses should be normalized during thepossible switches. The default settings are displayed in the panel above. It is not advised to normalizethe objective lens when switching between Focus and Exposure (this can affect the focus).NormalizeNormalizes the lenses selected by the check boxes to the right of the button.Switch Search to Focus via ExposureTo have settings more reproducible (especially when the Objective lens is normalized between Searchand Focus) it may help to proceed from Search to Focus via the Exposure state (the beam will remainblanked during all the transitions). The actual sequence followed is then Search > Focus > Exposure >Focus.User buttonsUnder User buttons, the user defines which <strong>Tecnai</strong> Control Pad User Buttons (L1 .. L3, R1 .. R3) areused by Low Dose and which button does what. If a user button name (L1..R3) is listed in the drop-downlist of a function, that function is assigned to a <strong>Tecnai</strong> User Button (otherwise the list will display None). Ifa new selection is made by selecting another entry in a list, the software will re-assign any functionpreviously assigned to the particular User Button (if present) by shifting it to the next empty User Button.Dose measurementIn the Exposure state you can measure the dose falling on the specimen. The dose is derived from thescreen current falling on the main screen and the magnification. The dose is reported in electons persquare nanometer per second. In order to derive the dose for the actual exposure, multiply by theexposure time used. If the small (focusing) screen is out, the diameter of the beam must be entered(unless it is larger than the main screen). If the small (focusing) screen is in, the beam must be largerthan the small screen for a proper measurement.Dose measurement is continuous if the Continuously check box is checked. However, the Dosemeasurement will only run in the Exposure state (if Low Dose is switched to another state, the dosemeasurement is stopped). You can stop a continuous dose measurement by pressing the (now yellow)button again. If the Continuously check box is unchecked, a single dose measurement will be done.The dose measurement assumes a fudge factor of 1.4 for the loss of backscattered electrons from themain or small screen (determined for 200 kV by comparison of the screen current and currentmeasurement with a proper Faraday cage). Should you find that the dose measurement on yourmicroscope deviates significantly from measurements by other methods, then it is possible to adjust thefudge factor in the registry (a string value in HKEY_LOCAL_MACHINE\Software\Philips\LowDose\BSFactor).

<strong>Tecnai</strong> on-line help Options 26Options Software version 21.7 TV / CCD tabThe TV / CCD tab of the Low Dose Control Panel contains the information that Low Dose uses withregard to cameras (TV and CCD). The system works in combination with the settings defined for theSearch, Focus and Exposure states. In some cases Low Dose automatically detects what cameras areon the system, while in other cases the user has to add some. The following are detected automatically:• If an off-axis TV is present in the microscope software configuration, the off-axis TV-rate camera boxis always checked (the user cannot remove the check mark). The same applies to the "TV-ratecamera present check box".• If a Gatan Imaging Filter is present, the GIF TV camera (retractable) is assumed to the present andthe box (bottom left) is checked. Once again, the user cannot remove this check.• All CCD cameras are automatically detected and their location is either retrieved from the storedmicroscope configuration (e.g. Tietz cameras) or from their name (e.g. Gatan). In the case when thenaming of a camera is possible, the names must be as follows: if the CCD camera is in the wideangleport (above the projection chamber), the name must have the character sequence "WA"; if thecamera is that of a GIF (Imaging Filter), the character sequence "GIF" must be in the name; if it is anoff-axis CCD, the character sequence "off-axis" must be present in the name. Upper- or lowercasedoes not matter. All cameras not having the above character sequences are assumed to be on onaxisbelow the projection chamber. Cameras identified are indicated by check marks in the boxes onthe right-hand side of the schematic microscope picture. Once again, these check marks cannot bechanged.

<strong>Tecnai</strong> on-line help Options 27Options Software version 21.7.1 Low Dose State mediaBecause CCD cameras are not always accessible (depending on whether the controlling software isrunning and/or the camera is switched on), the system used for selecting the media for the different LowDose states has been set up as flexibly as possible. The accessible media are always shown in themedia lists on the Settings tab. There are potentially ten media:• Screen - for Search and Focus• Plate - for Exposure• Four different TV positions - for Search and Focus• Four different CCD positions - for all statesA medium is identified by its position, not its name. Thus, if you can access a particular CCD throughdifferent controllers, the medium will be chosen on the basis of where it is located, even if you changecontroller. If a medium is not accessible (e.g. a CCD controller has not been started or shut down), it willdisappear from the lists. The selection in the list will temporarily default to screen or plate. When themedium becomes accessible, it will re-appear in the lists and the selection will change back to themedium as before - unless the user changes the medium selection in the list.The same thing logic applies to the selection when loading settings files. If the medium is available, it willbecome selected. If not, it will only become selected once the medium is available and the defaultmedium is selected in the meantime). Note that what is saved in the settings files are the media thatwould be selected when the proper CCD controller(s) is(are) present, and not necessarily what iscurrently displayed in the lists as chosen.An exampleIn the example we have a system with an Imaging Filter plus an off-axis TV camera. The user sets upthe system such that:• Search is on the off-axis TV camera. Because the off-axis shift in EFTEM is too limited at lowmagnifications, the Search magnification is chosen sufficiently high that the off-axis image shift canreach the camera (note that Low Dose does not change lens series - normal to and EFTEM or viceversa).• Focus is defined on the GIF TV-rate camera.• Exposure is on the GIF CCD camera.When changing states, Low Dose will automatically select the appropriate detector position, lift theviewing screen, and insert or retract the GIF TV camera. When DigitalMicrograph is not available, themedia will default to off-axis TV (since this does not rely on DigitalMicrograph), screen (the GIF TVcamera control is only through DigitalMicrograph) and plate (the CCD camera is not available). IfDigitalMicrograph becomes available again, the media will automatically be switched back to theprevious selection.TV-rate camera presentIf a TV-rate camera is present, it can be used in the Search and Focus states. Whenever possible, theLow Dose software will help in using the TV-rate camera, either by reminding the user to insert thecamera or by automatically lifting the screen (when the TV-rate camera is located underneath theviewing screen) and/or inserting the camera.CCD camera controllerIf a CCD camera is present, it can be used in the Search and Focus state for viewing and in theExposure state for recording. In all cases the Search, Focus and Acquire setups must be done in the<strong>Tecnai</strong> CCD/TV Control Panel. Low dose simply uses the predefined setups (Search = Search, Focus =Preview, Exposure = Acquire) and does not change any settings (except the exposure time used for theLow Dose Exposure). If more than one controller is present, you can change the controller to be used byselecting one from the drop-down list (this function is identical to that in the CCD/TV Control Panel). Thecontroller is not specific to Low Dose. Low Dose does not automatically select a controller.

<strong>Tecnai</strong> on-line help Options 28Options Software version 2TV rateIn the schematic diagram of part of the microscope there are four possible locations for TV-rate cameras,the Wide-Angle TV port (above the viewing screen), directly below the viewing screen (on- and off-axis)or further below (e.g. a GIF). If more than one TV-rate camera is present, select the particular option forthe TV-rate camera that will be used during Low Dose imaging.CCDIn the schematic diagram of part of the microscope there are four possible locations for CCD cameras,the Wide-Angle TV port (above the viewing screen), directly below the viewing screen (on- and off-axis)or further below (e.g. a GIF).

<strong>Tecnai</strong> on-line help Options 29Options Software version 21.8 Low Dose SettingsThe following values are stored with the Low Dose Settings, either as user settings or in a file :Options• Reset Search, Focus, Exposure settings for current mode or all• Peek enabled• TV/CCD use per state• TV/CCD settings• Multifunction knobs enabled or disabled in Search and Focus states• Normalizations selected for Low Dose state transitions• Switch from Search to Focus via Exposure• <strong>Tecnai</strong> User Button assignmentExposure settings• Exposure time• Waiting time after plate in• Pre-exposure used and time• Pre-exposure waiting time• Exposure series enabled/disabled• Exposure double enabled/disabled• Exposure series defocus values• Exposure double time values• For spotscan: use spotscan for exposure, and spotscan settings like dwell time and amplitudes,focus correction enabled/disabledSearch state• Mode• Magnification• Camera Length• Spotsize• Intensity• Focus of Objective lens (for HM image and LAD), of Diffraction lens for (HM D and LM Image)• Beam Shift• Image Shift• Diffraction ShiftFocus state• Substate (1 or 2)• Magnification• Camera Length• Spotsize• Intensity• Beam Shift (both substates separately)• Image Shift (both substates separately)

<strong>Tecnai</strong> on-line help Options 30Options Software version 2Exposure state• Mode• Magnification• Camera Length• Spotsize• Intensity• Focus of Objective lens (for HM image and LAD), of Diffraction lens for (HM D and LM Image)1.9 Low Dose calibrationsThe following values are stored with the Low Dose Calibrations, either as user settings or in a file :Image shift / A Tilt :• LM• HMBeam Blanker amplitudePeek amplitudeSpotscan beam shift• HM• LM• Nanoprobe

<strong>Tecnai</strong> on-line help Options 31Options Software version 22 Parfocal magnification series (FP5409/00)The parfocal magnification series consists of an adaptation of the <strong>Tecnai</strong> software that allows a fineadjustment of the focus setting, thereby reducing focus changes between different magnifications. Theadjustment consist of a user-calibrated change in the objective-lens current for each step of the SAmagnifications relative to the highest SA magnification. The calibration takes place in the HM-Imagealignment procedure, SA image subprocedure (which is modified relative to microscopes withoutparfocal magnification).For attaining the highest degree of reproducibility of focus settings, the following should be done:• During alignment and use the beam (Intensity setting) should be as similar as possible. This can beachieved by setting the beam to a defocus that has the total beam just covering the wholefluorescent screen. In the alignment procedure (and during use) this can be done byswitchingIntensity Zoom on (once it has been calibrated through the HM-Beam procedure, Spot-intensitycalibration subprocedure).• Normalize all lenses (especially the minicondenser, objective and projector) during the alignment andwhenever the magnification is changed. The latter can be set to be executed automatically onchange of magnification in the Normalizations Control Panel.

<strong>Tecnai</strong> on-line help Options 32Options Software version 23 TEM Photomontage (FP5456/00)3.1 IntroductionA photomontage consists of a series of images that together make one large image. The advantage of amontage over a lower-magnification image is the fact that the individual images of the montage arerecorded at higher magnification and thus allow resolving finer details. By photomontage it is possible,for example, to record detailed images and still allow an overview of the whole specimen areainvestigated. A photomontage can in principle be executed by hand. It is, however, difficult to ensure thatsufficient overlap exists between the images to avoid gaps in the montage and at the same timeminimize the number of images needed. The TEM Photomontage option makes it possible to define andautomatically execute recording of a complete photomontage.3.2 Getting startedThe TEM Photomontage option consists of two parts:• The Photomontage Control Panel.• The Photomontage Display window.To use the TEM Photomontage first use the <strong>Tecnai</strong> Workspace layout to drag the Photomontage ControlPanel into a workset. Go to the tab of the workset and press the Display button in the PhotomontageControl Panel. The Photomontage Display window will be loaded and its functionality become available.You can leave the Photomontage Control Panel in your workset for future use. The Display window willonly be loaded when the Display button is pressed.The major part of the Photomontage functionality is present only in the Display window. The ControlPanel also provides rapid access to the more often-used functions.3.3 Photomontage Control PanelPhotomontage ontrolpanels for Plate (left) andCCD (right) as recordingmedium.The Photomontage Control Panel contains two sets of functions for photomontage:• Functions that load the Photomontage Display window (a separate window that is typicallypositioned in the data space of the <strong>Tecnai</strong> User Interface) or determine its size.• Functions that give rapid access to often-used photomontage functions.

<strong>Tecnai</strong> on-line help Options 33Options Software version 2DisplayThe Photomontage Display window is only loaded on operator request. To load it press the Displaybutton. A message will appear that the Photomontage server is being loaded. Once the server isrunning, the window will be displayed, the Display button will turn yellow, and the message willdisappear. You are now ready to start working with Photomontage.If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded butsimply hidden. If you display it again (by once more pressing the button), it will still have kept all thephotomontage settings as set previously. The window is only unloaded when you close the <strong>Tecnai</strong> UserInterface.Size adjustableWhen this option is checked, the Display window can be positioned anywhere you like (the software willremember your settings and restore them) and you can define its size in the standard Windows way(click and drag on a border). If the option is off, the Display window will be fixed in size and position to fillthe data space of the <strong>Tecnai</strong> User Interface.The following controls will be enabled or disabled when their counterpart in the Display window isenabled or disabled.Add point / Acq imageFor Plate medium, adds a point to the series of boundary points that define the photomontage area. ForCCD, it starts the sequence for obtaining the reference image.Set magn.Starts the procedure to define the magnification to be used for the montage.Calib rot.Starts the rotation calibration procedure (Plate only) for the currently active magnification.ModifySwitches montage modification (Plate only) on or off. When modification is active, the Modify button willbe yellow.AutoStarts the Automatic Exposure procedure for Plate or CCD as medium . When Photomontage is in theAutomatic Exposure procedure, the Auto button will be yellow.ManualStarts the Manual Exposure procedure (Plate only). When Photomontage is in the Manual Exposureprocedure, the Manual button will be yellow.MountStarts the Mount procedure for CCD Photomontage. When Photomontage is in the Mount procedure, theMount button will be yellow.

<strong>Tecnai</strong> on-line help Options 34Options Software version 23.4 Photomontage displayThe Photomontage Display window is a window that is typically located in the data space of the <strong>Tecnai</strong>User Interface. It can either be freely positioned and sized or fixed. The choice between the two is madein the Photomontage Control Panel. The display contains two essential elements, the menu and thedisplay itself.TEM Photomontage has two major modes of operation :• Recording of the images on plate negatives. This mode of operation is the same as on older softwareversions, except that the possibility of manually recording CCD images is no longer available.• Recording of the images on CCD. This mode of operation is new to software version 2.1.8/3.0. It isonly available in combination with TIA CCD.Because the two modes of operation are substantially different, they are described separately below.adds a point to the series of boundary points that define the photomontage area. For CCD, it starts thesequence for obtaining the reference image.

<strong>Tecnai</strong> on-line help Options 35Options Software version 23.5 Recording on plate negativesPhotomontage with recording on plate will always use the stage for moving from one exposure toanother. The area covered by the montage is determined by driving the stage to a series of positions atthe perimeter and adding these to the boundary points.The main functionality of Photomontage resides in the menu of the display window. A typical sequencefor setting up and executing a plate-negative photomontage is as follows:• If necessary, check that all options are defined as needed.• Define the photomontage area by bringing a series of points on the perimeter of the area to thecenter of the screen with the specimen stage and using Add boundary point to enter it.• Select the magnification to be used for the photomontage.• If necessary, calibrate the rotation.• If necessary, modify the montage suggested by the software.• Execute the exposure of the montage, either automatically or manually.• Store and/or print the data for later reference.3.5.1 The displayThe window will display the photomontage, showing the area overlaid with the individual exposures.Initially the exposures are simply labelled by their sequence number. Once an exposure has beenrecorded, it number will be replaced by the exposure number (provided there is enough room for thelonger labels). The colors used for the display are freely user-configurable.MenuThe menu gives access to all photomontage functionality.

<strong>Tecnai</strong> on-line help Options 36Options Software version 2MontageThe montage is displayed in the window. The gray area is the area of the specimen covered. The greenrectangles are the individual exposures, labelled here still with sequence numbers.Stage axesThe x,y directions of the stage axes are indicated on the screen.Scale barThe scale bar gives an indication of the sizes of the photomontage area and individual exposures.Status barThe status bar lists on the left the current stage position. On the right it will display hints for gettingstarted, messages in case of errors, or the current setting of the magnification used for the montage andthe number of exposures in the montage.Note: The magnification listed is the so-called reference value (the value on the plate camera, that is,the one seen with the screen up). This fixed value is used to avoid confusion (which could occur whenphotomontage would continuously adjust values when the screen goes up or down). The values with thescreen down are roughly 10% lower than with the screen up.Border iconsThe border icons appear to behave as the standard Minimize, Maximize and Close buttons. The Closebutton, however, does not truly close the window but hides it. The window is closed only upon closingthe TEM User Interface. The Maximize button is only visible if the window size can be adjusted freely.3.6 The menuThe menu consists of a main menu with submenus:• File• Medium• Calibrate• Setup• Montage• Exposure• Help3.6.1 File menuThe File Menu provides the operations that are concerned with file operations, the settings and printing.New montageOpen listSave list,Save list asSave graphic,Save graphic asControl optionsLoad optionsSave optionsPrinter setupPrint listPrint graphicStarts a new montage (resetting all previous montage values).Not enabled in on-line version (off-line only)Saves the list of exposures with their X,Y locations and exposure numbers in atext file, Save as allows definition of different file nameSaves the current display as a bitmap, Save as allows definition of a different filename.Leads to the Control options dialog.Loads options from file.Saves options to file.Leads to printer setup dialog.Prints the list of exposures with their X,Y locations and exposure numbers.Prints the current graphical display.

<strong>Tecnai</strong> on-line help Options 37Options Software version 23.6.2 Medium menuWith the Medium Menu, the operator chooses which of the two media (Plate, CCD) is active. CCD willonly be enabled if TIA CCD is present and running.3.6.3 Calibrate menuThe Calibrate Menu has functions for CCD Photomontage only.3.6.4 Setup menuWith the Setup Menu, the operator defines the boundaries of the photomontage, sets the magnificationand, if needed, determines the rotation angle between the specimen-stage axes and the camera.Add boundary pointSet magnificationCalibrate rotationInsert the current specimen-stage X,Y location into the list of points that definethe photomontage boundary.Defines the magnification to be used for the photomontage. If the rotationcalibration has already been done and the rotation calibration option is off, thephotomontage will be calculated immediately after this procedure.Starts calibration procedure for determining the rotation angle between thephotomontage boundaries and the camera. The photomontage will be(re)calculated immediately after this procedure.3.6.5 Montage menuThe Montage Menu contains a number of functions that are related to the exposures: modifying thephotomontage and displaying a list of the montage X,Y locations.Modify montageInsertDeleteDelete from listUndo changesDisplay listBack to setupStarts or stops modification of the photomontage: mouse double-click or clickand drag, Cursor keys, Ctrl Cursor keys, Insert, Delete and Undo changesbecome active or inactive. When the menu item is checked, modification isactive. Modification will automatically be switched off when certain otherprocedures like Exposure are started.Inserts a new exposure at the cross set by mouse double click. The newexposure will be fit to the existing montage, not centered at the cross.Deletes the exposure at the cross set by double clicking the left-hand mousebutton.Allows deletion of one or more exposures by number from a list displayed.Undo changes will revert to the photomontage defined before Modify montagewas switched on.Shows the list of exposure, with sequence number, and X,Y locations, and ifavailable the exposure number. The list can be printed directly from here (aswell as from the File menu).Removes the currently defined montage and switches back to the situationwhere boundary points can be defined or deleted.3.6.6 Exposure menuThe Exposure Menu contains the functions that are related to the performing the exposure of thephotomontage.AutomaticManualStarts automatic exposure procedure.Starts manual exposure procedure.

<strong>Tecnai</strong> on-line help Options 38Options Software version 23.6.7 Help menuThe Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display thisHelp file in the <strong>Tecnai</strong> User Interface. About will display the About TEM Photomontage dialog listing thecurrent version number of the software. When reporting bugs, always include the version number in thereport.3.7 Control optionsThe software supports a range of options, defined in the Control options dialog. Options are stored asset for each user separately and recalled upon restart. Options can be saved to file and reloaded.Plate typeThe software supports rapid selection of exposure sizes from any of the three pre-defined plate cameraformats (the inch size, European cm format and Japanese cm format).The Camera dimensions consists of two values, stored separately for each camera, for the horizontaland vertical size of the exposure. In the case of the film cameras, horizontal is left to right for themicroscope operator (longest dimension for plate film), vertical is towards and away from the operator.It is advisable to check the values for the system used. Measure on a used negative the longest(= horizontal) and shortest (= vertical) exposed area (discounting the plate number). Subtract 2 to 3 mmto account for reproducibility of the plate camera.CCDNot relevant for plate photomontage.OverlapThe overlap between the exposures is the percentage of the horizontal or vertical axis that must becommon to both exposures to ensure fit of the photomontage without gaps. The overlap can be seteither automatically or at a fixed value. If done automatically the program will adjust the overlap for themagnification used (a higher value at higher magnification, typical values are 10% at 5000x or below,20% at 30kx, 30% at 50kx).Note: It is allowed to define a negative fixed overlap value. In that case the individual exposures will notoverlap but instead be spaced apart. In this way an area can be covered by a regular grid of exposureswithout requiring them to fill the whole area. This function can be used for example for statisticalanalysis.

<strong>Tecnai</strong> on-line help Options 39Options Software version 2A regular grid of non-overlapping exposures on an area.ExposureThe Exposure options concern whether a sound signal should announce start and finish of individualexposures and how long the software should wait after stage movement before taking an exposure.RotationIf the Rotation option Always calibrate is switched on, the software will start the rotation calibrationprocedure automatically whenever a new montage is defined (directly after Set magnification). Otherwisethe software will use a stored value for the current magnification. If no such value exists, the rotationmust be calibrated before the montage is calculated.Note: Although the magnifications are in principle rotation-free on most <strong>Tecnai</strong> instruments, deviationsup to several degrees can exist and these can affect the accuracy of photomontage, especially withsmall overlaps. The rotation calibration must be still be done therefore.ColorsThe four types of colors (Background, Area, Exposures, Labels) for the display can be freely selected.The selection is made in the Control options dialog or by clicking on the window with the right-mousebutton. The latter will popup a menu that allows definition of the colors.Note: There is one exception to free color selection: if hyperlabels are used the Label color cannot beblue (this is the flash color of the hyperlabel).Use hyperlabelsThe software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: whenthe cursor moves over them they flash their color to blue and a mouse-click brings up a dialog withinformation about the point, with additional functionality such as Go to or Delete). The labels are used inthe boundary point definition phase to display the boundary points themselves. Thereafter the labels arethose of the exposures.

<strong>Tecnai</strong> on-line help Options 40Options Software version 23.8 Setting up for a montageThe setup for a montage follows these steps:• Define the boundary points for the montage area.• Define the magnification to be used.• If required, calibrate the rotation between the stage axes and the image axes.3.9 Define boundary pointsThe boundary points define the perimeter of the photomontage area. The perimeter can be any shapedefined by a polygon of up to 500 points. These points themselves are determined simply by moving thespecimen stage so a point on the boundary lies in the center of the viewing screen. After a minimum oftwo points have been loaded, the program will display a gray area that represents the photomontagearea (for two points it will be a line).One aspect is critical in determining the boundary points. They must follow the perimeter of thephotomontage in their proper sequence (starting at one point and going around until the same startingpoint is encountered again). If a point is out of sequence the program may display the photomontagearea with gaps as in the left-hand image below.Improper sequence.Point 4 from left figure removed.The strange shape of the area in the left-hand picture is the result of intersecting lines and does notproperly represent the photomontage because point 4 was loaded between points 3 and 5 instead ofbetween points 1 and 2. If a point is found to be out of sequence, then use the hyperlabel option todelete that point.3.10 Set magnificationSet magnification is the Setup step where the magnification for the photomontage is defined. Theoperator is asked to set the microscope magnification to the desired value, after which the program will

<strong>Tecnai</strong> on-line help Options 41Options Software version 2read the value form the microscope. If no rotation calibration is necessary the software will proceed bycalculating the photomontage. First it quickly estimates the number of exposures required to cover themontage area. If the estimate indicates more exposures than the default plate stock of 56 exposures, awarning is given. The operator can then proceed or reset the magnification to a lower value.After the estimate the montage is calculated. Dependent on a variety of factors the suggested montagemay be good enough or it may require modification. In the former case you can proceed directly toautomatic exposure (plate camera only) of manual exposure.3.11 Calibrate rotationRotation calibration is the setup step wherein the program is informed about the rotation angle betweenthe specimen stage X,Y axes and the camera. This is done simply by moving a recognisable imagedetail from one screen marker on the left to one on the right .The Rotation calibration dialog during the first step. In thesecond step the feature selected is moved to the markeron the right-hand side.Once a rotation value has been determined for a particular magnification, it will be stored by thesoftware.3.12 Modifying a montageThe montage suggested by the software can be modified in various ways. In order to modify themontage, you have to switch modification on. During a modification procedure, the changes can beundone. The Undo will revert the montage to its configuration before modification was switched on. Youcan go through repeated cycles by switching modification on and off again.During modification mouse double-click and mouse click-and-drag operations on the window have thefollowing meaning.• Mouse double-click puts a cross marker at the point where the cursor was during the double click. Ifthe cross lies inside an exposure (don't go too far towards the edge; also don't click on the label if it isa hyperlabel, because then the hyperlabel dialog comes up) that exposure can be deleted. If thecross lies near a potential exposure location, that location can be inserted as a new exposure.• Mouse click-and-drag shifts the exposures relative to the montage area (the actual shift is onlydisplayed once the mouse button is released.

<strong>Tecnai</strong> on-line help Options 42Options Software version 2Exposure modification can be one of the following:• Shift the whole montage• Insert a new exposure (after a cross has been positioned with a double-click)• Delete an existing exposure (after a cross has been positioned with a double-click; by clicking on ahyperlabel and selecting Delete; or Deleting one ore more exposures from a list. In the list multipleexposures can be selected by Click - Shift-Click or Click - Ctrl+Click).Shifting the whole montage can be done in various ways:• Click the left-hand mouse button somewhere on the montage and drag the corresponding montagepoint to a new location. The new montage will be redrawn as soon as the mouse button is released.• Use the cursor (arrow) keys to move the montage up, down, left or right. Regular cursor keys moveone pixel, Pressing the Control (Ctrl) simultaneously with the cursor key will shift by ten pixels.3.13 Automatic exposureAfter a photomontage has been defined, it is possible to have the program record the whole montageautomatically. When Automatic exposure is started, the following dialog is brought up.The Automatic exposure progress dialog.At the top the software indicates the progress of the procedure (in this case no exposures have beenrecorded yet). An estimated total duration is given. This is subject to considerable uncertainty becausevarious procedures that can be time-consuming (stage movement, screen up/down) are only accountedfor in a general overhead on top of the exposure time.The Sound on checkbox allows changing the Sound option during the actual procedure.Underneath the progress bar various values are listed such as the currently available exposure stock,exposure number and exposure time. For the exposure time two options exist if automatic exposure timeis selected on the microscope. By default the software simply uses the exposure time as currentlymeasured (this implies that during the whole exposure sequence the viewing screen will remain up). Ifthe Measure for each checkbox is checked, the screen is lowered for each exposure to allow themicroscope to measure the exposure time for each individual exposure. Since the small focussing is notmotorized, the exposure time will always be read off the main screen then.Finally at bottom right there are two selections (for plate camera only) that allow automatic modificationof the film text on the plate. If the first is checked, a line is entered in the film text with 'Montage exposure#' where # is replaced by the sequence number of the exposure. If the second is checked a line is

<strong>Tecnai</strong> on-line help Options 43Options Software version 2entered with 'Loc X xxx.xx Y yyy.yy' where xxx.xx and yyy.yy are replaced by the stage x,y location ofthe exposure. Both options can be used together. Existing film text will be shifted down (if more than 4lines exist after insertion of the montage data, the excess lines will be lost and not restored afterwards).After the exposure procedure is stopped, the film text is restored to its original setting (minus any excesslines lost).Exposures that have been recorded will have their plate exposure numbered entered in the list and, ifsufficient space is available, their label on the screen is also replaced by the exposure number.Note 1: The procedure will stop or pause when an error is encountered, e.g. plate stock zero. In thatcase the operator can rectify the condition leading to the error and continuing with the procedure.Note 2: While exposing (both automatic and manual), the program uses a backlash correction procedureto make the positioning the exposures more accurate. However, because the backlash correction(probably) wasn't done during definition of the boundaries, there may be a systematic shift of the wholepattern of exposures of up to about 500 nm. For montages at high magnifications it is important thereforeto check the exposure locations (e.g. go to selected exposure locations through hyperlabels), especiallyif the edge of the area covered by the exposures is close (less than 1 um) to the intended photomontagearea.3.14 Manual exposureAfter a photomontage has been defined, the montage can be exposed manually. When Manualexposure is started, the following dialog is brought up.The Manual exposure progress dialog.At the top the software indicates the progress of the procedure (in this case no exposures have beenrecorded yet).Underneath the progress bar various values are listed such as the currently available exposure stock,exposure number and exposure time.At bottom right there are two selections (for plate camera only) that allow automatic modification of thefilm text on the plate. If the first is checked, a line is entered in the film text with 'Montage exposure #'where # is replaced by the sequence number of the exposure. If the second is checked a line is enteredwith 'Loc X xxx.xx Y yyy.yy' where xxx.xx and yyy.yy are replaced by the stage x,y location of theexposure. Both options can be used together. Existing film text will be shifted down (if more than 4 lines

<strong>Tecnai</strong> on-line help Options 44Options Software version 2exist after insertion of the montage data, the excess lines will be lost and not restored afterwards). Afterthe exposure procedure is stopped, the film text is restored to its original setting (minus any excess lineslost).Exposures that have been recorded will have their plate exposure numbered entered in the list and, ifsufficient space is available, their label on the screen is also replaced by the exposure number. IF theexposures are recorded on a CCD camera the exposure numbers given are 'CCD1', 'CCD2', etc.Note: While exposing (both automatic and manual), the program uses a backlash correction procedureto make the positioning the exposures more accurate. However, because the backlash correction(probably) wasn't done during definition of the boundaries, there may be a systematic shift of the wholepattern of exposures of up to about 500 nm. For montages at high magnifications it is important thereforeto check the exposure locations (e.g. go to selected exposure locations through their hyperlabels),especially if the edge of the area covered by the exposures is close (less than 1 um) to the intendedphotomontage area.

<strong>Tecnai</strong> on-line help Options 45Options Software version 23.15 Recording on CCD and automatic montageThe area covered by the montage is determined by recording a reference CCD image at a magnificationwhere the whole area of the montage is within the field of view and marking the perimeter of themontage in that image.Note 1: Two magnifications are involved in the montage, the magnification at which the reference imageis acquired and the magnification at which the individual montages images are acquired. In order toensure the software is able to record the montage images at the correct locations, it is very importantthat these two magnifications are accurately aligned to each other. To check, it is best to find arecognizable image feature, center that in the CCD (search) image at one magnification (mark theposition by putting in an image marker), then change to the other magnification (you may need to usenormalization to obtain accurate image positioning) and check that the feature is also exactly in thecenter at this magnification.Note 2: Recording and mounting a CCD montage requires a good deal of memory. If there are too manyimages the system will slow down very much. Before starting a montage, check the amount of memoryavailable in Task Manager (right-click on an empty part of the Taskbar and select Task manager, selectthe Mem usage in the Performance tab. After the montage exposure, check again. If the amount ofmemory remaining is not at least as much as was needed for the montage acquisition, do not mount themontage).Note 3: As an alternative to image acquisition into a data series in TIA and final assembly of the imagesinto a montage image by the photomontage software, you can acquire the images for storage in files. Inthis way you can acquire any number of images, unlimited by memory, but you cannot have the softwareassemble the images into a montage image.Supported file formats for image acquisition to file are Extended-header and Small-header binary (seesection 3.25) as well as MRC file format (see section 3.26), the latter either into separate images or asingle file containing the whole series.The main functionality of Photomontage resides in the menu of the display window. A typical sequencefor setting up and executing a plate-negative photomontage is as follows:• If necessary, check that the options are defined as needed.• Define the photomontage area by acquiring a CCD image - the reference image - that contains thewhole area that the photomontage should cover and using Acquire image to retrieve the image fromTIA.• Select the magnification to be used for the photomontage.• Execute the exposure of the montage.• Execute the montage mount.

<strong>Tecnai</strong> on-line help Options 46Options Software version 23.15.1 The displayThe window will display the reference image with the photomontage image boundaries overlaid.MenuThe menu gives access to all photomontage functionality.MontageA schematic view of the montage is displayed in the window. The reference image is in the background,with overlaid on it the boundary as defined in red outlined by white. The blue-white lines indicate are theindividual exposures. The individual montage images will be in an image series in TIA. After mountingthe mounted montage will also be in TIA. Both image series and montage are located in a displaywindow titled "Montage".Scale barThe scale bar gives an indication of the sizes of the photomontage area and individual exposures.Status barThe status bar lists on the left the current stage position. On the right it will display hints for gettingstarted, messages in case of errors, or the current setting of the magnification used for the montage andthe number of exposures in the montage.Note: The magnification listed is the so-called reference value (the value on the plate camera, that is,the one seen with the screen up). This fixed value is used to avoid confusion (which could occur whenphotomontage would continuously adjust values when the screen goes up or down). The values with thescreen down are roughly 10% lower than with the screen up.

<strong>Tecnai</strong> on-line help Options 47Options Software version 2Border iconsThe border icons behave as the standard Minimize, Maximize and Close buttons. The Close button,however, does not truly close the window but hides it. The window is closed only upon closing the <strong>Tecnai</strong>User Interface. The Maximize button is only visible if the window size can be adjusted freely.3.16 The menuThe menu consists of a main menu with submenus:• File• Medium• Calibrate• Setup• Montage (montage modification is not available for CCD montage)• Exposure• Mount• Help3.16.1 File menuThe File Menu provides the operations that are concerned with file operations, the settings and printing.New montageOpen listSave list,Save list asSave graphic,Save graphic asControl optionsLoad optionsSave optionsPrinter setupPrint listPrint graphicStarts a new montage (resetting all previous montage values).Not enabled in on-line version (off-line only)Saves the list of exposures with their X,Y locations and exposure numbers in atext file, Save as allows definition of different file nameSaves the current display as a bitmap, Save as allows definition of a different filename.Leads to the Control options dialog.Loads options from file.Saves options to file.Leads to printer setup dialog.Prints the list of exposures with their X,Y locations and exposure numbers.Prints the current graphical display.3.16.2 Medium menuWith the Medium Menu, the operator chooses which of the two media (Plate, CCD) is active. CCD willonly be enabled if TIA CCD is present and running.3.16.3 Calibrate menuThe Calibrate Menu has functions for CCD Photomontage only. The execution of a CCD montagerequires calibration of the TEM image, the image shift and stage shift. To avoid unnecessary duplication,Photomontage uses the calibration procedures and stored settings of TEM AutoAdjust. Whichcalibrations can done can be determined from the menu. Calibrations that are disabled require thecalibration above it to be done. The Setup menu will only be enabled if all three calibrations have beendone.MagnificationImage shiftStage shiftExecutes the magnification calibration.Executes the image-shift calibration.Executes the stage-shift calibration.

<strong>Tecnai</strong> on-line help Options 48Options Software version 2Calibrations have a set hierarchy (some calibrations can only be done once others have been done).They are specific to a number of conditions :• CCD camera used in combination with the microscope lens series (Normal or EFTEM)• High tension• Microscope mode (LM, Microprobe, Nanoprobe, Lorentz)For the microscope modes, only the Nanoprobe mode does not require full calibrations. For that modeonly the Focus/Stigmator calibration must be done separately (the rest of the calibrations if the same asin the Microprobe mode).Calibrations can only be done by TEM Experts (private calibrations for these users) and Supervisor,Service or Factory (dependent on availability, these calibrations are what basic microscope Users get.Calibration is done on the basis of a standard specimen, a cross-grating. This specimen has squares onit with a spacing of 463 nm.3.16.4 Magnification calibrationThe software will acquire an image of the cross-grating and analyse that to find the basic spacing. Theanalysis is done using an auto-correlation (at high magnifications) or an FFT (at low magnifications) inwhich the two basic vectors will be outlined by circles.An image of a cross-grating, showing the 463 nm squares.The result of the magnification calibration. The autocorrelationof the cross-grating image (zoomed in 2x) shows the centralpeak pointed at by the line (which has been added manually forclarity). The software adds the two circles around the two basevectors of the pattern. It is important that these circles arelocated at the correct positions and not e.g. at the diagonals ofthe squares. Additional circles are placed at the multiples of thebase vectors used to show how many base vectors were usedtogether for the analysis.

<strong>Tecnai</strong> on-line help Options 49Options Software version 23.16.5 Image-shift calibrationThe image-shift calibration procedure performs a calibration of the image shift on the CCD. Thiscalibration must be done for all magnifications in the required range (up to magnifications of ~150kx).Normally the whole range is calibrated in one procedure, but it also possible to (re)calibrate the imageshift for an individual magnification (e.g. when it appears that the calibration in the complete procedurewent wrong for one magnification, that magnification can be redone later).Note: If the image-shift calibration fails at low magnifications because of the presence of strongcontrast from grid bars, go to the center of the cross-grating specimen and use the triangle areain the "A" to do the image-shift calibration.3.16.6 Stage calibrationThe stage calibration calibrates the stage x and y shifts.3.16.7 Setup menuWith the Setup Menu, the operator defines the boundaries of the photomontage, sets the magnificationand, if needed, determines the rotation angle between the specimen-stage axes and the camera.Acquire imageSet magnificationCalibrate rotationStarts the retrieval of the reference image from TIA.Defines the magnification to be used for the photomontage.Not enabled for CCD. Calibrations are under the Calibrate menu.3.16.8 Exposure menuThe Exposure Menu contains the functions that are related to the performing the exposure of thephotomontage.AutomaticRepeat at currentstage positionManualStarts automatic exposure procedure.Repeats the automatic exposure but allows you to do this at the current stageposition (otherwise the stage will move back to the position where the montagewas originally defined). This option only becomes enabled once the automaticexposure has been done. It may be useful in cases where you simply want torepeat recording the same pattern more than once.Not available for CCD montage.3.16.9 Mount menuThe Mount Menu contains the functions that are related to the mounting of the photomontage.ExecuteShow logPressing execute will start the automatic mounting of the montage images.Will display a log containing information about the mounting of the images.

<strong>Tecnai</strong> on-line help Options 50Options Software version 23.16.10 Help menuThe Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display thisHelp file in the <strong>Tecnai</strong> User Interface. About will display the About TEM Photomontage dialog listing thecurrent version number of the software. When reporting bugs, always include the version number in thereport.3.17 Control optionsThe software supports a range of options, defined in the Control options dialog. Options are stored asset for each user separately and recalled upon restart. Options can be saved to file and reloaded.Plate typeNot relevant for CCD montage.CCDWith CCD acquisition, there are two ways to handle the images:• Collect the images into a data series in TIA. This series can then be used later as the basis forgenerating the montage image. The number of images strongly depends on image size and memoryavailable.• Collect the images as a file series. Each image is saved to file but not kept in a data series in TIA.While there is no limit to the number of images (except hard-disk space), you have to use othersoftware to assemble the images into a single montage image.• The CCD Store images only is disabled if you have already acquired images. To switch betweendata series in TIA and files, you have to redefine the montage (File, New montage).OverlapThe overlap between the exposures is the percentage of the horizontal or vertical axis that must becommon to both exposures to ensure fit of the photomontage without gaps. The overlap can be seteither automatically or at a fixed value. If done automatically the program will adjust the overlap for themagnification used.Note: It is allowed to define a negative fixed overlap value. In that case the individual exposures will notoverlap but instead be spaced apart. In this way an area can be covered by a regular grid of exposureswithout requiring them to fill the whole area. This function can be used for example for statisticalanalysis.

<strong>Tecnai</strong> on-line help Options 51Options Software version 2A regular grid of non-overlappingexposures on an area.ExposureThe Exposure options concern whether a sound signal should announce start and finish of individualexposures and how long the software should wait after stage movement before taking an exposure.RotationNot relevant for CCD montage.ColorsNot relevant for CCD montage.Use hyperlabelsThe software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: whenthe cursor moves over them they flash their color to blue and a mouse-click brings up a dialog withinformation about the point, with additional functionality such as Go to). The labels are those of theexposures.3.18 Setting up for a montageThe setup for a montage follows these steps:• Acquire the reference image.• Define the area for the montage in the reference image.• Define the magnification to be used.3.19 Acquire imageThe reference image that is used to define the area covered by the CCD montage must be acquired inTIA under conditions defined in the CCD/TV control panel of the TEM user interface. Select a suitablemagnification and press the Acquire button in in the CCD/TV control panel of the TEM user interface to

<strong>Tecnai</strong> on-line help Options 52Options Software version 2acquire a CCD image. If the image does not cover the whole montage area, modify the conditions andrepeat.When you select Acquire image in thePhotomontage menu, instructions willappear in the Photomontage window.Photomontage will always used the mostrecently acquired CCD image as thereference. Once you click on OK, theimage will be retrieved and displayed inthe Photomontage window. You can thenrequired to define the montage area.3.20 Define areaAfter the reference image has beenretrieved from TIA, the followinginstructions will be displayed.The operator is now required to definethe montage area by double-clicking withthe left-hand mouse button on thereference image. Each click will defineone boundary point. Make sure to goround the perimeter in a regularsequence (starting at one point and goingaround until close to the starting point). Itis possible to back up, deleting points bypressing the minus key on the keyboard.Pressing Enter finalizes the areadefinition and automatically closes theperimeter definition.To make changes to the area definitionafter pressing Enter, you have to reacquirethe image.

<strong>Tecnai</strong> on-line help Options 53Options Software version 2The area is now defined. The next step isto set the magnification for the montageitself.3.21 Set magnificationSet magnification is the Setup step where the magnification for the photomontage is defined. Theoperator is asked to set the microscope magnification to the desired value, after which the program willread the value from the microscope. The montage is then calculated

<strong>Tecnai</strong> on-line help Options 54Options Software version 23.22 Automatic exposureAfter a photomontage has been defined, the recording step is done fully automatic. The exposureconditions (image size, binning and exposure time) are as defined for CCD Acquire in the CCD/TVcontrol panel of the TEM user interface. When exposure is started, the following dialog is brought up.The exposure progress dialog.At the top the software indicates the progress of the procedure (in this case no exposures have beenrecorded yet). An estimated total duration is given once a few images have been recorded (prior to thatthe time cannot be estimated).The Sound on checkbox allows changing the Sound option during the actual procedure.3.23 Mounting the exposures into the montageAfter the images have been recorded, they can be mounted into the montage. The mounting happensautomatically, with the following restrictions:1. If the images do not overlap, they are mounted in an uniformly gray image of suitable dimensions(with some margins along the sides) in the nominal positions they have in the specimen.2. If there is an image overlap but the amount of overlap is too small to check the registration betweenthe individual images, the images are mounted according to the nominal positions in the specimen.The overlap areas are faded together to reduce the visibility of sharp boundaries where the imagesmay not match perfectly.3. If there is sufficient overlap, the registration between the individual images is measured by crosscorrelationand the images are mounted accordingly.Note: If the mounted image with check on image positions by cross-correlation (case 3) shows largeshifts or the faded image mounting (case 2) does not produce a good fit, there are two potentialproblems:• The calibrations are not good. Redo the calibrations.• The images are distorted (lens distortions). Because lens distortions are smaller closer to the centralaxis of the microscope, use smaller CCD areas (if necessary in combination with more images) torecord CCD montages.

<strong>Tecnai</strong> on-line help Options 55Options Software version 23.24 File formatsThe file formats supported by photomontage are :• Small-header binary : binary data are preceded by a 10-byte header containing the data type andx,y dimensions (first three items of the the extended-header format).• Extended-header binary : the binary data are preceded by an extended header that contains thedata type, image dimensions, and several types of information stored specifically for Focus seriesreconstruction (see extended-header definition below). Note that depending on the particularsoftware that writes the file, not all values are necessarily defined.• MRC file : see separate file format description3.25 Binary file formats3.25.1 Binary data type1 - unsigned 1-byte integer2 - unsigned 2-byte integer3 - unsigned 4-byte integer4 - signed 1-byte integer5 - signed 2-byte integer6 - signed 4-byte integer7 - 4-byte (single-precision) floating point8 - 8-byte (double-precision) floating point3.25.2 Extended-header format2-byte integer : data type of the image (see above)4-byte integer : image width in pixels4-byte integer : image height in pixels4-byte integer : total size of the header in bytes (including preceding values). In effect the offset tothe data.double : Microscope type 0 = T10 and T12, 1 = T20, 2 = T30double : Gun LaB6 = 0, FEG = 1double : Lens type HC = -2, BioTWIN = -1, TWIN = 0, STWIN = 1, UTWIN = 2double : D number of microscopedouble : High tension (kV)double : Focus spreaddouble : MTFdouble : Starting defocus (nm)double : Focus step (nm)double : DAC settingdouble : Focus value (nm)double : Pixel size (nm)double : Spherical aberration (mm)double : Semi-convergence (mrad)double : Info limit (nm-1)double : Number of imagesdouble : Image number in seriesdouble : Coma xdouble : Coma ydouble : Astigmatism 2 Xdouble : Astigmatism 2 Y

<strong>Tecnai</strong> on-line help Options 56Options Software version 2doubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoublethree doublesdoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoublefurther doubles: Astigmatism 3 X: Astigmatism 3 Y: Camera type serial number: Camera position (WA, MSC, GIF): Magnification: Camera length: STEM magnification: Lens series: Lorentz: Spot size: Microscope mode: Objective lens value: Currently undefined: Image shift X (m): Image shift Y (m): Image shift X (pixels): Image shift Y (pixels): Stage X (m): Stage Y (m): Stage Z (m): Stage A (rad): Stage B (rad): Image minimum level: Image maximum level: Image real (0) or reciprocal space: undefinedMany of the values stored in the extended header are used by the TrueImage reconstruction software asa first guess to optimize the reconstructed wave function.

<strong>Tecnai</strong> on-line help Options 57Options Software version 23.26 MRC file formatThe MRC file is a file with extension .mrc. It contains a primary header and an extended header asdescribed below, followed by the data. The file format is derived from the MRC file format which wasoriginated at the Medical Research Council in Cambridge, England.• The MRC format can contain a series of images in a single file. The format consists of:• A primary header. A 1024 byte section.• An extended header. Additional information about the individual images.• Image data.3.26.1 The primary header.Data type Name Byte Commentpositioninteger (4-byte) nx 0 number of columsinteger ny 4 number of rowsinteger nz 8 number of sections (i.e. images)integer mode 12 data type, 1 = pixel value stored as shortintegerinteger nxstart 16 lower bound of columsinteger nystart 20 lower bound of rowsinteger nzstart 24 lower bound of sectionsinteger mx 28 cell size in Angstroms (pixel spacing=xlen/mx)integer my 32integer mz 364-byte floating point xlen 404-byte floating point ylen 444-byte floating point zlen 484-byte floating point alpha 52 cell angles in degrees4-byte floating point beta 564-byte floating point gamma 60integer mapc 64 mapping colums, rows, sections on axis(x=1,y=2,z=3)integer mapr 68integer maps 724-byte floating point amin 764-byte floating point amax 804-byte floating point amean 842-byte integer ispg 88 space group number (0 for images)2-byte integer nsymbt90 number of bytesused for storingsymmetry operators4-byte integer next 92 number of bytes in extended header. This is animportant number. It defines the offset to thefirst image2-byte integer dvid 96 creator idchar (byte) extra[30] 98 extra 30 bytes data (not used)2-byte integer numintegers 128 number of bytes per section in extendedheader2-byte integer numfloats 130 number of floats per section in extended

<strong>Tecnai</strong> on-line help Options 58Options Software version 2header2-byte integer sub 1322-byte integer zfac 1344-byte floating point min2 1364-byte floating point max2 140 extra 28 bytes data4-byte floating point min3 1444-byte floating point max3 1484-byte floating point min4 1524-byte floating point max4 1562-byte integer idtype 1602-byte integer lens 1622-byte integer nd1 164 divide by 100 to get float value2-byte integer nd2 1662-byte integer vd1 1682-byte integer vd2 1704-byte floating point tiltangles[9] 172 used to rotate model to match rotated image4-byte floating point zorg 208 origin of image; used to auto translate model4-byte floating point xorg 212 to match a new image that has been translated4-byte floating point yorg 2164-byte integer nlabl 220 number of text labels with useful data (0-10)char data[10][80] 224 10 text labels with 80 characters3.26.2 The extended headerTotal number of extended headers (one for each image) is 1024.Please note: Some values have rather odd numbering. That is because prior usage determined thatcertain values were fixed too low and the addition of more types dictated the necessity for numbersbelow, while 0 is generally avoided. The 0 is typically an undefined number.Name Byte position Commenta tilt 0 Alpha tilt in degreesb tilt 4 Beta tilt in degreesx stage 8 Stage position in umy stage 12z stage 16x shift 20 Image shift position in umy shift 24defocus 28 Defocus in micrometersexposure time 32mean intensity 36tilt axis angle 40pixel size 44 Image pixel size in nanometersmagnification 48Microscope type 52 <strong>Tecnai</strong> 10 = -2, <strong>Tecnai</strong> 12 = -1,<strong>Tecnai</strong> 20 = 1, <strong>Tecnai</strong> 30 = 2, forTitan add 10 to HT equivalentGun type 56 W = -2, Lab6 = -1, FEG = 1Lens type 60 HC = -3, BioTWIN = -2, TWIN = -1, STWIN = 1, UTWIN = 2,XTWIN = 3

<strong>Tecnai</strong> on-line help Options 59Options Software version 2D number of microscope 64High tension (kV) 68Focus spread 72MTF 76Starting Df (nm) 80Focus step (nm) 84DAC setting 88Spherical aberration 92Semi-convergence 96Info limit (nm-1) 100Number of images 104Image number in series 108Coma 1 112Coma 2 116Astigmatism 2 1 120Astigmatism 2 2 124Astigmatism 3 1 128Astigmatism 3 2 132Camera type number 136Camera position 140 WA = 1, MSC = 2, GIF = 3Unused 144Unused 148

<strong>Tecnai</strong> on-line help Options 60Options Software version 24 TEM Grid Scanning (FP5457/00)4.1 IntroductionDuring an investigation of a specimen on a TEM grid, it is often difficult to keep track of grid holesalready investigated and grid holes still to be done. Furthermore, moving from one grid to another usuallyrequires lowering the magnification, moving the specimen stage to the new hole and increasing themagnification again for a more detailed investigation. Such systematic searches are made much easierby TEM Grid Scanning.Grid Scanning makes use of the fact that grids are regular arrays of square or hexagonal holes. Once afew essential points are known (through a calibration procedure in Grid Scanning), the software cancalculate the position of other grid holes and the specimen stage of the <strong>Tecnai</strong> microscope (theCompuStage) can be moved from one grid hole to another by a simple keyboard or mouse command.Holes passed during the Grid Scanning are displayed on the screen, making it easy to keep track ofareas already investigated. Interesting specimen locations can be stored with comments added. Storedlocations can be retrieved easily. Grid Scanning data can be saved in a data file for later retrieval. Ifreference points are defined, the software recalculates old to new locations, making it easy to retrievepreviously stored locations, even if the grid is later not exactly in the same position.4.2 Getting startedThe TEM Grid Scanning option consists of two parts:• The Grid Scanning Control Panel.• The Grid Scanning Display window.To use TEM Grid Scanning first use the <strong>Tecnai</strong> Workspace layout to drag the Grid Scanning ControlPanel into a workset. Go to the tab of the workset and press the Display button in the Grid ScanningControl Panel. The Grid Scanning Display window will be loaded and its functionality become available.You can leave the Grid Scanning Control Panel in your workset for future use. The Display window willonly be loaded when the Display button is pressed.The major part of the Grid Scanning functionality is present only in the Display window. The ControlPanel also provides rapid access to the more often-used functions.4.3 Grid scanning Control panelThe Grid Scanning Control Panel contains two sets of functionsfor grid scanning:• Functions that load the Grid Scanning Display window (aseparate window that is typically positioned in the data spaceof the <strong>Tecnai</strong> User Interface) or determine its size.• Functions that give rapid access to often-used Grid Scanningfunctions.

<strong>Tecnai</strong> on-line help Options 61Options Software version 2DisplayThe Grid Scanning Display window is only loaded on operator request. To load it press the Displaybutton. A message will appear that the Grid Scanning server is being loaded. Once the server is running,the window will be displayed, the Display button will turn yellow, and the message will disappear. Youare now ready to start working with Grid Scanning.If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded butsimply hidden. If you display it again (by once more pressing the button), it will still have kept all the GridScanning settings as set previously. The window is only unloaded when you close the <strong>Tecnai</strong> UserInterface.Freely sizeableWhen this option is checked, the Display window can be positioned anywhere you like (the software willremember your settings and restore them) and you can define its size in the standard Windows way(click and drag on a border). If the option is off, the Display window will be fixed in size and position to fillthe data space of the <strong>Tecnai</strong> User Interface.The following controls will be enabled or disabled when their counterpart in the Display window isenabled or disabled.NextMoves the stage to the next hole.PreviousMoves the stage back to the previous hole.CenterMoves the stage back to the center of the current hole.AddAdds a stored location for the current stage position. If text has been inserted for a label, this text willautomatically be added to the location. If labels are not used, the label text is disabled. If labels are usedand the location label field in the Control Panel is empty, a dialog will be displayed in the Grid ScanningDisplay in which the label can be entered. (So the difference is that the label in the Control Panel isdefined before storing the location while in the display it is done afterwards.)Location labelText entered here will be added as a label to a location stored.

<strong>Tecnai</strong> on-line help Options 62Options Software version 24.4 Grid scanning displayThe Grid Scanning Display window is a window that is typically located in the data space of the <strong>Tecnai</strong>User Interface. It can either be freely positioned and sized or fixed. The choice between the two is madein the Grid Scanning Control Panel. The display contains two essential elements, the menu and thedisplay itself.The main functionality of Grid Scanning resides in the menu of the display window. A typical sequencefor setting up and executing a Grid Scanning is as follows:• If necessary, check that all options are defined as needed (some options cannot be changed laterunless New grid is select first in the File menu).• Define the grid hole parameters by bringing three special points on a grid hole and its neighbor to thecenter of the screen with the specimen stage during the calibration procedure.• Start moving from gird hole to grid hole, storing locations along the way.• If the data for the grid must be stored for later retrieval with the grid again in the microscope, definethe reference points.• Store and/or print the data for later reference.4.5 The displayThe window will display the a schematic outline of the grid (circle) with the stage axes. The currentlocation of the stage is indicated by a solid square (red in the image below). Grid holes passed andstored locations are also displayed, the grid holes in their proper orientation relative to the stage axes.Stored locations are indicted by their serial number. The colors used for the display are freely userconfigurable.

<strong>Tecnai</strong> on-line help Options 63Options Software version 2MenuThe menu gives access to all Grid Scanning functionality.Status informationGrid Scanning displays some status information in a legend at top left of the display. This includes thenumber of holes passed and of stored locations.Status barThe status bar lists on the left the current stage position. In the center it displays the major optionschosen for the grid scan. Error messages can be displayed on the right.Border iconsThe border icons appear to behave as the standard Minimize, Maximize and Close buttons. The Closebutton, however, does not truly close the window but hides it. The window is closed only upon closingthe <strong>Tecnai</strong> User Interface.4.6 The menuThe menu consists of five main menus with submenus:• File• Setup• Hole• Location• Help4.6.1 File menuThe File Menu provides the operations that are concerned with file operations, the settings and printing.New gridOpenSave,Save asControl optionsLoad optionsSave optionsPrinter setupPrint listPrint graphicStarts a new grid scan (resetting all previous montage values).Opens an existing grid scanning file.Saves the grid scanning data in a file, Save as allows definition of different filename.Leads to the Control options dialog.Loads options from file.Saves options to file.Leads to printer setup dialog.Prints the list of stored locations and labels.Prints the current graphical display.

<strong>Tecnai</strong> on-line help Options 64Options Software version 24.6.2 Setup menuWith the Setup Menu, the operator defines the parameters of the Grid Scanning, defines (or redefines incase of data reloaded from file) the reference points, and starts a new spiral scan.Calibrate gridReference pointsShow ref.pt. dataNew spiralInsert the current specimen-stage X,Y location into the list of points that define theGrid Scanning boundary.Starts procedure for storing reference points.After reference points from an old file have been found again, the software candisplay the relation between old and new reference points, such as rotation angleand relative shifts.Allows starting of a new spiral scan at a user-defined location.4.6.3 Hole menuThe Hole Menu contains the items that control movement of the specimen stage: to the next hole, backto the previous hole and back to the centre of the current grid hole. Where the next hole will be isdependent on the scan type chosen.Next holePrevious holeHole centerStart/resume autoStop autoMoves stage to next hole. How the movement is executed depends on the type ofscan chosen.Moves stage back to previous hole.Moves the specimen stage back to the centre of the current grid hole.In automatic movement mode the scan can be (re)started with start/resume auto.In automatic movement mode the scan can be stopped with stop auto.4.6.4 Location menuThe Location Menu contains a number of functions that are related to the locations stored.AddDeleteDisplay listShow numberAdds the current stage location to the stored locations list.Deletes one or more locations from the stored locations list.Displays a list with all stored locations (X, Y, label) and allows printing of the list.Shows a list of stored locations (with number and label), allowing selection of onelocation for which the data (X,Y, label) will be shown. A button Goto allows movingthe stage to the stored location.4.6.5 Help menuThe Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display thisHelp file in the <strong>Tecnai</strong> User Interface. About will display the About TEM Grid Scanning dialog listing thecurrent version number of the software. When reporting bugs, always include the version number in thereport.

<strong>Tecnai</strong> on-line help Options 65Options Software version 24.7 Control optionsThe software supports a range of options, defined in the Control options dialog. Options are stored asset for each user separately and recalled upon restart. Options can be saved to file and reloaded.Grid typeThe software supports two types of grid, with square and with hexagonal (honeycomb) holes. Identify thetype of grid before the calibration is startedScan patternThe scan pattern is one of four:A Spiral scan is a scan where the movement goes around in a spiral around thestarting point.Spiraling around the first hole (in the centre of the grid at the intersection of theblack lines that represent the X and Y axes), an increasing number of holes ispassed (follow the red lines with arrows). Passed holes are shown in their properorientation in green, the current stage position is shown by the red square.A Linear scan is a scan where the holes are arranged in lines thatstretch from one side of the grid to the other. These lines themselvesare accessed once again in a spiral.On a linear scan an increasing number of holes is passed (follow thered lines with arrows). Passed holes are shown in their properorientation in green, the current stage position is shown by the redsquare.A Cursor scan is a scan where the movement depends on the cursor keys pressed bythe operator (up, down, left, right).Starting at the first hole an increasing number of holes is passed (follow the red lineswith arrows) by pressing one of the keyboard cursor keys. The meaning of up, down,left, right is as seen on the display (not direction of motion on the microscope'sfluorescent screen). Passed holes are shown in their proper orientation in green, thecurrent stage position is shown by the red square.

<strong>Tecnai</strong> on-line help Options 66Options Software version 2A Random scan is a scan where the software calculates grid-holepositions on the basis of random moves in the X,Y direction. In this way itis possible to remove operator bias from the procedure and thus toreduce the number of grid holes that must be scanned for statisticalconfidence.Each hole passed is based on a random selection of X and Y position.Movement from one hole to the next is outlined by the red line andarrows.Scan typeThe Scan type can be either manual or automatic. In manual scan, the operator must command thesoftware to move the specimen stage from one hole to the next. In automatic scan, the software waitsuntil the delay time has passed and then moves the stage to the next hole.OptionsRelocatable gridA Relocatable grid is a specially shaped grid that has an asymmetric 'ear'that fits into the single-tilt specimen holders delivered since 1993. The 'ear'takes care that the grid can be loaded into the specimen in only one way,without rotation and very little sideways movement. The relocatable grid thusmakes it easy to remove specimens from the microscope holder and puttingthem back in and still retrieve stored locations easily.If relocatable grids are used, switch on the option under Control Options.Grid Scanning will then make it possible to drive back directly to the oldlocation of the first reference point which should be very close to the currentlocation of that point.Use labelsThe label refers to user-written comments (up to a maximum of 100 characters) than are stored togetherwith stored locations. If Use labels is checked the software will bring up a dialog allowing insertion of thelocation label.Use hyperlabelsThe software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: whenthe cursor moves over them they flash their color to blue and a mouse-click brings up a dialog withinformation about the point, with additional functionality such as Go to or Delete).Use Function keysFunction keys can be used in addition to menu commands to control Grid Scanning. The followingfunctions are used:• Up, down, left, right arrows in cursor scan (these have no menu equivalent).• F4: Add location• F6: Next hole• F8: Previous hole

<strong>Tecnai</strong> on-line help Options 67Options Software version 2Use Other keysAdditionally a few other keyboard keys can be used:• Enter key: Add location• Space key: Next holeColorsThe four types of colors (Background, Foreground, Holes, Locations, Current location) for the displaycan be freely selected. The selection is made in the Control options dialog or by clicking on the windowwith the right-mouse button. The latter will popup a menu that allows definition of the colors.Note: There is one exception to free color selection: if hyperlabels are used the Location color cannot beblue (this is the flash color of the hyperlabel).4.8 Calibrate gridCalibration is the setup step wherein the software is informed about the dimensions and orientation ofthe grid. This is done by moving three points to the center of the screen with the specimen stage. Thesoftware will show a dialog with one of the following schematic drawings of a part of a grid.The three calibration points lie on opposite sides of a grid hole (1, 2) and in the next grid hole (3). Thechoice of the points doesn't matter (for instance, 1 and 3 can be closest to the operator, 2 furthest away);what counts is their relation.After the third point has been entered the software knows the size of a grid hole, the orientation and theshift necessary to go from one hole to the next.The software then will drive the stage to a grid hole 750 µm away from the current position, where apoint similar to number 1 must be found. From the position of this point, the software fine-tunes thecalibration. If no fourth point can be identified with certainty, simply press OK without moving the stage.4.9 Reference pointsReference points are locations on the specimen that are easily recognized and can be used as referencewhen the specimen is removed from the specimen holder and later put back in again. By finding thereference points again, the software can recalculate the old locations to new ones.Reference points should be located close to but not at the periphery of the specimen. Locations furtherfrom the center increase the accuracy of relocation but if a reference point later lies outside the reach ofthe stage, then it is not possible to recalculate the old locations to new ones. Because specimens areoften shifted up to 0.2 mm when re-inserted into the specimen holder, keep the reference points lessthan 800 µm away from the center.Setting the reference points can be done at any stage of Grid Scanning (but before the data are savedinto a file, otherwise the reference points are not included in the file). When an old file containing

<strong>Tecnai</strong> on-line help Options 68Options Software version 2reference points is opened, the software will ask to redo the reference points immediately. If thereference points are not redone at this stage, the software will display the stored locations and tracks intheir original configuration. The reference points can then still be done later.Setting reference points for new dataDuring the procedure wherein new reference points are defined, the operator is askedto drive the stage to 2 or 3 points that can be recognized. It is suggested to avoidpoints that lie on the opposite side of the center as this will make it much more difficultto decide later whether the specimen has been reloaded in the same way or upsidedown. If at least two points are defined lying at an angle of about 120°, then it will beeasier to decide. Three points make recognition of an upside-down position of thespecimen even easier. If no third reference point is to be set, then simply press OKwhile keeping the specimen stage at the same location as for point 2. The third point will then beignored.Redoing reference points for old dataIf a file containing reference points is reopened, the software will suggest to gothrough the procedure for relocating the old reference points. The software will showthe distribution of the old reference points and list the label of each point in turn. Thereference point locations will be updated with the new ones, once a reference pointhas been found again.During this procedure this software can assist in finding back the old reference points through the Assistfunction. The Assist function will list a number of possibilities depending on which reference points mustbe found again:• Move the specimen stage to the old location for point 1.• Move the specimen stage in a circle, around 0,0 for point 1, around 1 for point 2, etc.• Calculate alternative locations for points 2 and 3 on the assumption that the specimen is upsidedown.After finding reference point 1, the software will calculate what the most likely new location is forreference point 2 and ask if the stage must be driven there. The same is done for point 3 (if a thirdreference point had been set originally).For each reference point found back as well as the whole set of the reference points the software will gothrough a number of consistency checks (distances between old points and new points comparable,angles from the center of gravity of the old three-point triangle and old points and the center of gravity ofthe new three-point triangle and new points comparable).The software can handle rotation, shift and mounting upside down of the specimen. The software alsocalculates a 'stretch' parameter that is related to the percentage difference in dimensions between oldand new reference points. The software cannot however accommodate severe distortions due tobending of the specimen, so some care should be taken to ensure the integrity of the specimen. Thevarious values can be displayed under Show ref. pt. data.

<strong>Tecnai</strong> on-line help Options 69Options Software version 24.10 Show ref.pt. dataAfter reference points from an old file have been found again, the software can display the relationbetween old and new reference points, such as rotation angle and relative shifts. The data are displayedas follows:• Old reference points (1, 2, 3) X,Y locations.• New reference points (1, 2, 3) X,Y locations.• Specimen position normal or upside down.• Rotation angles for all three points and individual angles.• Relative shift.• Stretch factors.The two sets of reference points define two triangles. The first effect may be that one triangle is mirroredrelative to the other (when the grid is upside down). In the calculation of the data the software will first'undo' the mirror operation. After removing any mirror, we again have two triangles which are now(usually) rotated relative to each other. The software next 'undoes' the rotation. The final result will betwo triangles that are somewhat shifted relative to each other.Rotation anglesThe software calculates the rotation angles for four points on each triangle: the center of gravity and thethree reference points individually. The values for all angles should be consistent (larger deviations forindividual points may occur when the points lie too close to 0,0 since the angles are defined as betweenthe lines through New - 0,0 and old - 0,0).Relative shiftThe shift is can also be calculated for the centers of gravity of the triangles.Stretch factorsThe distances between reference points in the old and new triangles should be similar (e.g. Old 1 to 2should be of the same length as New 1 to 2). Strong deviations in the stretch factors from 1.0 are anindication that either one or more reference points is wrong or the grid has been distorted considerablyduring unloading/loading.If there are serious errors in the reference points, note down the positions of the new reference pointsmost likely to be correct and open the file again. Go through the reference points procedure again to findthe proper reference points.4.11 New spiralIn spiral scanning it may be desirable to start a new spiral at a new location, for example to skip areasempty of specimen, as when several tissue sections lie dispersed on a grid with empty space in betweenor the support foil of part of the grid has disappeared. To start a new spiral scan, drive the stage to thecenter of the grid hole that will form the start of the new spiral.

<strong>Tecnai</strong> on-line help Options 70Options Software version 25 TEM Smart Tilt (FP5454/00)Diffraction experiments can be difficult, especially in combination with the non-eucentric β tilt.Inexperienced users often find it difficult to predict by how much the stage must be tilted to bring acertain feature of the diffraction pattern (zone axis or systematic row) into alignment with the optical axis.TEM Smart Tilt combines a range of tools that assist in diffraction experiments:• A representation of the <strong>Tecnai</strong> viewing screen with the orientation of the tilt axes and tilt anglesindicated. The display is coupled directly to the active camera length on the microscope.• The possibility to double-click with the left-hand button of the mouse anywhere on the 'viewingscreen' displayed and the feature corresponding to that location will be brought to the center of thediffraction pattern by tilting.• Accurate measurement of tilts necessary to bring certain features in the diffraction pattern along theoptical axis or tilt them half-way ('Bragg' tilting to go from a diffracted beam in Bragg condition to thesystematic row). After Bragg tilting it is possible to tilt left or right around the systematic rowafterwards in user-defined tilt steps.• Rapidly toggle between focussed diffraction pattern and shadow image for both SAED and CBED.5.1 Getting startedThe TEM Smart Tilt option consists of two parts:• The Smart Tilt Control Panel.• The Smart Tilt Display window.To use TEM Smart Tilt first use the <strong>Tecnai</strong> Workspace layout to drag the Smart Tilt Control Panel into aworkset. Go to the tab of the workset and press the Display button in the Smart Tilt Control Panel. TheSmart Tilt window will be loaded and its functionality become available. You can leave the Smart TiltControl Panel in your workset for future use. The Display window will only be loaded when the Displaybutton is pressed.The major part of the Smart Tilt functionality is present only in the Display window. The Control Panelalso provides rapid access to the more often-used functions.5.2 Smart Tilt Control PanelThe Smart Tilt Control Panel contains two sets of functions forSmart Tilt:• Functions that load the Smart Tilt Display window (aseparate window that is typically positioned in the data spaceof the <strong>Tecnai</strong> User Interface) or determine its size.• Functions that give rapid access to often-used Smart Tiltfunctions.

<strong>Tecnai</strong> on-line help Options 71Options Software version 2DisplayThe Smart Tilt Display window is only loaded on operator request. To load it press the Display button. Amessage will appear that the Smart Tilt server is being loaded. Once the server is running, the windowwill be displayed, the Display button will turn yellow, and the message will disappear. You are now readyto start working with Smart Tilt.If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded butsimply hidden. If you display it again (by once more pressing the button), it will still have kept all theSmart Tilt settings as set previously. The window is only unloaded when you close the <strong>Tecnai</strong> UserInterface.Freely sizeableWhen this option is checked, the Display window can be positioned anywhere you like (the software willremember your settings and restore them) and you can define its size in the standard Windows way(click and drag on a border). If the option is off, the Display window will be fixed in size and position to fillthe data space of the <strong>Tecnai</strong> User Interface.The following controls will be enabled or disabled when their counterpart in the Display window isenabled or disabled.FullStarts the Full (zone-axis) tilt procedure.BraggStarts the Bragg tilt procedure.LeftTilts one tilt step to the left.RightTilts one tilt step to the right.SwitchStarts the switch direction procedure.Tilt stepDefines the step used for tilting right and left.FocusSets beam or diffraction pattern focus to the focussed setting.DefocusSets beam or diffraction pattern focus to the defocussed setting (shadow image).Flap-out buttonThe flap-out leads to the Smart Tilt Options tab.

<strong>Tecnai</strong> on-line help Options 72Options Software version 25.3 Smart Tilt OptionsThe Smart Tilt Options contains several settings for using SmartTilt.Use compucentric tiltingSmart Tilt fully supports compucentric tilting (tilting where the non-eucentric behavior of the β tilt axis iscompensated). The compucentric tilting checkbox allows selection or deselection of compucentric tilting.This choice is a system-wide setting. The same setting may be selected elsewhere, e.g in theCompucentricity Control Panel itself.Buttons visibleThe Smart Tilt display has the possibility to display a toolbar with buttons for often-used functions. TheButtons visible option decides if this toolbar is visible or not.Show reminderThe various Smart Tilt procedure rely on a properly centered diffraction pattern at the start. The operatorcan either make sure that the pattern is always properly centered. Or the software can remind theoperator in each.User buttonsYou can assign some of the Smart Tilt functions to User Buttons (L1..L3, R1..R3) on the <strong>Tecnai</strong> ControlPads. This may make it easier to control, especially when intensively observing the diffraction pattern onthe viewing screen. Each function can be assigned to one of the User Buttons by selecting one from thedrop-down list. If None is selected, the function is not connected to a User Button. When a function isassigned to a button already occupied, the function of the occupied button will be switched automaticallyto en empty user button.The buttons are disconnected as long as Smart Tilt is not loaded or when it is hidden (e.g. by pressingthe Display button).Focus CBED and SAEDFor the focus/defocus function of Smart Tilt two choices exist, one being CBED (Convergent BeamElectron Diffraction), the other SAED (Selected Area Electron Diffraction). For CBED the Intensity (C2) isused to switch between focussed diffraction pattern and shadow image. For SAED it is the diffractionfocus that is used.

<strong>Tecnai</strong> on-line help Options 73Options Software version 25.4 Smart Tilt displayThe Smart Tilt Display window is a window that is typically located in the data space of the <strong>Tecnai</strong> UserInterface. It can either be freely positioned and sized or fixed. The choice between the two is made in theSmart Tilt Control Panel. The display contains two essential elements, the menu and the display itself.The main functionality of Smart Tilt resides in the menu of the display window.5.5 The displayThe window will display a schematic representation of the <strong>Tecnai</strong> viewing screen (but in gray instead ofyellow to reduce ambient lighting near the microscope).MenuThe menu gives access to all Smart Tilt functionality.Toolbar buttonsToolbar buttons giving rapid access to often-used functions can be found on the left-hand side of theSmart Tilt display (the display of the toolbar is optional). If there is sufficient room the buttons will belarge, otherwise they are small (as shown here). From top to bottom the buttons are Full tilt, Bragg tilt,Left, Right, Switch direction, Undo, Redo, Tilt step, Focus and Defocus.DisplayThe display is a representation of the viewing screen of the <strong>Tecnai</strong> microscope with its inscribed circlesand plate markers. On top of this, Smart Tilt displays the tilt axes in their orientation and circlesrepresenting tilt angles. The tilt axes are shown in red, the blue circles on the screen mark tilt angles andthe green line (if present) marks the direction of the diffraction spot used for Bragg angle tilting.Convention: The direction of the tilt axes (+/-) is displayed in such a way that the center of a Laue circlewill move in the direction of the marker on the screen if that particular direction and sign of tilt is chosen.

<strong>Tecnai</strong> on-line help Options 74Options Software version 2By double clicking with the left-hand mouse button on the area covered by the screen, the software isinstructed to change the tilt of the CompuStage by the values corresponding to the point where themouse button was clicked.Convention: The tilt under mouse-button control is such that the point corresponding to the cursorlocation is brought to the center of the diffraction pattern. Note that this tilt is in the opposite direction tothe movement indicated by the tilt axes on the screen.Status barThe status bar lists on the left the current stage position. In the center it displays the major optionschosen for the grid scan. Error messages can be displayed on the right.Border iconsThe border icons appear to behave as the standard Minimize, Maximize and Close buttons. The Closebutton, however, does not truly close the window but hides it. The window is closed only upon closingthe <strong>Tecnai</strong> User Interface.5.6 The menuThe menu consists of five main menus with submenus:• File• Calibrate• Tilt• Focus• Help5.6.1 File menuThe File Menu provides the operations that are concerned with file operations and toolbar display.Load calibrationsSave calibrations,Save calibrations asButtons visibleOpens an existing Smart Tilt calibration file.Saves the Smart Tilt calibration data in a file, Save as allows definition ofdifferent file name.Determines whether the toolbar buttons are visible (menu item checked) ornot.5.6.2 Calibrate menuIn the Calibrate Menu the calibration procedures are found. The calibrations required by Smart Tilt aretwo-fold:• The apparent rotation between the diffraction shift and the viewing screen for the different cameralengths.• The calibration of the diffraction shift (rotation and distance) against the alpha and beta tilts.Both types of calibrations are necessary for proper operation of Smart Tilt. Calibrations can be saved ina file and reloaded. Calibrations are also automatically stored for the user once a procedure has beenfinished successfully.All camera lengthsCurrent camera lengthAlpha tiltBeta tiltCalibration procedure going through all camera lengths.Calibration procedure for the current camera length.Calibration of the alpha tilt against the diffraction shift.Calibration of the beta tilt against the diffraction shift.

<strong>Tecnai</strong> on-line help Options 75Options Software version 25.6.3 Tilt menuThe Tilt Menu contains the items that control the tilting operations. During tilting operations the softwarekeeps track of (a limited amount of) past stage positions (approximately 50) so a tilting operation canalways be undone. Once one or more operations is undone, it is also possible to step through themforward again.Show reminderZone axisBraggSet stepLeftRightSwitchUndoRedoDisplay listCopy (as text)CompucentricThe tilting operations rely on a measurement of the diffraction shift. Startingposition must therefore always be a well-centered diffraction pattern. If thereminder is on, the operator will be reminded before each diffraction-shiftmeasurement to check the centering of the diffraction pattern.Starts the Full (zone-axis) tilt procedure.Starts the Bragg tilt procedure.Sets the tilt step used for Left and Right tilting. The tilt step is the absolutechange in angle in space (so made up of the proper combination of alpha andbeta tilt).Tilts around the systematic row (defined by the Bragg tilt or Switch directionoperation) to the left.Tilts around the systematic row (defined by the Bragg tilt or Switch directionoperation) to the right.Changes the direction of the systematic row. This procedure is similar inoperation to the Bragg tilt except that only the measurement is executed andno change of tilt is made.Switches back to previous stage position.Goes forward to next stored stage position.Display the list of past tilting operations. Any of the items can be selected byclicking. A right-hand mouse-button click will popup a menu with Hide list(equivalent to using Display list in main menu again), Go to selectedposition, Copy (as text).Copies contents of tilt list as text to the clipboard. It can then be pasted intoany program that accepts text input.Enables (checked) or disables compucentric tilting. Function is disabled whenTEM Compucentricity is absent.5.6.4 Focus menuThe Focus Menu contains a number of functions that are related to the use of the focus functionality forrapid toggling between focussed diffraction pattern and shadow image. The Focus and Defocus settingsmust be defined before these functions can be used. It is advised to define these on a regular basis (e.g.each microscope session).FocusDefocusCBEDSAEDDefine focusDefine defocusToggles to focus condition.Toggles to defocus (shadow image) condition.Select CBED focus/defocus (Intensity is focussing lens).Select SAED focus/defocus (Diffraction lens used for focussing).Defines focus condition (use Intensity for CBED, Diffraction focus for SAED).Defines defocus condition (use Intensity for CBED, Diffraction focus forSAED).

<strong>Tecnai</strong> on-line help Options 76Options Software version 25.6.5 Help menuThe Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display thisHelp file in the <strong>Tecnai</strong> User Interface. About will display the About TEM Smart Tilt dialog listing thecurrent version number of the software. When reporting bugs, always include the version number in thereport.5.7 Camera length calibrationThe rotation calibration consists of a simple procedure to determine the orientation of the diffractionpattern relative to the viewing screen. The procedure for a single camera length consists of two steps. Inthe first step, the diffraction pattern must be centered accurately on the screen with the Multifunction X,Yknobs. Once that is done the following dialog appears.The central beam in the diffraction pattern must now be shifted to the marker indicated (or as far aspossible in the direction of the marker). Note that the distance shifted is not relevant, it is the directionthat is important.In the procedure for all camera lengths, the software goes through the same procedure for all cameralengths.5.8 Alpha and beta tilt calibrationIn order for the program to display the orientation of the tilt axes on the screen and determine the tiltsrequired when the diffraction shift is used, it is necessary to calibrate the tilt axes. In these procedures(unlike the rotation calibration), it is not only the direction that is important but also the magnitude of theshift.The tilt calibration procedure consists of the following steps (all executed after instructions from theprogram):• Tilt a crystal to a zone axis that is well-defined and will be recognisable from the Kikuchi pattern evenwhen tilted off axis.• Center the diffraction pattern accurately.• Tilt the zone axis away as far as possible (the higher the tilt away from zone axis, the more accuratethe calibration will be) with one tilt axis only, while still being able to recognise it.• With the diffraction shift recenter the zone axis (the intersection of the Kikuchi bands) on the screen.

<strong>Tecnai</strong> on-line help Options 77Options Software version 2This procedure provides the program with the necessary data to transform a diffraction shift (orientationand value) to a tilt (direction and value).A suitable specimen for the procedure is one that will show strong Kikuchi bands and one that isn't bent(bending of the specimen will change the apparent tilt of the specimen and thus lead to an incorrectcalibration). The silicon substrate of semiconductor specimens (somewhat thicker areas tend to showgood Kikuchi bands and are also usually straight, unlike the thin edge) has been found to be quite good.5.9 Full (zone axis) tiltThe displacement of the diffraction pattern is a measure of tilt. By centering a feature in the diffractionpattern on the viewing screen with the diffraction shift the software can convert the tilts required to bringthe particular feature (e.g. the center of a zone axis) to the screen center. In the case of Full tilt, the tiltsare set in such a way that the particular feature is centered fully.Sequence of diffraction patterns outlining the Full tilt method:Starting point is a pattern where a zone axis is clearly recognisable (atthe intersection of the Kikuchi bands) but off-center. The center of theviewing screen is marked by the black cross.Next the pattern has been shifted with the diffraction shift so now thezone axis has been centered on the screen. The center of the viewingscreen is marked by the black cross.The displacement necessary to center the pattern is then translated intotilts and the displacement is reset to zero and as a result the zone axisis now centered. The center of the viewing screen is marked by theblack cross.

<strong>Tecnai</strong> on-line help Options 78Options Software version 25.10 Bragg tiltThe displacement of the diffraction pattern is a measure of tilt. By centering a feature in the diffractionpattern on the viewing screen with the diffraction shift the software can convert the tilts required to bringthe particular feature (e.g. a diffracted beam in Bragg condition) to the screen center. In case of theBragg angle tilt, the tilts are set in such a way that only half of the angle from diffracted beam totransmitted beam is done (that is, if a beam is in the Bragg condition, the pattern after tilting will be in thesymmetrical condition).Besides bringing a crystal from the Bragg condition into the symmetrical condition, this option alsodefines the direction for right and left-perpendicular tilting (tilting around the systematic row that containsthe diffracted beam just used for setting the Bragg angle). The direction of the original diffracted beam isindicated on the viewing-screen representation by a green line from the center.Sequence of diffraction patterns outlining the Bragg angle method:Starting point is a pattern where a diffracted beam is in the Braggcondition (note placement of the Kikuchi lines). The center of theviewing screen is marked by the black cross.Next the diffracted beam is centered on the screen with the diffractionshift. The center of the viewing screen is marked by the black cross.The displacement necessary to center the diffracted beam is thentranslated into tilts, the displacement is reset to zero, and as a result thepattern on the right shows the pattern centered on the systematic row.The center of the viewing screen is marked by the black cross.Thereafter it is possible to select left or right perpendicular tilt which willtilt the crystal around the systematic row containing the diffracted beam.

<strong>Tecnai</strong> on-line help Options 79Options Software version 25.11 Focussed diffraction and shadow imageThe shadow image is often a handy tool during tilting. In normal operation it is unfortunately alwaysnecessary to refocus the diffraction pattern (for SAED: Selected Area Electron Diffraction) or the beam(for CBED: Convergent Beam Electron Diffraction) when the shadow image has been used. Not only isthis cumbersome, it can also require changing to different conditions before changing back to thediffracting conditions required. Smart Tilt allows the operator to set a focus and a defocus condition (onthe Intensity for CBED, on the diffraction lens for SAED) and then toggle rapidly and accurately betweenthem.Shadow image: an explanationThe shadow image essentially is a mixture of diffraction and image information. In a focussed diffractionpattern (either SAED or CBED), there is no image information. The diffraction information in the patternis therefore at its optimum, but it is difficult to determine where exactly the information is coming from. Inthe shadow image all diffraction disks (even the SAED pattern no longer has real spots) show a smallimage containing the information contributing to the diffraction disk. This image information makes it thuspossible to see where the diffraction pattern is coming from or to track and correct for sidewaysmovement during tilting.In the shadow image Fresnel contrast can be visible. This Fresnel contrast (not the diffraction contrast -an area scattering strongly will remain dark in the transmitted beam) depends on whether the shadowimage is underfocus or overfocus, both in SAED and CBED. Going from under- to overfocus results in aFresnel contrast reversal as well as a flip of the image visible in the disks by 180°. In order to getconsistent results, one should always choose one or the other.

<strong>Tecnai</strong> on-line help Options 80Options Software version 26 TEM Compucentricity (FP5455/00)6.1 IntroductionThe β tilt of the double-tilt holder is not eucentric. Its motion therefore results (in the ideal case) in achange in the X and Z values. (In non-ideal cases also Y and α are changed a little.) This non-eucentricbehaviour can make it hard to keep track of a feature of interest in the specimen during tilting. TEMCompucentricity makes it possible to reduce these non-eucentric motions and thereby makes it easier touse the β tilt.Schematic diagram of the effect of the β tilt. The thick black lines show the locations of the X and Z stageaxes, while the dotted black line indicates the eucentric height. The central blue circle is the β tilt axis,which is displaced from its 'ideal' X 0, Z 0 location. The dotted blue circles indicate the path traveled by apoint when tilted around β. Tilting around β for the point lying on the smaller dotted blue circle requires asmall correction to X and Z (red arrow lines) to bring the area back to the same position in space (but adifferent stage position). For the point lying on the larger dotted blue circle the correction (red arrowlines) is much larger because it is further away from the tilt axis itself. The corrections are appliedautomatically when compucentric tilting is used.Compucentricity consists of:• Establishing the current stage position and the required new tilts.• Calculating what corrections should be made to the stage axes to keep the feature of interestcentered on the screen.• Moving the CompuStage to the new position.Since specimen holders deviate from the 'ideal' model, a correction can (and for good results should) bemade for this 'non-ideal' behaviour through a calibration procedure. Data are stored for each specimenholder separately. Prior to calibration holders must therefore be identified by name.

<strong>Tecnai</strong> on-line help Options 81Options Software version 26.2 Getting startedThe TEM Compucentricity option consists of two parts:• The Compucentricity Control Panel.• The Compucentricity server. This (hidden) server is accessible to other software that supportscompucentric tilting like TEM Smart Tilt.To use the TEM Compucentricity first use the <strong>Tecnai</strong> Workspace layout to drag the CompucentricityControl Panel into a workset. Go to the tab of the workset with the Compucentricity Control Panel. Selectan existing holder or add a new one. If necessary calibrate the holder. From there on compucentric tiltingis available.Note: Due to the construction of the CompuStage and the nature of the Goto procedure, the Y axis willchange when the Z is changed. In order to ensure getting the correct stage position after compucentrictilting, the tilting is done in two stages, the first setting the X, Z, α and β positions, and the second the Yposition.6.3 Compucentricity Control PanelThe Compucentricity Control Panel provides the controlsnecessary for using TEM Compucentricity. In essence thismeans identifying a holder data set and selecting it. Holder datasets do not necessarily have to be physically separate holders.The same physical holder can have several different holder datasets in the list (e.g. each user his/her own set).Holder listThe holder list gives an overview of all holders currently defined. The list gives the names and theWindows NT user name of the person who defined the holder. The holder list can be sortedalphabetically on holder name and user name by clicking on the buttons Holder and User at the top ofthe list. Repeated clicking on the same button reverses the sorting order. The Select button doesn'tbecome enabled until one holder in the list is selected.One entry in the list is always Default. This entry defines the 'ideal holder' which has its β tilt axis definedas 0 for all parameters.SelectThe Select button can be clicked once a selection in the holder list has been made. When the Selectbutton is pressed the calibration parameters of that holder are entered into TEM Compucentricity. Toreset the parameters to 0, select the Default holder and press Select.Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabsData, Calibrate and Cal. data are not accessible.

<strong>Tecnai</strong> on-line help Options 82Options Software version 2RemoveThe Remove button allows removal of the holder selected from the list. Holders can only be removed bytheir owners (the user with the same name as in the list) or the supervisor. The software will ask forconfirmation before a holder is removed.CopyThe Copy button allows copying the parameters from one holder data set to a new holder data set. Firstenter a new name for the holder. Then select a holder from the list and press Copy.Holder nameA new holder data set is defined by entering a name in the edit control at the bottom of the Control Paneland either pressing Add or Copy.AddWhen a name in entered for the holder in the edit control at the bottom the Add button becomes active.When the button is pressed a new data set is entered in the list. Add is equivalent to a copy of theDefault holder data set.Flap-out buttonThe flap-out leads to the Compucentricity Options, Data, Calibrate and Cal. data tabs.6.4 Compucentricity OptionsThe Compucentricity Options tab provides a checkbox to selector deselect compucentric tilting. This choice is a system-widesetting. The same setting may be selected elsewhere, e.g in theSmart Tilt Control Panel. When the choice is on, tilting operationsby software that supports compucentric tilting will usecompucentricity, otherwise tilting will be done without corrections.6.5 Compucentricity DataThe Compucentricity Data tab provides information on thecalibration data for the holder selected. For the Default holder thetab is disabled (all settings are zero by definition).

<strong>Tecnai</strong> on-line help Options 83Options Software version 2Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabsData, Calibrate and Cal. data are not accessible.The holder data consist of the following parameters:• X shift: the displacement of the β tilt axis in the X direction of the stage.• Z shift: the displacement of the β tilt axis in the Z direction of the stage.• A skew: the rotation of the β tilt axis in the direction of the α tilt axis of the stage.• C skew: the rotation of the β tilt axis in the vertical direction (a non-existent γ tilt axis of the stage).• Residual: a measure of the deviation of all the individual position measurements of the calibrationsfrom the fitted data.• Points: the number of X, Y, Z, α points for which a calibration has been done (with a maximum of10).• Tilts: the number of tilts for each calibration. For all microscopes this number will be 11 except for U-TWIN instruments where it is 9 (because of the more limited tilt range of the U-TWIN lens).• X, Y, Z locations of the calibration points in a list.The data can be copied by either clicking with the right-hand mouse button in the tab (and selectingCopy to text) or clicking inside the tab and pressing Ctrl+C. The data are then copied to the clipboard astext and can be pasted into any software that accepts text input.6.6 Compucentricity CalibrateThe Calibrate tab contains the controls needed to perform acalibration of the β tilt axis of a holder. The calibration procedureconsists of defining a series of locations of a point on thespecimen for a range of β tilt values, starting at 0°, then on to -5°,-10°, etc. unto -25° (-20° for U-TWIN), then back to +5°, +10° andso on to +25° (once again +20° for U-TWIN). At each tilt theoperator must recenter the feature in the specimen accurately atthe center of the screen and at the eucentric height.Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabsData, Calibrate and Cal. data are not accessible.Note 1: It is important that all points are correct. Since it may be impossible to achieve the correcteucentric height (due to the limitations of the Z range) at high β tilts and high values of X (further fromzero than -500 µm or +500 µm), calibration should in general not be attempted too far away from thecenter of the stage. The accuracy of the calibration depends, however, on the location where thecalibration is executed, with improved accuracy if the calibration point is close to the area wherecompucentric tilting will be done. The worst case is to calibrate close to the β tilt axis (where thecompensations are small so stage position measurement errors can dominate over the actualcompensations) and then tilt much further away from the actual position of the tilt axis. A much bettergeneral procedure is to combine calibrations at about -400 µm and +400 µm from the β tilt axis position.A general procedure for establishing a suitable calibration would be as follows:

<strong>Tecnai</strong> on-line help Options 84Options Software version 2• Find a recognizable specimen area at X ~ 0 and Y ~ 0. Execute a calibration. The data for the holderwill now list where the X position of the β tilt axis lies.• Find a recognizable specimen area at approximately X shift -400 µm. Select Replace to replace theearlier calibration (whose X and Z positions are usually reasonable but the A and C skews tend to belarger than realistic) and perform a new calibration.• Find another recognizable specimen area at approximately X shift +400 µm. This time select Add (orCompare) and Add another point to the calibration.Note 2: All β tilts set by the calibration procedure will be executed compucentrically. That means that theimage feature selected should stay reasonably close to the center of the screen, unless the currentholder calibration is far off (which may be the case when starting from the default settings).Start buttonThe Start button starts the calibration procedure. The software instructs the operator to set the β tilt closeto 0 and center the recognizable image feature.Instructions panelThe instructions panel gives instructions during execution of the calibration procedure.Continue buttonThe Continue buttons leads to the next step of the calibration procedure. When the procedure is finished,the calibration parameters and residual are calculated, dependent on the option Replace, Add orCompare chosen. After the procedure the stage is reset to the position (and tilts) at which the calibrationprocedure was started.Cancel buttonThe Cancel button cancels the calibration procedure. The stage is reset to the position (and tilts) atwhich the calibration procedure was started.ReplaceIf a calibration already exists, the Replace option is enabled. When this is selected the new calibrationwill replace the earlier calibration (all points).AddIf a calibration already exists, the Add option is enabled. When this is selected the new calibration will beadded to the earlier calibration. If ten points were already present, the first point is deleted and all pointsare shifted down one to make room for the data of the new point.CompareIf a calibration already exists, the Compare option is enabled. When this is selected the two newcalibrations will be calculated, one in which the new calibration is used only (Replace) and one in which itis added to the earlier calibration (Add). The Cal. data tab will become active and the data are displacedside by side. Choose an option (Replace or Add) and press Continue.

<strong>Tecnai</strong> on-line help Options 85Options Software version 26.7 Compucentricity Cal. dataThe Compucentricity Cal. data tab is displayed automatically atthe end of a calibration procedure when the Compare option waschosen (it can also be inspected afterwards). It compares thecalibration parameters and residual for the case where the newcalibration would replace all existing data (left) and where itwould be added (right). The operator can now choose to Replaceor Add and press Continue to finish the calibration.Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabsData, Calibrate and Cal. data are not accessible.

<strong>Tecnai</strong> on-line help Options 86Options Software version 27 TEM k-Space Control (FP5453/00)7.1 IntroductionTEM k-Space Control is software that uses an input set of crystal-lattice parameters and lattice type to• Create a stereographic projection of the crystal’s zone axes• Simulate the electron diffraction patterns and Kikuchi lines that would be seen in the TEM when thecrystal is tilted to various orientations.The Kikuchi- and diffraction-pattern simulations can aid the identification of diffraction patterns from thecrystal’s zone axes in the TEM.In conjunction with the automatic control of the CompuStage and the ability for on-line diffraction patternmeasurement of the microscope, TEM k-Space Control becomes a powerful tool for crystallographicresearch with the TEM. The orientation of the crystal can be determined with respect to the tilt axes ofthe CompuStage and the crystal can then be tilted by the software to any zone axis within the range ofthe specimen tilts.7.2 Getting startedThe TEM k-Space Control option consists of two parts:• The k-Space Control Control Panel.• The k-Space Control Display window.To use TEM k-Space Control:• Open the <strong>Tecnai</strong> Workspace layout (bottom-right selection list) and drag the k-Space Control ControlPanel into a workset.• Go to the tab of the workset and press the Display button in the k-Space Control Control Panel. Thek-Space Control window will be loaded and its functionality become available.You can leave the k-Space Control Control Panel in your workset for future use. The Display window willonly be loaded when the Display button is pressed.7.3 General introductionThe flow of working with k-Space Control falls into three separate parts:1. Definition of the crystal structure2. Determination of the orientation of the crystal3. Changing the orientation7.3.1 Definition of the crystal structureIn order for crystallographic calculations to be performed, the crystal structure (lattice parameters andtype of structure - Primitive, Face-centered, etc.) must be known. These data are entered by the user,either by typing and selecting the relevant data or by loading from an existing crystal structure file. Thecrystal structure must therefore be known beforehand (k-Space Control is NOT a tool for matchingcrystal structure from a database with data derived from diffraction on the microscope).

<strong>Tecnai</strong> on-line help Options 87Options Software version 27.3.2 Determination of the orientation of the crystalWhen the data for the crystal structure have been entered, the software will assume a 'generic'orientation. Next the real orientation of the crystal must be determined. k-Space Control provides twomethods for determining the orientation, one called two-zone indexing and the other manual indexing.With the two-zone method, we use two zone axes with their stage tilts for defining the orientation of thecrystal. With the manual method, we use a single zone axis with an indication of the rotation of thediffraction pattern. You can use either of these methods or start with manual indexing and then use twozoneindexing for fine-tuning. The manual method is usually faster (only a single zone axis needed) butless accurate (because of the estimation of the rotation or the diffraction pattern). The manual methodcan therefore be used quite effectively to do a rough orientation and on the basis of that select two zoneaxes for the two-zone indexing method.k-Space Control provides assistance with the determination of the orientation by:• Automatic indexing of the zone axis on the basis of the on-line measurement or manual entry of twodiffraction vectors and their angle.• Display of the diffraction patterns and Kikuchi maps.7.3.3 Changing the orientationThere are three methods to change the orientation of the k-Space Control display:• Define a zone axis to go to, enter the zone axis in the Zone edit control and press Tilt to.• Enter values for a and b CompuStage tilts under Stage grid a and Stage grid b and press Set.• Click with the left-hand mouse button on a point inside the k-Space Control display. This point(orientation) will be brought to the center.Please note: The intersection of Kikuchi lines on the display may not coincide with the displayedlocations of the zone axes. In that case the location of the zone axis is correct. The intersections of theKikuchi lines may come out in the wrong position because the software simplifies Kikuchi lines to straightbands. Clicking on a Kikuchi-line intersection will therefore not necessarily bring the intersection tocenter, because it is the crystallographic direction that is centered.To change the orientation of the CompuStage to bring it into agreement with the orientation as seen onthe k-Space Control display, press the Stage to button.7.4 Display elementsOne the k-Space Control window a number of elements can be selected for display:• Lab grid• Stage grid• Zone axes with or without indices• Kikuchi map• Diffraction pattern• Lattice• Markers7.4.1 Lab gridThe Lab grid displays a stereographic projection with lines at 10° intervals. For more information onstereographic projections, please refer to the standard crystallography literature textbooks (or, if you canget hold of a copy: J.W. Edington, Practical electron microscopy in materials science, Monograph Two.Electron diffraction in the electron microscope).

<strong>Tecnai</strong> on-line help Options 88Options Software version 27.4.2 Stage gridThe stage grid is a grid of lines at 10° spacings that displays the tilt angles for the a and b tilt axes of thespecimen stage. The maximum tilts are read directly from the microscope. In general this will be ±30° forthe b tilt. For the a tilt this will generally be ±80°, which is the range accessible to the a tilt itself but formost microscopes it will be much more limited in practice due to space limitations in the gap between theobjective-lens pole pieces. A practical limit is also set by the shadowing caused by the specimen-holdercup at high tilt angles (in general above ~50° the shadow cast by the specimen-holder cup completelyblocks the electron beam). S-TWIN pole pieces generally do not allow more than ~40° of a tilt and U-TWIN pole pieces no more than 20°. Because it is difficult to predict what the effective limit will be for acertain combination of X, Y, Z, a and b, the stage limits in k-Space Control are set to the limits allowedby the stage. In practice you may find that you cannot reach all orientations.7.4.3 Zone axesOverlaid on the stereographic projection can be shown the positions of the zone axes. Zone axes can bedisplayed with a label showing their indices or without (dependent on the status of the Display checkbox). The position of the zone axes on the stereographic projection is accurate (unlike that ofintersections of Kikuchi lines, see below).Zone axes with indices up to a maximumvalue of 4 shown with a small font size (8).Kikuchi map are also shown. The lab gridis shown in red, the stage grid at anorientation of 60° in blue.

<strong>Tecnai</strong> on-line help Options 89Options Software version 2Zone axes with indices up to a maximumvalue of 1 shows with a large font size(20). Kikuchi map are also shown. The labgrid is shown in red, the stage grid at anorientation of 60° in blue.Zone axes indication without the indexlabels at a higher camera constant.7.4.4 Kikuchi mapKikuchi lines can be displayed in various ways. The user can choose the maximum index of the Kikuchilines (up to 6) and whether the intensity of the Kikuchi lines is scaled to their intensity on the viewing

<strong>Tecnai</strong> on-line help Options 90Options Software version 2screen of the microscope. The maximum index for the Kikuchi lines and the scaling are changed on theDisplay tab of the Control Panel flap-out. Note that the Kikuchi lines themselves are not indexed.The Kikuchi line index determines up to what values of the hkl indices Kikuchi lines will be displayed.The acceptable maximum values will generally depend on the effective camera constant and whetherthe lines are shown with relative intensities or bright (see below). At low values, Kikuchi lines up to highindices will tend to fill the display completely and give a very messy result, while at high cameraconstants the use of low maximum indices may result in the conspicuous absence of lines expected. Ingeneral, a value of 3 is useful for lower camera constants.Kikuchi map shown with indices up to amaximum value of 6, with relative (scaled)intensities. The lab grid is shown in red,the stage grid at an orientation of 60° inblue. The labels indicate the zone axes.Kikuchi map shown with indices up to amaximum value of 6, with all Kikuchi lineintensities the same (making it impossibleto distinguish individual lines). The lab gridis shown in red, the stage grid at anorientation of 60° in blue. The labelsindicate the zone axes.

<strong>Tecnai</strong> on-line help Options 91Options Software version 2Kikuchi map now shown with indices up toa maximum value of only 3, with all Kikuchiline intensities the same. The lab grid isshown in red, the stage grid at anorientation of 60° in blue. The labelsindicate the zone axes.A display with only a Kikuchi map withrelative intensities.Please note: The intersection of Kikuchi lines on the display may not coincide with the displayedlocations of the zone axes. In that case the location of the zone axis is correct. The intersections of theKikuchi lines may come out in the wrong position because the software simplifies Kikuchi lines to straightbands.

<strong>Tecnai</strong> on-line help Options 92Options Software version 27.4.5 Diffraction patternThe spot diffraction pattern can be displayed with the zone axis shown at the center. Note that a cameraconstant larger than 10 is necessary to display a pattern (when the spots are too close together andoverlap they disappear). The spot pattern can be quite sensitive to small deviations of the crystal fromthe zone axis (in order to set the crystal exactly to the zone axis, the Tilt to function can be used). Thedisplay of the labels of the spot indices can be suppressed by removing the check from the DiffractionDisplay check box.Please note: The diffraction pattern displayed is based on the crystallographic parameters as input. Thismay deviate from the true diffraction pattern as seen on the microscope because the k-Space Controlsoftware does not account for structure factors, multiple scattering and possible forbidden diffractions.7.4.6 LatticeThe display can be set so that a projection of the crystal unit cell is shown. In the center, the unit cell isoutlined and circles display the generalized positions in the unit cell (so at the corners for Primitive,corners and faces for Face-centered, etc.). For unit cells beyond the central one (their number dependson the setting of the Lattice points on the Display tab of the Control Panel flap-out) only the circles areshown and no further lines.A lattice display with a value of 1 selectedfor an all-face centered cubic structure in orientation.

<strong>Tecnai</strong> on-line help Options 93Options Software version 2The same structure now with a latticedisplay up to value 3.7.4.7 MarkersThe markers show the positions of the reference axes used during two-zone indexing. The axes aredisplayed by squares with a number, easiest seen when zone axis and Kikuchi Line display is off.Displaying the reference axes makes it easy to tilt back to these axes.The marker for zone memory 2 which represents the secondary zoneaxis from Two-Zone Indexing. The position of the marker differs fromthe zone axis (102) to which it corresponds for the reason detailedbelow.The position of one of the markers may deviate from the exact position of the zone axis to which iscorresponds because the primary zone axis is assumed to be absolute and any difference between thecalculated and measured angles is taken up by giving the secondary zone axis some freedom to move(along the line connecting the two zone axes). Thus, in the example shown above, the 102 axis isdisplayed at its crystallographic correct position while the marker shows the measured stage position.

<strong>Tecnai</strong> on-line help Options 94Options Software version 27.5 Two-zone indexingWith the two-zone axis indexing method, the orientation of a crystal is defined by identifying two zones.In each case, the crystal must be tilted by the operator to a first zone axis. The possible indices of theaxis must then be identified. The software reads the tilt settings for the axis. Then the crystal must betilted by the operator to a second zone axis (note: the larger the difference in tilt, the more accurate willbe the determination of the orientation) which must then be identified. Usually two zone axes aresufficient for determining an orientation, but there may be cases where a third axis is needed. (E.g. whenin a cubic crystal the and axes are identified, the axis can be on either side of theplane through the two axes. In that case a third axis is required for confirmation.) If more than two axesare needed, the two-zone method can be extended simply by choosing a third zone axis.In Two-zone indexing, a number of dialogs is used:• Two-zone indexing dialog• Zone-axis calculator dialog• Match constraints dialogAfter these dialogs have been considered, a step-by-step procedure for Two-zone indexing is given.7.5.1 Two-zone indexing dialogWhen Two-Zone Indexing is selected, the software will display the following dialog:CalculatorPressing the Calculator button leads to the Calculator dialog for the zone memory selected.Zone memoryA total of five memories is available for zone axes, which can be used in any combination (but thesoftware always uses only a combination of the two axes currently selected). The memory is selected viathe spin buttons next to the Calculator buttons for the primary and secondary zone axes. The spin

<strong>Tecnai</strong> on-line help Options 95Options Software version 2buttons work such that the selection for the other zone axes (that is, secondary for the primary-axis spinbutton and vice versa) is always skipped. It is thus not possible to have the primary and secondary zoneaxes on the same zone memory. If you want to use zone memory 2 as the primary axis, while it currentlyis at the secondary axis, and another as the secondary, then first change the secondary zone from 2 toanother value and then the primary axis can be changed to select 2.Zone axisThe two zone axes used for the indexing are displayed in the edit controls just below the “Calculator”buttons. The zone index can be changed by clicking on the required index in the list underneath or bytyping in the index.. The special keyboard keys are used to enter negative values.Zone axes listThe zone axis lists contain the possible indices of the zone axes, as determined via the zone axisCalculator. In the current version of the software, there is no particular sequence in the lists and allentries will be found to occur more than once. To determine if a second axis is compatible with a solutionafter a first axis has been defined (click in the primary zone axis list), click somewhere in the secondaryaxis zone list, then use the up or down arrows to change the selection. The 'Degrees between zone'value will be updated after each change. when the value matches that measured with the stage, thematch is correct.View patternWhen the View pattern button is pressed, the diffraction display on the right will show the diffractionpattern for the primary of secondary zone axis. If the camera constant is on manual scaling, the valuewill be set to a camera constant of 10 to allow viewing of the pattern.Diffraction patternThe diffraction pattern is displayed to the right. This pattern either corresponds to the pattern determinedby the current orientation of the Lab grid or to the pattern of the zone axis selected for which the 'Viewpattern' button was pressed. By viewing the diffraction pattern it is, for example, possible to compare thedisplayed pattern with that on the microscope. In this it is possible to distinguish between solutions thatare mirror images of each other.Degrees between zonesAfter 'Degrees between zones' the difference in angle between the two zones used for two-zoneindexing are displayed. These tilts are calculated when the selection in the edit controls is changed (bymake a change in the selection in one of the lists)..Degrees between stageAfter 'Degrees between stage' the difference in angle between the stage tilts for the two zones used fortwo-zone indexing are displayed. These tilts are read automatically from the microscope.SuggestionAfter two zone axes have been defined through the calculator, the software will determine a matchbetween zone axes, based on the angle measured with the stage. The match is usually only one of a setof possible permutations. For example, the match suggested with 00-1 and 02-1 is equally valid to thematch selected (of 100 and 102).IndexPressing Index exits from the Two-zone indexing dialog, while changing the orientation as defined by theselection for the two zone axes.Note: The position of the primary zone axis is assumed to be absolute (so it is made to coincide exactlywith the stage position measured for it) and any difference between the calculated and measured angles

<strong>Tecnai</strong> on-line help Options 96Options Software version 2is taken up by giving the secondary zone axis some freedom to move (along the line connecting the twozone axes). Such differences can occur because of the accuracy of the measurement of the stageposition, including the effects of backlash on the tilts (which may result in deviations up to 0.2° or so) orbecause the crystal is bent and the second zone axis orientation was not determined from the exactsame place in the crystal.CancelPressing Cancel exits from the Two-zone indexing dialog, while leaving the orientation as it was.7.5.2 Zone axis calculator dialogWhen one of the Calculator buttons is pressed, the software brings up the Zone axis calculator dialog:MeasurementThe Zone Axis Calculator can use the same method for measuring in diffraction as the standardMeasuring control panel. Please see the description there as to the execution of such measurements.The results of the measurements are automatically inserted into the edit controls for d1, d2 and theinterplanar angle.d1d1 is the first d-spacing used for indexing of the zone axis. The value for the d-spacing (in nanometers)can be determined with the on-line measurement or entered by hand.d2d2 is the second d-spacing used for indexing of the zone axis. The value for the d-spacing (innanometers) can be determined with the on-line measurement or entered by hand.RatioWhen Calculate is pressed, the ratio between the two d-spacings as measured is calculated. This valuecan be compared to the calculated value.

<strong>Tecnai</strong> on-line help Options 97Options Software version 2Interplanar angleThe interplanar angle is the angle between the two d-spacings. The value for the interplanar angle (indegrees) can be determined with the on-line measurement or entered by hand.SetupPressing the Setup buttons brings up the Match constraints dialog in which the limits on deviations areset for the match between d-spacings and interplanar angle.CalculateWhen the Calculate button is pressed, the d1, d2 and interplanar angle are matched against calculationsof d-spacings an their interplanar angles, and possible matching zone axes are determined.Calculated valuesUnder calculated values are listed the matches of the calculations:• Indices for the best matches for d1 and d2• Calculated d-spacings for d1 and d2• Calculated interplanar angle between matches for d1 and d2 spacings• Ratio between matches for d1 and d2 spacingsResultant zoneThe resultant zone list contains all matching zone axes. The contents of this list is copied to the Twozoneindexing dialog when OK is pressed.OKPressing OK exits from the Zone-axis calculator dialog, while keeping the newly defined match for thezone axis. The data like d-spacings, interplanar angle and stage tilts are stored in the zone memory. Thestage axes are read when the OK button is pressed. It is important, therefore, to keep in mind that thestage tilts at that particular moment must be such that they coincide with the orientation of the zone axis.CancelPressing Cancel exits from the Zone axis calculator indexing dialog, without changing any of the values.7.5.3 Match constraints dialogAbsolute Distance Error LimitThe absolute distance error limit determines by how much a calculated d-spacing may differ from themeasured one for it to be accepted as a possible match for one of the d-spacings.Ratio Distance ErrorThe ratio distance error limit determines by how much the calculated ratio between d-spacing 1 and d-spacing 2 may deviate from the ratio of the measured spacings.

<strong>Tecnai</strong> on-line help Options 98Options Software version 2Angular Error LimitThe angular error limit determines by how much the interplanar angle between the two d-spacingscalculated may differ from the measured angle.OKPressing OK exits the match constraints dialog while keeping any changes made to the values of thematch constraints.CancelPressing Cancel exits the match constraints dialog without changing the values of the match constraints.7.6 Two-zone indexing procedure in stepsThe two-zone indexing procedure can be executed as follows:• Find a suitable first zone on the crystal with the stage tilts.Intermezzo: Making it easier to find a suitable second zone axis• Identify the zone axis and use the Manual indexing method to set the rough orientation of the crystal.• Store the stage position in the Stage control panel.• Change the orientation to a different zone axis on the k-Space display.• Press the Stage To button in the k-Space Control Panel to tilt the real crystal to the new orientation.• Adjust the stage tilts to bring the required zone axis to the center.• Store this stage position also in the Stage control panel.• Go back to the stored position for the first zone axis with the Move To or Go To function of the stage.• When the second zone axis is needed, use the same functions to recall that stage position.End of intermezzo• Press the 2-Zone button in the Crystal tab of the flap-out of the k-Space Control panel.• In the Two Zone Indexing dialog, select a zone-axis memory for the primary zone axis with the spinbuttons and press Calculator for the Primary zone axis.

<strong>Tecnai</strong> on-line help Options 99Options Software version 2• In the Zone Axis Calculator, use the measuring procedure to define two d-spacings and theirinterplanar angle or enter the values by hand (the latter as in the example dialog below).• If necessary, check the Match constraints by pressing the Setup button.• Press the Calculate button. Calculated values will now be inserted and the Resultant Zone axis listwill be filled.• Press OK to exit from the Zone Axis Calculator dialog and go back to the Two-Zone Indexing dialog.

<strong>Tecnai</strong> on-line help Options 100Options Software version 2The Two-Zone Indexing dialogafter the first zone axis hasbeen established. Note thatthe diffraction pattern visiblestill corresponds to the defaultcrystal orientation (100) andnot yet to the 00-1 axis.• If it is required to display the diffraction pattern for the select one axis, Press View Pattern for thePrimary zone axis.The Two-Zone Indexing dialogafter pressing View Pattern.The camera constant hasbeen changed to 10 and thediffraction pattern now visiblecorresponds to the 00-1 axis.

<strong>Tecnai</strong> on-line help Options 101Options Software version 2• Change the stage orientation to a second zone axis.• In the Two Zone Indexing dialog, select a zone-axis memory for the secondary zone axis with thespin buttons and press Calculator for the Secondary zone axis.• In the Zone Axis Calculator, use the measuring procedure to define two d-spacings and theirinterplanar angle or enter the values by hand (the latter as in the example dialog below).• Press the Calculate button. Calculated values will now be inserted and the Resultant Zone axis listwill be filled.• Press OK to exit from the Zone Axis Calculator dialog and go back to the Two-Zone Indexing dialog.The Two-Zone Indexing dialogafter the second zone axishas been established.

<strong>Tecnai</strong> on-line help Options 102Options Software version 2• If it is required to display the diffraction pattern for the select one axis, Press View Pattern for theSecondary zone axis.The Two-Zone Indexing dialogdisplaying the diffractionpattern for the 02-1 zone axis.• If necessary, change the selected primary and secondary zone axes.• Press Index and the orientation of the crystal in the k-Space display will be changed to the orientationestablished.If necessary, it is possible to refine the orientation by using the k-Space display to tilt to a more suitablezone axis, storing the relevant zone axis through the Stage Control Panel, then re-entering the Two-Zone Indexing dialog and either replacing one or both the existing zone axes in the existing zonememories or changing to different zone memories and storing the results of the new zone axes there.7.7 Manual indexingThe manual indexing requires setting the crystal to a known zone axis and identifying a g vector (indicesof the diffraction spot). The direction of this g vector is defined relative to the zero-offset angle which inturn is defined by the top of the display (north). Negative crystallographic indices can be set by using thespecial keyboard keys.

<strong>Tecnai</strong> on-line help Options 103Options Software version 2The Manual Indexing dialog.7.8 Special keyboard keysIn order to allow display of the special notations that crystallography uses for zone axes, with the ‘bar’ 1,etc. (with the - sign on top of the number), a special font (called k_font) has been developed for k-SpaceControl. This special font replaces a number of standard characters with ‘bar’ 1, ‘bar’ 2. The followingcharacters on the keyboard give access to the special k-Space Control characters:Normal character How to get k_font! Shift + 1 ‘bar’ 1@ Shift + 2 ‘bar’ 2“ Shift + ‘ ‘bar’ 2# Shift + 3 ‘bar’ 3$ Shift + 4 ‘bar’ 4% Shift + 5 ‘bar’ 5^ Shift + 6 ‘bar’ 6These characters are the first six characters in the normal fonts (it can be checked in the Windowsprogram Character Map, available under Accessories). Except for the ‘bar’ 2 as “, most of thesecharacters are above the corresponding number on many keyboards. (Note: there is no real consistencyin this, due to the fact that many countries have their own keyboard lay-out).The same characters are also used when the keypad keys are used (when Num Lock is on). So thekeypad key 1 gives the 'bar one' character while the key above the Q gives the normal one.The special font is used automatically where needed, as e.g. in the Zone edit control on the k-SpaceControl Panel and various dialogs of the k-Space Display.

<strong>Tecnai</strong> on-line help Options 104Options Software version 27.9 k-Space Control Control PanelThe k-Space Control Control Panel contains two sets of functionsfor k-Space Control :• Functions that load the k-Space Control Display window (aseparate window that is typically positioned in the data spaceof the <strong>Tecnai</strong> User Interface) or determine its size.• Functions that control k-Space Control.DisplayThe k-Space Control Display window is only loaded on operator request. To load it press the Displaybutton. Once the server is running, the window will be displayed, and the Display button will turn yellow.You are now ready to start working with k-Space Control.If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded butsimply hidden. If you display it again (by once more pressing the button), it will still have kept all the k-Space Control settings as set previously. The window is only unloaded when you close the <strong>Tecnai</strong> UserInterface.SizeableWhen this option is checked, the Display window can be positioned anywhere you like (the software willremember your settings and restore them) and you can define its size in the standard Windows way(click and drag on a border). If the option is off, the Display window will be fixed in size and position to fillthe data space of the <strong>Tecnai</strong> User Interface.The following controls will remain disabled until a crystal structure has been defined through New on theCrystal tab of the flap-out.Stage toWhen Stage to is pressed, the CompuStage will be tilted to the currently active orientation on the k-Space Control display. If the orientation entered is out of reach for the stage tilts, the Stage to button willbe disabled. The function is enabled once the orientation of a crystal has been determined.ZoneIn the Zone edit control the crystallographic notation of a zone axis can be entered. Individual indicescannot exceed a single character (so the 1001 for ten-zero-one is not allowed). Negative indices areentered using the special keyboard keys.Tilt toWhen Tilt to is pressed, the stereographic projection is tilted to the zone axis indicated in the Zone editcontrol. There are no limitations imposed on the tilting. The tilt applies only to the display, not to thestage. The function is enabled once the orientation of a crystal has been determined.

<strong>Tecnai</strong> on-line help Options 105Options Software version 2Camera constantThe magnification of the stereographic projection and the other elements displayed depends on thecamera constant. This value can either be defined by hand, using the camera constant slider, orautomatic (in which case it is linked to the active camera length). The range of camera constants usedby k-Space Control is 1 to 20.The camera constant (the camera length*the electron wavelength) is in mm*nm, so a camera constant of1 means a distance of 1 mm corresponds to 1 nm d spacing (on the printed page; due to monitor sizedifference these values may not be exact on the monitor display).The camera constant (L*λ) forms part of the formula for converting distances in a diffraction pattern to dspacings:D*d = L*λ or d = (L*λ ) / Dwhere D is the distance of the diffraction spot in the pattern to the origin of the pattern (the transmittedbeam). Thus while 1 mm at a camera constant of 1 means 1 nm d spacing, 2 mm means 0.5 nm dspacing (as D occurs in the denominator).Table of (approximate) camera lengths against camera constant for different accelerating voltages.Cam. const. 120 kV 200 kV 300 kV1 300 mm 400 mm 500 mm5 1500 mm 2000 mm 2500 mm10 3000 mm 4000 mm 5000 mm20 6000 mm 8000 mm 10 000 mmNote: For displaying the diffraction pattern, it is necessary to choose a larger camera constant,otherwise the spots will be too close together and invisible. If the diffraction pattern display is on and nopattern is visible, increase the camera constant.The display at the smallest cameraconstant.

<strong>Tecnai</strong> on-line help Options 106Options Software version 2The display at a camera constant of ~4shows a detail of that in the image above.AutoThe magnification of the display of the stereographic projection and the other elements displayeddepends on the camera constant. This value can either be defined by hand, using the camera constantslider, or automatic (in which case it is linked to the active camera length). If Auto is checked, theadjustment is automatic, otherwise it must be set with the Camera constant slider.Stage grid aThe Stage grid a value defines the tilt angle given to the stage a axis of the k-Space display. The stagegrid tilt is changed to the value defined when the the Set button is pressed.Stage grid bThe Stage grid b value defines the tilt angle given to the stage b axis of the k-Space display. The stagegrid is changed to the value defined when the the Set button is pressed.SetThe stage grid tilts are changed to the values defined for stage grid a and b when the the Set button ispressed.Flap-out buttonThe flap-out leads to the k-Space Control Crystal, Info, Display and View tabs.

<strong>Tecnai</strong> on-line help Options 107Options Software version 27.10 k-Space Control CrystalThe k-Space Control Crystal Control Panel contains severalfunctions for using k-Space Control.NewIn order to start working on a new crystal or phase, the software must know the crystal structure. To thispurpose the lattice parameters and crystal-structure type must be entered in the Crystal data dialog. Theprogram works on a triclinic basis so for crystals with higher symmetry, the symmetry derives simplyfrom the lattice parameters (all angles 90° and dimensions the same for cubic, all angles 90° and a=b fortetragonal, etc.). For hexagonal crystals the rhombohedral notation must be used.The Crystal datadialog.For simplified entering of the data, a set of crystal structures has been predefined (cubic, tetragonal,etc.). These are used simply to avoid defining unnecessary entries (e.g. when cubic is selected allangles will be set to 90° and b and c will be equal to a). These crystal structures are not imposed on theunderlying calculations (as stated above, all is done on a generalized, triclinic basis). Thus a selection ofcubic with a value of 0.52 for a is equivalent to selecting triclinic and entering the 0.52 for a,b and c andsetting all angles to 90° (but selecting the cubic crystal structure is less work than entering all the valuesby hand).

<strong>Tecnai</strong> on-line help Options 108Options Software version 2Print SetupWhen the Print Setup button is pressed, the k-Space Control software displays the printer setup dialog(e.g. for selecting the paper size).PrintPrinting causes the current display to be printed on the printer selected (if necessary, changes to printersetup can be made under the Printer setup button). Note that printing is done on a white background sothe color selection may need to be adjusted.OpenVia the Open dialog an existing oriented crystal structure can be read from disk. Crystal structure fileshave the extension .xtl. In the crystal structure file, the program saves the lattice parameters and thecrystal-structure type.SaveOnce defined, crystal data can be saved for later recall. If no filename has been defined yet, the programdisplays the Save As dialog in which the filename can be set. If a filename has already been defined,then using Save will overwrite the existing file while under Save As a new filename can be given. Beforeoverwriting an existing file, the software will ask for confirmation.Save AsOnce defined, crystal data can be saved for later recall.For working with k-Space Control, the orientation of the crystal must be determined. For thedetermination, two indexing methods are available, the Two-zone and the Manual method.2-ZoneWhen the 2-zone button is pressed, the Two-zone indexing method of determining the orientation of acrystal, is started.ManualWhen the Manual button is pressed, the Manual indexing method of determining the orientation of acrystal is started.If the Compucentricity option is present on the microscope, the tilting can be done with the aid ofCompucentricity whereby the non-eucentric behavior of the β tilt axis is corrected.Use compucentric tiltingThe Use Compucentric tilting checkbox is used to select or deselect compucentric tilting. This choice is asystem-wide setting. The same setting may be selected elsewhere, e.g in the Compucentricity or SmartTilt Control Panels. When the choice is on, tilting operations by software that supports compucentrictilting will use compucentricity, otherwise tilting will be done without corrections.

<strong>Tecnai</strong> on-line help Options 109Options Software version 27.11 k-Space Control InfoThe k-Space Control Info Control Panel contains informationconcerning the crystal currently defined in k-Space Control.ParametersUnder Parameters the crystal lattice parameters are listed.TypeUnder Type the type of crystal lattice is listed.IndexingUnder Indexing the method used to determine the crystal orientation is listed. If the two-zone methodwas used, the primary and secondary zone axes used for the indexing are listed.MatrixThe orientation matrix of the crystal is defined as the matrix that relates the crystal axes to the lab grid(see also V. Randle, 1992, Microtexture determination and its applications, The Institute of Materials,London). The orientation matrix can be copied to the clipboard by clicking with the right-hand mousebutton on the Info Control Panel and selecting Copy matrix to clipboard in the popup menu.The format of the output is a nine numbers separated by tabs on a line and each line separately. Youcan copy these directly into programs like Microsoft Excel and you will get 3x3 cells filled with the matrixvalues.

<strong>Tecnai</strong> on-line help Options 110Options Software version 27.12 k-Space Control DisplayThe k-Space Control Display Control Panel contains severalsettings for the k-Space Control display.Crystal indicesCrystal indices determines the maximum value used for the uvw indices of the zone axes displayed (anumber between 1 and 6). The zone axes can be displayed with or without the index label (depends onthe Display check box).Display (crystal indices)The status of the Display check box determines whether the zone axes displayed on the k-Space Controldisplay are labeled with their uvw index (check box checked) or shown without label.DiffractionDiffraction determines the maximum value used for the hkl indices of the diffraction spots displayed (anumber between 1 and 6). The diffraction-pattern spots can be displayed with or without the index label(depends on the Display check box).Display (diffraction)The status of the Display check box determines whether the spots of the diffraction pattern are shownwith or without label.Lattice pointsThe Lattice points value determines the number of lattice points used on the display (a number between1 and 6). For lower numbers fewer unit cells are shown. Only the central unit cell is indicated by lines.Kikuchi line indexThe Kikuchi line index determines up to what values of the hkl indices Kikuchi lines will be displayed.The acceptable maximum values will generally depend on the camera constant and whether the linesare shown with relative intensities or bright (see below). At low values, Kikuchi lines up to high indiceswill tend to fill the display completely and give a very messy result, while at high camera constants theuse of low maximum indices may result in the conspicuous absence of lines expected. In general, avalue of 3 is useful for lower camera constants. A value of 6 can be used if the Bright option is switchedoff.

<strong>Tecnai</strong> on-line help Options 111Options Software version 2BrightKikuchi lines can be displayed with relative intensities (where Kikuchi lines that have lower intensities inthe microscope are displayed with lower intensities on the k-Space Control display) or as bright. In thelatter case all Kikuchi lines will have the same (green) intensity.Note that the relative intensities are not based on the structure factors that normally also determine theintensities of Kikuchi lines but on a general approximation.Stage orientationGenerally, the orientation of the stage tilt axes does not correspond to the a tilt being E-W and the b tiltN-S as shown on the k-Space Control display. On most instruments the majority of camera lengthstypically have a running about NNW-SSE and b ENE-WSW. You can adjust the stage grid display toreflect this deviation in orientation by entering a rotation angle for the stage orientation.How to determine the stage orientationThe following procedure provides a method for determining which value to enter for the stage orientation:• Insert into the microscope a type of specimen that is relatively flat (not strongly bent) and with wellvisibleKikuchi lines.• Tilt the specimen to a clearly visible zone axis.• Press the New button and enter some kind of structure (it doesn't really matter what).• Press the Manual button and enter some values for the zone and g vector (once again, it isn'timportant what you enter, as long as it is something possible).• The k-Space Control display will now show some kind of zone axis at the current stage position.• Change the a tilt angle of the stage back and forth to get a feeling of the direction in which the a tiltaffects the diffraction pattern (for the reference camera length ~500mm on many microscopes thiswill be close to NNW-SSE). Enter a value for the stage orientation (the angle rotates the tilts anticlockwisefrom 0 where the a tilt is horizontal), e.g. 60°, and press the Enter button. The display ofthe stage grid will now be in the new direction.• Change to a larger camera constant (10) or reasonably high camera length (~1000mm, with Autochecked).• Click on the display in the direction of the plus or minus a tilt (just a little to the NNW or SSE in theexample used here). The zone axis will be displaced form the center of the display.• Now press the Stage to button. The a tilt should now be adjusted so that the zone axis becomesvisible on the viewing screen displaced in the same direction as on the k-Space Control display.• If the zone axis has moved into the wrong direction, add 180° to the stage orientation value (so itwould become 240° in the example used here).• If necessary, check again.

<strong>Tecnai</strong> on-line help Options 112Options Software version 27.13 k-Space Control ViewThe k-Space Control View Control Panel contains severalsettings for the k-Space Control elements displayed. All colors(except for Kikuchi lines which is currently fixed to green) aredefined by clicking on the colored rectangle to the right of theelement. This brings up a standard color selection dialog whereinyou can either select one of the predefined colors or add acustom color.Lab gridThe Lab grid is the grid displaying the stereographic projection.The color can be selected by clicking on the colored rectangle tothe right. Whether the Lab grid is displayed or not depends onthe status of the check box (checked = display).Stage gridThe Stage grid is the grid displaying the orientation and tilts of the CompuStage a and b tilts. The colorcan be selected by clicking on the colored rectangle to the right. Whether the Stage grid is displayed ornot depends on the status of the check box (checked = display).KikuchiKikuchi refers to the color of the Kikuchi lines. This is currently fixed to green. Whether the Kikuchi mapis displayed or not depends on the status of the check box (checked = display).Zone axesZone axes defines the color for the zone axis symbols. The color can be selected by clicking on thecolored rectangle to the right. Whether the zone axes and their indices are displayed or not depends onthe status of the check box (checked = display).Diffraction patternIndices defines the color for the spots of the diffraction pattern. The color can be selected by clicking onthe colored rectangle to the right. Whether the diffraction is displayed or not depends on the status of thecheck box (checked = display).LatticeLattice defines the color for the display of the schematic lattice. The color can be selected by clicking onthe colored rectangle to the right. Whether the lattice is displayed or not depends on the status of thecheck box (checked = display).MarkersMarkers defines the color for the markers that indicate the position of the zone axes used for defining thecrystal orientation with the Two-zone indexing method. The color can be selected by clicking on thecolored rectangle to the right. Whether the markers are displayed or not depends on the status of thecheck box (checked = display).Font sizeUnder font size you select the size of the font used for displaying the zone axis and diffraction spotsindices.

<strong>Tecnai</strong> on-line help Options 113Options Software version 28 TEM Free Lens Control (FP5458/00)8.1 IntroductionFree Lens Control allows control over individual lenses of the <strong>Tecnai</strong> microscope. The followinglimitations apply.Safety• The spot size (first condenser lens or C1) cannot be taken lower than the value for spot size number1.• On 200 kV systems the spot size (first condenser lens or C1) cannot be taken lower than the valuefor spot 5 when the high tension is higher than 120 kV and no holder is present in the CompuStage.• On 200 and 300 kV systems the spot size (first condenser lens or C1) and intensity (secondcondenser lens or C2) are blocked when the gun is in conditioning.Operation• All lens settings range from 0 to 100%, except for the first condenser lens which has a minimumvalue as decribed above and the minicondenser lens which ranges from -100 to 100%.• The changes in lens settings are not persistent. If microscope settings are changed that affect lenssettings (e.g. the switch between Microprobe mode and Nanoprobe mode), the Free Lens settingsare overruled. In order to avoid interference as much as possible, the relevant knobs on the ControlPads of the microscope are disconnected. Thus the Intensity knob is disconnected when C2 ischanged, the Focus knob when the objective lens is changed, and the Magnification knob when oneof the projection system lenses(Diffraction, Intermediate, Projector 1, Projector 2) is changed. Theseknobs are reconnected when Free Lens Control is deactivated. There are, however, many controlsthat can interfere with Free Lens Control and not all can be blocked.• As long as a particular lens has not been changed through Free Lens Control, its particularmicroscope control is not disconnected. Thus, if you only change settings of the projection system,the control of the condenser system (spot size, intensity) can still be done with the normalmicroscope controls.

<strong>Tecnai</strong> on-line help Options 114Options Software version 28.2 Free Lens Control PanelActivateWhen the Activate button is pressed the Free Lens Control isswitched on (the button becomes yellow) or off.Get from 'scopeWhen the Get from 'scope button is pressed, the current lenssettings are copied from the microscope to the Free Lenssettings. This function can be used before activating Free LensControl to make the current lens settings on the microscope thestarting settings for working with Free Lens Control.For each lens the following controls are presentMicroscope valueThe microscope value lists the current lens value as it is on the microscope. The display will update eachtime a lens setting is changed, also when Free Lens Control is not active.Free Lens Control valueThe Free Lens Control value in the edit control displays the value used for a particular lens in Free LensControl. You can change the value by typing another value. The value is transmitted to the Free LensControl slider when the Enter button is pressed. If Free Lens Control is active, the new value is then seton the microscope (otherwise only the slider setting changes).Enter buttonWhen the Enter button is pressed, the Free Lens Control value is transmitted to the Free Lens Controlslider. If Free Lens Control is active, the new value is then set on the microscope (otherwise only theslider setting changes).Free Lens Control sliderThe Free Lens Control slider controls the setting of the particular lens. When Free Lens Control is activeany change of the slider is transmitted immediately to the microscope. The step sizes (small step by

<strong>Tecnai</strong> on-line help Options 115Options Software version 2clicking on the left or right arrows, large step by clicking left or right of the thumb tab - the 'drag'rectangle) are controlled by the step size control to the right of the slider.Free Lens Control slider step size controlThe Free Lens Control slider step size control changes the size of the small and large step used by theslider. The meaning of the ranges (in percent) is as follows for all lenses except the minicondenser andobjective lens:Step Small step Large step1 0.00001 0.0012 0.00003 0.0033 0.0001 0.014 0.0003 0.035 0.001 0.16 0.003 0.3For the objective lens all steps are ten times smaller, for the minicondenser they are two times smaller.Flap-out buttonThe flap-out button leads to the Registers tab of the Free Lens Control Control Panel.8.3 Free Lens RegistersIntroductionFree Lens settings can be stored in up to 10 registers. Thesettings of the registers are stored for each individual user whenthe user logs off and reloaded when the user logs on again. Setsof registers can also be stored in a file and loaded from file.Register listThe register list contains the labels of the registers defined by theuser. The label of a register can be anything (up to 255characters) except empty. If you do not specify a label yourself,the software will automatically call the register 'Register' with aserial number when a new register is added.Registers are selected by clicking on the entry in the list. Thelabel of the selected entry is copied automatically to the registerlabel edit control.Set to 'scopeWhen a register has been selected, its settings can betransferred to Free Lens Control by pressing the Set to 'scopebutton. If Free Lens Control is active, these settings (all lensestogether) are transferred immediately to the microscope. If FreeLens Control is not active, the settings are transferred to thesliders but not the microscope. To set only part of the registersettings, first deactivate Free Lens Control, then use Set to'scope. Re-activate Free Lens Control and press the Enterbuttons of the lenses that must be set on the microscope totransfer those settings to the microscope.

<strong>Tecnai</strong> on-line help Options 116Options Software version 2Register labelThe register label edit control contains the label of a register. It can be either a label typed in for a newregister or the register currently selected. The label of a register can be anything (up to 255 characters)except empty. If you do not specify a label yourself, the software will automatically call the register'Register' with a serial number when a new register is added. The software does not check for labelcontents (that is, whether two registers have the same label).AddWhen the Add button is pressed, a new register is added to the currently defined registers. The settingsof the new register are those currently defined for Free Lens Control. Registers can be addedirrespective of whether Free Lens Control is off or on.OverwriteWhen the Overwrite button is pressed, the currently selected register is overwritten with new settings.These settings are those currently defined for Free Lens Control. Registers can be added irrespective ofwhether Free Lens Control is off or on.DeleteWhen the Delete button is pressed, the currently selected register is deleted. The software asks forconfirmation.OpenWhen the Open button is pressed, the Open file dialog is displayed in which a file can be selected (theextension for Free Lens Control files is .frl). The contents of this file are then loaded into the registers(overwriting anything that is there).Save, Save asThrough the Save or Save as buttons the contents of the registers can be saved to a file (the extensionfor Free Lens Control files is .frl). When the Save button is used and a file name has been definedpreviously, the contents of the existing file are overwritten. When no file name has been defined or theSave as button is used, the Save file dialog is displayed, allowing definition of the file name under whichthe settings are stored. When the file name is the same as an existing file, the software will ask if that filemust be overwritten.

<strong>Tecnai</strong> on-line help Options 117Options Software version 29 Magnification Calibration (FP5458/50)9.1 Magnification Calibration Control PanelThe Magnification Calibration Control Panel semi-automaticallycalibrates:• the HM magnifications on an embedded CCD camera• the LM magnifications on an embedded CCD cameraTheMagnification Calibration Control Panel semi-automaticallycalibrates:• the D camera lengths on an embedded CCD camera• the HM-STEM magnification• if Microprobe STEM is installed, the calibration of theMicroprobe STEM against the normal (Nanoprobe) STEMThe values measured are entered into the magnification tables of DigitalMicrograph and/or TIA CCDor in the TIA STEM magnification table. The control panel is accessible only to Supervisor, Service andFactory.Note: The magnifications as used by Magnification Calibration for CCDs always are the so-calledreference magnifications. These are the magnifications as displayed by the microscope when the mainviewing screen is up. This applies even to CCD cameras located in the wide-angle position (above theviewing screen at the top of the projection chamber), because it is the reference magnifications that areused by the CCD camera software to determine the effective magnifications from the magnificationtables. There thus is an apparent discrepancy between the magnification value displayed by themicroscope and by the Magnification Calibration software as well as the CCD magnification tables forwide-angle CCD cameras.Calibration selection listThe calibration selection list allows selection of the calibrations to be executed. Currently the list onlycontains calibration of:• the HM (Mi, SA and Mh) magnifications• the Mi plus SA magnifications• the LM magnifications• the HM-STEM magnifications in TIA• if Microprobe STEM is installed, the calibration of the Microprobe STEM against the normal(Nanoprobe) STEM(the difference between the first two being that it is not necessary to switch to another specimen for highresolutionimaging for the latter calibration).InstructionsThe instructions field will display instructions on how to proceed or a status description of the functioncurrently running or finished.Start buttonThe Start button starts the calibration procedure. The software will link up to DigitalMicrograph or TIAand when successful, will display the name of the camera, the lens series (TEM or EFTEM) and high

<strong>Tecnai</strong> on-line help Options 118Options Software version 2tension setting. If TIA CCD is present on a system with Gatan cameras, the software will also requirethat TIA is running (otherwise the TIA CCD magnification table will not be set).The Start button will change to a yellow color and its caption toStop. Pressing the Start button when it is yellow will stop thecalibration procedure.Starting the calibration procedure will open a log in a separate window. The log shows what is beingdone and what the results are. It can be saved in text format (under the File menu of the log window).The log can also be printed (if a printer is available to the system). Text files can also be opened in thelog file window.Next buttonDuring the calibration procedure the operator will be expected to do certain things as setting upillumination, specimen area and focus, as well as change specimen (later in the procedure). Once thesethings have been done (as given by the instructions displayed), pressing the Next button will continuewith the procedure.Flap-out buttonThe flap-out button leads to the Report, File and Options tabs of the Automated Experiments ControlPanel.9.1.1 The HM magnification calibration procedureThe calibrations procedure consists of a calibration for each magnification of the series. The followingprocedure is used:• Direct cross-grating calibration of the lowermost SA magnification.• Calibration of the image shift at the lowermost SA magnification (this does not calibrate amagnification, but the image shift itself, used later).• Calibration of the Mi magnifications with the cross-grating method.• Calibration of the remaining SA magnifications, going up in magnification. The software detects themagnification at which the cross-grating method does not work anymore and displays a warning inthat sense. Then the software automatically switches to the image shift method. Once themagnification is high enough for high-resolution images (determined from the measured pixel size),the software will switch to the lattice-image method.• Calibration of the Mh magnifications with the lattice-image method.

<strong>Tecnai</strong> on-line help Options 119Options Software version 29.1.2 The Mi plus SA magnification calibration procedureThe calibrations procedure consists of a calibration for each magnification of the series. The followingprocedure is used:• Direct cross-grating calibration of the lowermost SA magnification.• Calibration of the image shift at the lowermost SA magnification (this does not calibrate amagnification, but the image shift itself, used later).• Calibration of the Mi magnifications with the cross-grating method.• Calibration of the remaining SA magnifications, going up in magnification up to the point where themagnification on the CCD camera is larger than 250kx. The software detects the magnification atwhich the cross-grating method does not work anymore and displays a warning in that sense. Thenthe software automatically switches to the image shift method which will be used for the remainingSA magnifications.9.1.3 The LM magnification calibration procedureThe calibrations procedure consists of a calibration for each magnification of the series down to amagnification of 200x on the CCD. The procedure uses an analysis of the FFT of the cross-grating todetermine the magnifications.9.1.4 The D camera length calibration procedureThe calibrations procedure consists of a calibration for the camera lengths of the series (in the case ofEFTEM the lowermost camera lengths are excluded from the calibration because they often are notusable - the projection system reduction is so strong that it becomes impossible to get the electron beamthrough the differential pumping aperture). The following procedure is used:• Direct calibration of the diffraction rings of a cross-grating at a camera length selected by software onthe basis of CCD camera size and high tension.• Calibration of the diffraction shift at that camera length (this does not calibrate a camera length, butthe diffraction shift itself, used later).• Calibration of the lowermost camera lengths with the diffraction shift (the rings are too close to thecenter of the diffraction pattern).• Calibration of the rings for the camera lengths where the rings are well enough separated from thecenter but not so large that they are no longer visible on the CCD.• Calibration of the higher camera lengths with the diffraction shift (the rings are too large to fit on theCCD).If possible, the procedure will be run in the Nanoprobe mode, because the large illuminated area forparallel illumination in Microprobe is so large that it often leads to the appearance of severe distortions inthe center of the pattern. It is important to focus the pattern so the rings becomes as sharp as possible.The focusing in Nanoprobe should be done with the Intensity - provided the camera lengths are focusedprior, which should have been done in the Image HM-TEM Camera Length alignment procedure.Note: The software is not suitable for calibration of the LAD (LM) camera lengths nor for the Lorentzmode camera lengths.

<strong>Tecnai</strong> on-line help Options 120Options Software version 29.1.5 The HM-STEM magnification calibration procedureThe calibrations procedure consists of a calibration for selected magnifications (unlike the TEMmagnifications, which are made from discrete lens settings with magnification-dependent random errors,the STEM magnifications are controlled by the amplitude of the scan which quite linear, so only a fewmagnifications suffice). The following procedure is used:• Direct cross-grating calibration of a lower HM-STEM magnification (not necessarily the lowermostbecause this may be too low to resolve the grating squares effectively). If the image distortion is toohigh, the software will automatically adjust the distortions in an iterative procedure until the distortionsare less than 0.1% (vector lengths of the grating square dimensions) and 0.1° (angle betweenvectors).• Calibration of the image shift at the same HM-STEM magnification (this does not calibrate amagnification, but the image shift itself, used later).• Calibration of several additional magnifications. The software detects the magnification at which thecross-grating method does not work anymore and automatically switches to the image shift method.The STEM calibration procedure will be run for a total STEM rotation angle of 0°. This means that thefree (user) STEM rotation is set to 0°, and the aligned value (which corrects for the mismatch betweenstage axes and STEM image axes) is also temporarily set to 0° (the latter value is reset at the end of theprocedure to the starting value). Effectively this means that the smallest distortions and most accuratemagnifications are achieved at the same STEM rotation angle. For this purpose it is necessary for usersto set the free rotation to the compensating value of the alignment value (which is included in thecalibration report).Note: The STEM magnifications are strongly dependent on the pivot points, perpendicular correctionsand distortion adjustment. For STEM magnifications as accurate as possible, the same STEM settingsshould be used as during the calibration. It is therefore advised to adjust the pivot points andperpendicular corrections carefully before the calibration and store these settings in an alignment file(after the calibration, because that may have included adjustment of the STEM distortion) and havingusers always use those alignments. If the STEM distortions initially were a long way off, stop theprocedure after the distortion adjustment and check the pivot points and perpendicular corrections, andthen restart the procedure.9.1.6 The Microprobe STEM magnification calibration procedureMicroprobe STEM is the STEM mode in which the illumination system is in Microprobe (theminicondenser lens is set to positive). This mode is used for special applications in which the size of thebeam is not decisive, but rather the illumination angle (which is smaller in Microprobe), which in turngives smaller diffraction disks. Microprobe STEM can be reached from normal STEM by switching fromNanoprobe to Microprobe. Because TIA is not aware of normal (Nanoprobe) or Microprobe STEM, itdoes not have separate magnification tables. For Microprobe STEM we therefore have to make sure thatthe internal calibration in the <strong>Tecnai</strong> software is defined such that the same nominal magnification innormal and in Microprobe STEM gives the same actual magnification. This is done by the MicroprobeSTEM calibration procedure.

<strong>Tecnai</strong> on-line help Options 121Options Software version 2The following procedure is used:• Direct cross-grating calibration of a lower HM-STEM magnification (not necessarily the lowermostbecause this may be too low to resolve the grating squares effectively). If the image distortion is toohigh, the software will automatically adjust the distortions in an iterative procedure until the distortionsare less than 0.1% (vector lengths of the grating square dimensions) and 0.1° (angle betweenvectors).• Direct cross-grating calibration of a comparable Microprobe STEM magnification. If the imagedistortion is too high, once again the software will automatically adjust the distortions in an iterativeprocedure until the distortions are less than 0.1% (vector lengths of the grating square dimensions)and 0.1° (angle between vectors).These STEM calibration procedure will be run for a total STEM rotation angle of 0°. This means that thefree (user) STEM rotation is set to 0°, and the aligned value (which corrects for the mismatch betweenstage axes and STEM image axes) is also temporarily set to 0° (the latter value is reset at the end of theprocedure to the starting value). Effectively this means that the smallest distortions and most accuratemagnifications are achieved at the same STEM rotation angle.Note: Both types of STEM-mode magnifications are strongly dependent on the pivot points,perpendicular corrections and distortion adjustment. For STEM magnifications as accurate as possible,the same STEM settings should be used as during the calibration. It is therefore advised to adjust thepivot points and perpendicular corrections carefully before the calibration and store these settings in analignment file (after the calibration, because that may have included adjustment of the STEM distortion)and having users always use those alignments. If the STEM distortions initially were a long way off, stopthe procedure after the distortion adjustment and check the pivot points and perpendicular corrections,and then restart the procedure.The Microprobe STEM magnification calibration does not result in a calibration file. The calibration valuefor the Microprobe STEM magnification is directly set to the <strong>Tecnai</strong> software and also entered as acalibration value in the system branch of the microscope settings in the registry.9.1.7 Measurement on cross-gratingThe magnification calibration is done on the basis of a standard specimen, a cross-grating. Thisspecimen has squares on it with a spacing of 463 nm. The software will acquire an image of the crossgratingand analyse that to find the basic spacing. The analysis is done either using an auto-correlationor an FFT (at high and low magnifications, respectively) in which the two basic vectors will be outlined bycircles. Additional circles may be present further away from the center to show the position of themultiple of the base vector that is actually used for further refinement of the measurement.

<strong>Tecnai</strong> on-line help Options 122Options Software version 2An image of a cross-grating, showing the 463 nm squares.The result of the magnification calibration. The autocorrelationof the cross-grating image (zoomed in 2x) shows the centralpeak pointed at by the line (which has been added manually forclarity). The software adds the two circles around the two basevectors of the pattern. It is important that these circles arelocated at the correct positions and not e.g. at the diagonals ofthe squares.9.1.8 Measurement with image shiftThe image shift of the <strong>Tecnai</strong> is largely free of hysteresis and can be used to calibrate distances, fromwhich magnifications can be determined. The cross-grating method is usable only at lowermagnifications where a sufficient number of grid squares is present in the image. The lattice-imagemethod can only be used at high magnifications where lattice fringes become visible. The indirect imageshiftmeasurement must therefore be used in the gap left by these two methods, at intermediatemagnifications.Before the image shift can be used to calibrate magnifications, it must first be calibrated itself. This isdone at the lowermost SA magnification, immediately after the magnification there has been calibratedwith the cross-grating. (The software thus does not rely on any existing - potentially inaccurate or evenwrong - image shift calibrations in the system.)The procedure followed during image shift calibration is as follows:• Apply a negative shift from the current image position and acquire image 1, with a negative backlashcorrection (first more negative, then to the desired position).• Apply a similar but now positive shift from the original position and acquire image 2.• Apply a positive shift from the original image position and acquire image 3 (position the same asimage 2), with a positive backlash correction (first more positive , then to the desired position).• Apply a similar but now negative shift from the original position and acquire image 4 (positions thesame as image 1).

<strong>Tecnai</strong> on-line help Options 123Options Software version 2• Apply a negative shift from the original image position and acquire image 5 (position the same asimage 1), with a negative backlash correction (first more negative, then to the desired position).• The time at which each acquisition is finished is stored.• Determine the drift (if any) by measuring the shift between images 5 and 1 and normalising to thetime in between the acquisition of these images.• Determine the shift between images 1 and 2, and between 3 and 4, corrected for drift. These shiftsgive the two vectors used for the calibration.9.1.9 Measurement on lattice imageA high-resolution image contains lattice lines whose apparent spacing is an accurate reflection of themagnification. Although the cross-grating is made of gold-palladium particles that will display a highresolutionimage, the spacings found in the Fourier Transform of the image (the diffractogram) are poorlydefined, mostly because of lattice relaxation and particle size effects. In addition, the spacing of goldpalladiumis not known accurately (there is no guarantee that the alloy composition is constant from onecross-grating to another). Because of this, the magnification calibration uses one of two other types ofeasily available specimens:• Silicon in [110] orientation.• Single-crystal gold in [001] orientation.When appropriate in the procedure (dependent on magnification), the software will ask the operator toinsert one of these specimens, orient it, and focus so a lattice image becomes visible. For silicon both directions (0.31 nm) must be visible and for gold the (0.204 nm). In the latter case, makesure that the are well visible. If the are weak or invisible and the spacings (0.144nm) well visible, the software may confuse these and give the wrong calibration.The software automatically will detect if silicon or gold is present (on the basis of the interplanar angles)and perform the calibration.9.1.10 Diffraction-ring analysisDiffraction by the particles on a cross-grating results in rings in the diffraction pattern. The softwareanalyses the diffraction pattern, finding the center and then determining the distance to four rings (0.23,0.20, 0.14, 0.12 nm: note that these are not the spacings of gold, because the cross-grating is made witha gold-palladium alloy). If the pattern is not suitably centered, the software will center the diffractionpattern itself first. The ring used for the calibration is that of the 0.23 nm distance. The other rings areonly used for crosscheck. In principle the 0.141 and 0.12 nm rings will give the same camera length asthe 0.23 nm, while the 0.20 ring usually gives a value that is a bit off (because it forms a shoulder on the0.23 nm ring, it is less well-defined). The distance of the rings is determined in six directions,perpendicular to the blooming (positive and negative direction) and at the four 45° angles. The errordisplayed is the standard deviation of the six distances. The log will list in how many profiles a particularring was found.NOTE: ON 16-BITS CCD CAMERAS SEVERE OVEREXPOSURE IN THE CENTER IS NOTNECESSARY AND SHOULD BE AVOIDED TO PREVENT DAMAGE TO THE CAMERA.Important: The illumination conditions for diffraction-ring analysis must be set such that there issufficient intensity in the rings, especially the most important one (0.235 nm). Especially on CCDcameras with a limited bit depth (12- or 14-bits), this means that the center of the diffraction pattern mustbe overexposed which will give blooming (the center shows a central overexposed blob, with two tails,usually up and down, a bit like the image of an aircraft propeller). The blooming is necessary and doesnot present a problem, neither for the CCD (which will NOT be damaged) nor for the ring analysis (whichknows that blooming can be present and takes care of that). If you reduce the illumination intensityso that blooming is avoided, the rings analysis may fail.

<strong>Tecnai</strong> on-line help Options 124Options Software version 2The images above show a diffraction pattern with a suitable amount of blooming (top left). The rings areinvisible until the contrast levels in the image are adjusted (top right). After the analysis the software willconvert the image to the log of itself, set the contrast levels and insert circles for the rings detected.

<strong>Tecnai</strong> on-line help Options 125Options Software version 29.1.11 Measurement with diffraction shiftThe diffraction shift of the <strong>Tecnai</strong> is largely free of hysteresis and can be used to calibrate diffractionspacings, from which camera lengths can be determined. The ring analysis method is usable only atcamera lengths where the rings in the diffraction pattern are well-separated from the center and are nottoo large for the CCD. The indirect diffraction-shift measurement must therefore be used where the ringsanalysis cannot be done, that is for the lower and higher camera lengths.Before the diffraction shift can be used to calibrate camera lengths, it must first be calibrated itself. Thisis done at a suitable camera length, immediately after the camera length there has been calibrated withthe rings. (The software thus does not rely on any existing - potentially inaccurate or even wrong -diffraction shift calibrations in the system.)The procedure followed during diffraction shift calibration is as follows:• Apply a negative shift from the current diffraction-pattern position and acquire image 1, with anegative backlash correction (first more negative, then to the desired position).• Apply a similar but now positive shift from the original position and acquire image 2.• Apply a positive shift from the original diffraction-pattern position and acquire image 3 (position thesame as image 2), with a positive backlash correction (first more positive , then to the desiredposition).• Apply a similar but now negative shift from the original position and acquire image 4 (positions thesame as image 1).• Determine the shift between images 1 and 2, and between 3 and 4. These shifts give the two vectorsused for the calibration.Since drift is not an issue in diffraction, no drift correction is done in the diffraction-shift based calibration.9.2 Magnification Calibration Report Control PanelThe Report tab of the Magnification Calibration Control Panelcontains the controls used for report generation. All reports are inAcrobat Reader (pdf) file format.Two types of report can be created:• A tabular overview of the results of a calibration sessions, displaying the TEM magnification, themeasured value(s) according to the method used, which value is used for the magnification table, theassociated consistency error, and the magnification factor from plate camera to CCD or the ratiobetween nominal and measured STEM magnifications..• A graphical overview of the results of (at most) the last twelve calibrations done.

<strong>Tecnai</strong> on-line help Options 126Options Software version 2Note: the STEM magnification is a rather arbitrary value, based on the approximation that a full STEMframe should correspond to a frame covering 100x100mm at the same TEM magnification. Significantdeviations form this arbitrary value can be found. However, such deviations are unimportant. The onlyreally relevant value is the pixel size, which is what is calibrated.Create reportWhen a calibration procedure has been executed successfully, the Create report button will becomeenabled and a report can be made of the calibration just done.Auto open reportWhen the Auto open report checkbox is checked, the report will be opened automatically in AcrobatReader (provided this is installed on the system). Otherwise the software will display a message whenthe report has been created successfully.Report from fileReports can be created from the files stored automatically by the software when a calibration procedureahs been executed successfully. Select a file from the list underneath and press the Report from filebutton.History reportA historical overview can be made of the (at most) twelve last calibrations done - for the combination ofcamera and microscope conditions. Click on one of the files in the list (this will define which conditionswill be used for the selection). press the History report button. The software will find the result of the lasttwelve calibrations and display these in a graph.Calibrations file listThe result of each successful calibration is always stored to file. These files are located in the folder<strong>Tecnai</strong>\Data\Magnification Calibration and have the extension tcl. It is advised to remove old files after asuitable period. When there are too many files present (more than 100), a warning will be displayed toremove some. These files can be read in by the software to create reports. The files are sorted inchronological order (the most recent at the top). The four columns list the creation date, the name of theCCD camera, microscope lens series, and high-tension setting.

<strong>Tecnai</strong> on-line help Options 127Options Software version 29.2.1 Tabular format reportThe tabular format list the camera and microscope settings at the top. Below that follows a table with thefollowing columns:• TEM - the magnification reported by the <strong>Tecnai</strong> microscope (the reference magnification on the platecamera, displayed by the microscope when the viewing screen is up).• X-grating - the results of the magnification calibration from the cross-grating directly.• Im. shift - the result of the magnification calibration performed using the image shift of themicroscope.• Lattice - the result of the magnification calibration performed with high-resolution images of gold [001]or silicon [110].

<strong>Tecnai</strong> on-line help Options 128Options Software version 2• User - the value used for the magnification table. In most cases there will be only one calibrationmethod for each magnification, but in some cases there may be more than one. In that case the usedmagnification indicates which of the method results is used (the other method then gives a crosscheck).• Error (%) - each result is based on the measurement of two vectors (see the description of themethods). The error indicates the consistency in the measurement of the two vectors and is NOT anindication of the absolute accuracy achieved.• CCD/TEM - the ratio between the measured magnification on the CCD camera and the TEMreference magnification. On a perfect system, these values would all be the same.

<strong>Tecnai</strong> on-line help Options 129Options Software version 29.2.2 History format reportThe history format list the camera and microscope settings at the top. Below that follows a graphdisplaying the CCD to TEM ratio or nominal to measured ratio for STEM images for each magnification.The last calibration is shown with filled red circles, the other calibrations have other symbols (unfilled,change of color and symbol type). A 2% error bar is shown in the top right-hand corner of the graph (notin the picture above). Note that the data of the above example are not real data.The graph allows rapid assessment of the quality of each calibration (measurement consistent withearlier or later calibrations) as well as (any) change in time of calibrations performed.

<strong>Tecnai</strong> on-line help Options 130Options Software version 29.3 Magnification Calibration File Control PanelThe File tab of the Magnification Calibration Control Panelcontains the controls used for setting magnification tables fromstored calibrations.Set Mag Table from FileWhen a calibration file is selected in the Calibrations file list and DigitalMicrograph is running, it ispossible to set the magnification table from the data in the file. The software will if TIA CCD (whenpresent) should be updated as well and give a warning when TIA is not running. Next the softwarechecks that the camera currently selected in DigitalMicrograph matches that in the calibrations file. If not,the software will switch cameras. The calibration data are then loaded into the magnification tables.Calibrations file listThe result of each successful calibration is always stored to file. These files are located in the folder<strong>Tecnai</strong>\Data\Magnification Calibration and have the extension tcl. It is advised to remove old files after asuitable period. When there are too many files present (more than 100), a warning will be displayed toremove some. These files can be read in by the software to create reports. The files are sorted inchronological order (the most recent at the top). The four columns list the creation date, the name of theCCD camera, microscope lens series, and high-tension setting.9.4 Magnification Calibration Options Control PanelThe Options tab of the Magnification Calibration Control Paneldefines the automatic behavior during focusing.Focus on :For focusing there is a choice between the main screen (the main screen is lowered automatically toallow focusing) and the CCD camera (Preview mode; CCD image acquisition in the Preview mode willstart automatically). To use the latter, set the CCD Preview mode to a suitable setting (continuousacquisition, half or quarter size, exposure time ~0.25 sec).

<strong>Tecnai</strong> on-line help Options 131Options Software version 2With a GIFWith a GIF there is a choice for focusing between the TV camera (the camera is inserted automatically toallow focusing) and the CCD camera (Preview mode; CCD image acquisition in the Preview mode willstart automatically). To use the latter, set the CCD Preview mode to a suitable setting (continuousacquisition, half or quarter size, exposure time ~0.25 sec).

<strong>Tecnai</strong> on-line help Options 132Options Software version 210 TEM AutoGun (FP5460/20)10.1 AutoGun proceduresThe AutoGun Control Panel provides a number of automated procedures. A short description of theseprocedures is given below.10.1.1 Auto ConditoningConditioning can be used to stabilize the high tension. It is mostly used after an exchange of a filamentor on higher-voltage (200kV or higher) instruments. Conditioning takes the high tension up in controlledsteps while monitoring the vacuum pressure in the gun. If the pressure remains more than 2 log stepsabove the previous attained level, the procedure will wait for it to drop again. If the pressure exceeds thepreviously attained value by a large amount, the procedure will step the high tension back a bit and wait.Conditioning goes up to the specified level with the delay step, both as defined by the user.On 100 and 120kV conditioning takes place by slowly increasing the high tension up to the maximum ofthe instrument (these microscopes do not have the ability to increase the high tension beyond thehighest value). Once the user-defined goal has been reached, the conditioning procedure is finished. Ifthe currently active high-tension setting is not one of the fixed high-tension steps, the high tension will beset to the closest fixed high-tension step below the current value.On 200 and 300kV instruments, the conditioning will take place in the normal regime at low high tensionsettings, but switch to true conditioning when an overvoltage (a high tension value beyond the normalmaximum of 200 or 300kV) is required. After the user-define goal has been reached, the procedure willkeep the high tension at that value for 10 more minutes for stabilization. Afterwards the high tension isset to the closest fixed high-tension step equal to or below the current value.The starting point for the conditioning depends on the current high-tension status :• If the high tension is off, it will be changed to lowermost fixed high-tension step.• If the high tension is on, the current high tension value defines the starting point.During conditioning the control panel displays how much time conditioning is estimated to take. Theestimate is based on the assumption that there will be no delays due to vacuum pressures increases.Conditioning is accessible only to Expert user level or higher.10.1.2 Auto SaturationThe saturation of thermionic filaments is often problematic. Many microscope users tend to try andremove all structure visible in the focused beam (while especially LaB6 filaments tend to have a structurethat cannot be removed or only by strongly oversaturating the filament). The resulting overheating leadsto considerable reductions in filament life time. The correct saturation point of a filament is the pointwhere further increases in heating current do not lead to significant increases in beam current. Once thebeam is defocused for normal operation, any structure visible in the focused beam disappears.The AutoGun software has a procedure for automatically saturating the filament to the correct point. Thefinal measurement is always made using the screen current as a function of filament step, combined withrefinement of the gun tilt. The typical pattern seen is an S-shaped curve when the filament setting is

<strong>Tecnai</strong> on-line help Options 133Options Software version 2plotted against screen current (with some additional complications possible). The saturation point lies atthe top of this curve.If there is no screen current detectable at the start of the autosaturation procedure, a first analysis will bebased on the emission current. At some point, the software will know, on the basis of the emissioncurrent present, that it should be possible to detect light. it will then continue with the gun tilt to find thelight, then reduce the filament setting until the screen current is gone, and then increase the filamentsetting again while monitoring the screen current.The emission setting at which the procedure will aim depends on the selection of the two options:• Not to exceed the maximum emission current given.• And/or use the currently active emission setting.If both options are active, the option not to exceed the maximum emission current wins if there is aconflict (the currently active emission setting is too high and gives too much emission current). The twooptions actually lead only to three possible combinations. For safety the combination that both are off ineffect means that the software will still use the given maximum as the limit (and is thus the same as notto exceed on).10.1.3 Auto AlignThe gun alignment can be performed automatically. The alignment is split into three possibleprocedures:• Gun tilt - the alignment that is the most frequently needed.• Gun tilt plus gun shift - since the gun shift is much more stable, this alignment is really only neededafter a change of filament (thermionic gun).• Gun tilt, gun shift plus spot size-dependent gun shift - the full procedure which is really only neededafter a filament change.Notes:• The alignments make use of the measurement of the current on the large fluorescent screen. Thisscreen must there be down and the small screen out. If the beam current is less than the minimumdetectable screen current, the procedure will either terminate or (in the spot size-dependent gunshift) skip the smaller spot sizes.• Some of the measurements take place at high magnifications and require a reasonably focusedbeam. The latter is set through the focused beam function that is derived from the spot-size intensitycalibration (in the HM Beam and LM Beam alignment procedures). Because on a FEG the size of thebeam additionally depends on the extraction voltage and gun-lens setting, the software will ask theuser to focus the beam. The aforementioned calibration should nevertheless be done correctly,because the software only asks for one spot, the others should then automatically be focused aswell.• For a FEG, the is one more important alignment - the gun tilt pivot points. These cannot be alignedon the basis of the screen-current measurement and are therefore not included.• For a FEG the alignment depends on the gun settings, so all alignment values may change as afunction of extraction voltage and gun-lens setting - though in general the same sensitivity is presentas with the thermionic filament, with gun tilt most frequently needed and spot size-dependent gunshift the least.• The accessibility of alignments depends on user level. For 'Users' only the gun-tilt procedure isaccessible (the inherent assumption is that the remainder of the alignment is derived from Supervisoror higher user levels). All other levels have access to all three procedures.

<strong>Tecnai</strong> on-line help Options 134Options Software version 210.2 AutoGun (User) – (Thermionic)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, filament, alignment and vacuum controls.Note: The AutoGun control panel covers all available functionality of the normal Filament (User)control panel (and has more). It is suggested that you remove the normal Filament (User) controlfrom your user interface setup and insert the AutoGun control panel instead.LightThe Light button starts the procedure to make light :• Check high tension status and switch high tension on if needed.• Check the status of the filament and switch it on if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM range with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. This means that the column valves areclosed and the filament is switched off.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the gun-tilt alignment procedure. The color of the button indicateswhether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 135Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising :• 40, 60, 80 and 100 kV for <strong>Tecnai</strong> 10• 20, 40, 60, 80, 100 and 120 kV for <strong>Tecnai</strong> 12• 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> 20• 50,100,150,200, 250 and 300 kV for <strong>Tecnai</strong> 30FilamentThe Filament button switches the filament on or off. The color of the button indicates the filament status(yellow = on). The button is only enabled when the high tension is on.SaturateThe Saturate button starts or stops the filament autosaturation procedure. The color of the buttonindicates whether the procedure is active or not (yellow = active). The button is only enabled when thehigh tension is on.Current filament settingThe value gives the currently active filament setting (corresponding to the value displayed underneaththe progress bar).User filament settingThe User filament index is set with the spin control. Spin the value to the required setting. The valuecannot exceed the filament limit set by the Supervisor. When the Enter button is enabled (which occursafter the value has been changed by the user), the value currently displayed can be sent to the server bypressing the button (as long as the button is enabled, the server has not been updated).Filament setting displayThe Filament setting display shows the currently active setting of the filament. The value is displayed innumerical format underneath as well as in a progress bar. The progress bar always has a range from 0to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by ared bar coming from the right.Emission currentThe emission current display shows the value of the emission current.Emission settingThe emission setting can be changed with the spin control within the range 1 to 6.

<strong>Tecnai</strong> on-line help Options 136Options Software version 210.3 AutoGun (Expert) – (Thermionic)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, filament, alignment and vacuum controls.Note: The AutoGun control panel covers a major part of the functionality of the normal Filament(Expert) and High tension (Expert) control panels (and has more). It is suggested that you removethese control from your user interface setup and insert the AutoGun control panel instead.The functionality not covered is that related to the ability to set the high tension to other valuesthen the preselection (Free High Tension control).LightThe Light button starts the procedure to make light:• Check high tension status and switch high tension on if needed.• Check the status of the filament and switch it on if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM range with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means thatthe column valves are closed.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the alignment procedure selected from the drop-down list on the right.The color of the button indicates whether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 137Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising :• 40, 60, 80 and 100 kV for <strong>Tecnai</strong> 10• 20, 40, 60, 80, 100 and 120 kV for <strong>Tecnai</strong> 12• 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> 20• 50,100,150,200, 250 and 300 kV for <strong>Tecnai</strong> 30The actual high tension value is displayed on the right. Normally this is the same as the value from thedrop-down list, but during conditioning it may differ.FilamentThe Filament button switches the filament on or off. The color of the button indicates the filament status(yellow = on). The button is only enabled when the high tension is on.SaturateThe Saturate button starts or stops the filament autosaturation procedure. The color of the buttonindicates whether the procedure is active or not (yellow = active). The button is only enabled when thehigh tension is on.Current filament settingThe value gives the currently active filament setting (corresponding to the value displayed underneaththe progress bar).User filament settingThe User filament index is set with the spin control. Spin the value to the required setting. The valuecannot exceed the filament limit set in the flap-out. When the Enter button is enabled (which occurs afterthe value has been changed by the user), the value currently displayed can be sent to the server bypressing the button (as long as the button is enabled, the server has not been updated).Filament setting displayThe Filament setting display shows the currently active setting of the filament. The value is displayed innumerical format underneath as well as in a progress bar. The progress bar always has a range from 0to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by ared bar coming from the right.Emission currentThe emission current display shows the value of the emission current.Emission settingThe emission setting can be changed with the spin control within the range 1 to 6.Flap-out buttonThe flap-out button leads to the Control and Saturate tabs of the AutoGun Control Panel.

<strong>Tecnai</strong> on-line help Options 138Options Software version 210.4 AutoGun Control (Expert) – (Thermionic)The AutoGun Control Panel provides a number of systemsettings concerning the filament as well as access to theConditioning procedure.FilamentThe filament type can either be Tungsten or LaB 6 . The type of filament can only be chosen by theSupervisor.Filament limitThe filament limit is the value up to which the filament setting can be increased before it stopsautomatically. The value can be adjusted by Expert user and Supervisor.Condition statusIt is not always possible to start the conditioning procedure. The Condition status indicates whyconditioning is not possible (apart from another procedure currently running). If conditioning is possible,the condition status is not visible and the Condition button is enabled.ConditionPressing the Condition button starts or stops the Conditioning procedure. The color of the buttonindicates the status (yellow = active).Condition toThe value for condition to is the goal used by the conditioning procedure. For Experts the suggestedvalue is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it isthe maximum high tension.Condition delay stepThe condition delay step defines the number of seconds the conditioning procedure will before it takesanother step (subject to the vacuum pressure being low enough).Filament hour counterThe filament hour counter displays the date the filament was installed and the number of hours thefilament has been in operation.

<strong>Tecnai</strong> on-line help Options 139Options Software version 210.5 AutoGun Saturate (Expert) – (Thermionic)The AutoGun Saturate Control Panel provides the settings thatcontrol the autosaturation procedure.Do not exceedWhen checked, the 'Do not exceed' (a defined emission current) option will make sure that the emissioncurrent with the saturated filament does not exceed the maximum given. Suitable settings are 10 µA forLaB 6 and 30 µA for Tungsten filaments.Notes:• The software subtracts any dark emission current (emission current reading with the filament off)from the measured emission current when determining the allowed maximum.• If both options are on, the instruction not to exceed the maximum wins in case of a conflict (currentemission setting gives too much emission current).• For safety reasons (to prevent filaments burning out because of too high emission), the softwaretreats the case with both options off as being the same as 'Do not exceed' on.Keep current emission settingWhen checked, the 'Keep current emission setting' options will aim at saturating the filament for thecurrently active emission (Wehnelt) setting.

<strong>Tecnai</strong> on-line help Options 140Options Software version 210.6 AutoGun (Supervisor) – (Thermionic)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, filament, alignment and vacuum controls.Note: The AutoGun control panel covers a major part of the functionality of the normal Filament(Supervisor) and High tension (Expert) control panels (and has more). It is suggested that youremove these control from your user interface setup and insert the AutoGun control panelinstead.The functionality not covered is that related to the ability to set the high tension to other valuesthen the preselection (Free High Tension control).LightThe Light button starts the procedure to make light:• Check high tension status and switch high tension on if needed.• Check the status of the filament and switch it on if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM range with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means thatthe column valves are closed.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the alignment procedure selected from the drop-down list on the right.The color of the button indicates whether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 141Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising :• 40, 60, 80 and 100 kV for <strong>Tecnai</strong> 10• 20, 40, 60, 80, 100 and 120 kV for <strong>Tecnai</strong> 12• 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> 20• 50,100,150,200, 250 and 300 kV for <strong>Tecnai</strong> 30The actual high tension value is displayed on the right. Normally this is the same as the value from thedrop-down list, but during conditioning it may differ.FilamentThe Filament button switches the filament on or off. The color of the button indicates the filament status(yellow = on). The button is only enabled when the high tension is on.SaturateThe Saturate button starts or stops the filament autosaturation procedure. The color of the buttonindicates whether the procedure is active or not (yellow = active). The button is only enabled when thehigh tension is on.Cold run upThe Cold run up button starts or stops the cold run up procedure, which is a combination of conditioning,filament autosaturation and full gun alignment. After the procedure has finished it will close the columnvalves and switch the filament off again. The procedure is meant to be run after exchange of a filamentand the button is only enabled when the filament hour counter has been reset to 0 or a gun air has beendone. The color of the button indicates whether the procedure is active or not (yellow = active).Current filament settingThe value gives the currently active filament setting (corresponding to the value displayed underneaththe progress bar).User filament settingThe User filament index is set with the spin control. Spin the value to the required setting. The valuecannot exceed the filament limit set in the flap-out. When the Enter button is enabled (which occurs afterthe value has been changed by the user), the value currently displayed can be sent to the server bypressing the button (as long as the button is enabled, the server has not been updated).Filament setting displayThe Filament setting display shows the currently active setting of the filament. The value is displayed innumerical format underneath as well as in a progress bar. The progress bar always has a range from 0to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by ared bar coming from the right.

<strong>Tecnai</strong> on-line help Options 142Options Software version 2Emission currentThe emission current display shows the value of the emission current.Emission settingThe emission setting can be changed with the spin control within the range 1 to 6.Flap-out buttonThe flap-out button leads to the Control and Saturate tabs of the AutoGun Control Panel.10.7 AutoGun Control (Supervisor) – (Thermionic)The AutoGun Control Panel provides a number of systemsettings concerning the filament as well as access to theConditioning procedure.FilamentThe filament type can either be Tungsten or LaB 6 (CeB 6 can be treated as equal to LaB 6 ). Theinstrument has two sets of values for controlling the filament. These are exchanged when the otherfilament type is chosen. The type of filament can only be chosen by the Supervisor from the drop-downlist.Filament limitThe filament limit is the value up to which the filament setting can be increased before it stopsautomatically. The value can be adjusted by Expert user and Supervisor. Note that the Supervisorsetting is automatically used for the 'User' user level.Condition statusIt is not always possible to start the conditioning procedure. The Condition status indicates whyconditioning is not possible (apart from another procedure currently running). If conditioning is possible,the condition status is not visible and the Condition button is enabled.ConditionPressing the Condition button starts or stops the Conditioning procedure. The color of the buttonindicates the status (yellow = active).Condition toThe value for condition to is the goal used by the conditioning procedure. For Experts the suggestedvalue is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it isthe maximum high tension.

<strong>Tecnai</strong> on-line help Options 143Options Software version 2Condition delay stepThe condition delay step defines the number of seconds the conditioning procedure will before it takesanother step (subject to the vacuum pressure being low enough).Filament hour counterThe filament hour counter displays the date the filament was installed and the number of hours thefilament has been in operation.Reset countPressing the Reset count button resets the filament counter to zero (that is, the date becomes today andthe hour counter becomes zero). This function is only accessible to the Supervisor. Pressing the Resetcount button should be done after the filament has been exchanged.10.8 AutoGun Saturate (Supervisor) – (Thermionic)The AutoGun Saturate Control Panel provides the settings thatcontrol the autosaturation procedure. Note that the settingsdefined by the Supervisor automatically apply to the 'User' userlevel.Do not exceedWhen checked, the 'Do not exceed' (a defined emission current) option will make sure that the emissioncurrent with the saturated filament does not exceed the maximum given. Suitable settings are 10 µA forLaB 6 and 30 µA for Tungsten filaments.Notes:• The software subtracts any dark emission current (emission current reading with the filament off)from the measured emission current when determining the allowed maximum.• If both options are on, the instruction not to exceed the maximum wins in case of a conflict (currentemission setting gives too much emission current).• For safety reasons (to prevent filaments burning out because of too high emission), the softwaretreats the case with both options off as being the same as 'Do not exceed' on.Keep current emission settingWhen checked, the 'Keep current emission setting' options will aim at saturating the filament for thecurrently active emission (Wehnelt) setting.

<strong>Tecnai</strong> on-line help Options 144Options Software version 210.9 AutoGun (User) – (FEG)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, FEG, alignment and vacuum controls.Note: The AutoGun control panel covers all available functionality of the normal FEG (User)control panel (and has more). It is suggested that you remove the normal FEG (User) control fromyour user interface setup and insert the AutoGun control panel instead.LightThe Light button starts the procedure to make light:• Check high tension status and switch high tension on if needed.• Check the status of the FEG and switch to Operate if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means thatthe column valves are closed.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the gun-tilt alignment procedure. The color of the button indicateswhether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 145Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> F20 and 50,100,150,200,250 and 300 kV for <strong>Tecnai</strong> F30).OperateThe Operate button switches between the Operate and Standby FEG states. The button is only enabledwhen the FEG is on.Current extraction voltageThe value gives the currently active extraction voltage (corresponding to the value displayed on theprogress bar).User extraction voltageThe Extraction voltage is set with the spin control. Spin the value to the required setting. The value of theExtraction voltage cannot exceed the Extractor limit set by the Supervisor. When the Enter button isenabled (which occurs after the value has been changed by the user), the value currently displayed canbe sent to the server by pressing the button (as long as the button is enabled, the server has not beenupdated).Extraction voltage displayThe Extraction voltage display shows the value of the extraction voltage. The value is displayed innumerical format on the left as well as in a progress bar. The progress bar always has a range of 1000Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a redbar coming from the right.Emission currentThe FEG Emission current display shows the value of the emission current.Gun lensThe Gun lens setting can be changed with the spin control within the range 1 to 8.

<strong>Tecnai</strong> on-line help Options 146Options Software version 210.10 AutoGun (Expert) – (FEG)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, FEG, alignment and vacuum controls.Note: The AutoGun control panel covers a major part of the functionality of the normal FEG(Expert) and High tension (Expert) control panels (and has more). It is suggested that you removethese control from your user interface setup and insert the AutoGun control panel instead.The functionality not covered is that related to powering the FEG up or switching it off and theability to set the high tension to other values then the preselection (Free High Tension control).LightThe Light button starts the procedure to make light:• Check high tension status and switch high tension on if needed.• Check the status of the FEG and switch to Operate if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means thatthe column valves are closed.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the alignment procedure selected from the drop-down list on the right.The color of the button indicates whether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 147Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> F20 and 50,100,150,200,250 and 300 kV for <strong>Tecnai</strong> F30).OperateThe Operate button switches between the Operate and Standby FEG states. The button is only enabledwhen the FEG is on.Current extraction voltageThe value gives the currently active extraction voltage (corresponding to the value displayed on theprogress bar).User extraction voltageThe Extraction voltage is set with the spin control. Spin the value to the required setting. The value of theExtraction voltage cannot exceed the Extractor limit set in the flap-out. When the Enter button is enabled(which occurs after the value has been changed by the user), the value currently displayed can be sentto the server by pressing the button (as long as the button is enabled, the server has not been updated).Extraction voltage displayThe Extraction voltage display shows the value of the extraction voltage. The value is displayed innumerical format on the left as well as in a progress bar. The progress bar always has a range of 1000Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a redbar coming from the right.Emission currentThe FEG Emission current display shows the value of the emission current.Gun lensThe Gun lens setting can be changed with the spin control within the range 1 to 8.Flap-out buttonThe flap-out button leads to the Control tab of the AutoGun Control Panel.

<strong>Tecnai</strong> on-line help Options 148Options Software version 210.11 AutoGun Control (Expert) – (FEG)The AutoGun Control Panel provides access to the extractionlimit as well as the Conditioning procedure.Extraction limitThe filament limit is the value up to which the filament setting can be increased before it stopsautomatically. The value can be adjusted by Expert user and Supervisor.Condition statusIt is not always possible to start the conditioning procedure. The Condition status indicates whyconditioning is not possible (apart from another procedure currently running). If conditioning is possible,the condition status is not visible and the Condition button is enabled.ConditionPressing the Condition button starts or stops the Conditioning procedure. The color of the buttonindicates the status (yellow = active).Condition toThe value for condition to is the goal used by the conditioning procedure. For Experts the suggestedvalue is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it isthe maximum high tension.Condition delay stepThe condition delay step defines the number of seconds the conditioning procedure will before it takesanother step (subject to the vacuum pressure being low enough).Operate and standby hoursThe FEG Timers control panel displays the date the timers were started (FEG tip installation) and thehours the FEG tip has run in Standby and Operate modes. The timers can be reset only by service.

<strong>Tecnai</strong> on-line help Options 149Options Software version 210.12 AutoGun (Supervisor) – (FEG)The AutoGun Control Panel provides functionality to control theelectron gun and related functions that are required to switch'light' on or off on the microscope. It combines functionality fromthe high-tension, FEG, alignment and vacuum controls.Note: The AutoGun control panel covers a major part of the functionality of the normal FEG(Expert) and High tension (Expert) control panels (and has more). It is suggested that you removethese control from your user interface setup and insert the AutoGun control panel instead.The functionality not covered is that related to powering the FEG up or switching it off and theability to set the high tension to other values then the preselection (Free High Tension control).LightThe Light button starts the procedure to make light:• Check high tension status and switch high tension on if needed.• Check the status of the FEG and switch to Operate if needed.• Check the status of the Column valves and open these if needed.• Switch to a low magnification in the HM with suitable illumination settings.If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the statuswill display why.When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means thatthe column valves are closed.When the make light (or switch off) is active, the button will be orange (a transitional status).AlignThe Align button starts or stops the alignment procedure selected from the drop-down list on the right.The color of the button indicates whether the procedure is active (yellow).Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because thebeam moves when the pivot points are changed. AutoGun has a special feature that will detect thechange in pivot point and automatically compensates the gun-deflection settings to keep the beamcentered. This makes the alignment of the gun tilt pivot much easier. You do not have to activateanything in AutoGun, just make sure it is in the user interface.StatusThe status label provides feedback on status and operation of the AutoGun functionality.

<strong>Tecnai</strong> on-line help Options 150Options Software version 2High tensionPressing the High tension button switches the high tension on and off. The color of the button indicatesthe high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list boxon the right. The High tension button has three possible settings:• The high tension is enabled but off : the button is 'normal' gray.• The high tension is on : the button is yellow.• The high tension is disabled : the text in the button is gray.The high tension is enabled through the High tension enable button on the System On/Off Panel.High-tension selectionThe high tension setting is by clicking in the drop-down list box and selecting the required setting (arange of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for <strong>Tecnai</strong> F20 and 50,100,150,200,250 and 300 kV for <strong>Tecnai</strong> F30).OperateThe Operate button switches between the Operate and Standby FEG states. The button is only enabledwhen the FEG is on.Current extraction voltageThe value gives the currently active extraction voltage (corresponding to the value displayed on theprogress bar).User extraction voltageThe Extraction voltage is set with the spin control. Spin the value to the required setting. The value of theExtraction voltage cannot exceed the Extractor limit set in the flap-out. When the Enter button is enabled(which occurs after the value has been changed by the user), the value currently displayed can be sentto the server by pressing the button (as long as the button is enabled, the server has not been updated).Extraction voltage displayThe Extraction voltage display shows the value of the extraction voltage. The value is displayed innumerical format on the left as well as in a progress bar. The progress bar always has a range of 1000Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a redbar coming from the right.Emission currentThe FEG Emission current display shows the value of the emission current.Gun lensThe Gun lens setting can be changed with the spin control within the range 1 to 8.Flap-out buttonThe flap-out button leads to the Control tab of the AutoGun Control Panel.

<strong>Tecnai</strong> on-line help Options 151Options Software version 210.13 AutoGun Control (Supervisor) – (FEG)The AutoGun Control Panel provides access to the extractionlimit as well as the Conditioning procedure.Extraction limitThe filament limit is the value up to which the filament setting can be increased before it stopsautomatically. The value can be adjusted by Expert user and Supervisor. For 'User' user level theextraction limit as set by the Supervisor is used.Condition statusIt is not always possible to start the conditioning procedure. The Condition status indicates whyconditioning is not possible (apart from another procedure currently running). If conditioning is possible,the condition status is not visible and the Condition button is enabled.ConditionPressing the Condition button starts or stops the Conditioning procedure. The color of the buttonindicates the status (yellow = active).Condition toThe value for condition to is the goal used by the conditioning procedure. For Experts the suggestedvalue is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it isthe maximum high tension.Condition delay stepThe condition delay step defines the number of seconds the conditioning procedure will before it takesanother step (subject to the vacuum pressure being low enough).Operate and standby hoursThe FEG Timers control panel displays the date the timers were started (FEG tip installation) and thehours the FEG tip has run in Standby and Operate modes. The timers can be reset only by service.

<strong>Tecnai</strong> on-line help Options 152Options Software version 211 AutoAdjust (FP5460/10)11.1 AutoAdjust (User) Control PanelThe AutoAdjust Control Panel provides a number of automatedfunctions:• AutoFocus with or without autostigmation• Auto beam center• Auto rotation center• Auto eucentric heightFocusWhen the Focus button is pressed, the AutoFocus procedure is started when the button is gray. Whenthe procedure is already running (the button is yellow), the procedure is canceled.The Focus button is enabled when :• No other procedure is running.• The currently active microscope mode is compatible with the AutoFocus procedure (TEM imaging orNanoprobe imaging at magnifications

<strong>Tecnai</strong> on-line help Options 153Options Software version 2TuneWhen the Tune button is pressed, the tuning procedure selected in the drop-down list is started when thebutton is gray. When the procedure is already running (the button is yellow), the procedure is canceled.The available tuning procedures are :• All - in sequence - eucentric height, beam center, rotation center, focus and stigmation• Eucentric height• Beam center• Rotation center• Tune selectionThe drop-down list allows selection of one of the tuning procedures.Very important: The procedures available depend on the status of the calibrations done.Eucentric height cannot be performed if no calibrations have been done. Focus and rotationcenter can be done but will require more effort, because the software must iterate themeasurements in order to achieve the desired result. Also, the focus will be calibrated for thecurrently active magnification, but for nay other magnification, it will need to iterate again.StatusThe Status field shows information about the currently active procedure, feedback on possible failures,and instructions to be carried out.NextWhen the user must carry out some instruction (as given in the Status field), the Next button becomesenabled. Press the Next button to continue (after having carried out the instructions) or the Focus orTune button to cancel further execution.FlapoutThe flapout button leads to the Settings tab of the AutoAdjust Control Panel.

<strong>Tecnai</strong> on-line help Options 154Options Software version 211.2 AutoAdjust (Expert) Control PanelThe AutoAdjust Control Panel provides a number of automatedfunctions:• AutoFocus with or without autostigmation• Auto beam center• Auto rotation center• Auto eucentric heightFocusWhen the Focus button is pressed, the AutoFocus procedure is started when the button is gray. Whenthe procedure is already running (the button is yellow), the procedure is canceled.The Focus button is enabled when :• No other procedure is running.• The currently active microscope mode is compatible with the AutoFocus procedure (TEM imaging orNanoprobe imaging at magnifications

<strong>Tecnai</strong> on-line help Options 155Options Software version 2The drop-down list allows selection of one of the tuning procedures.StatusThe Status field shows information about the currently active procedure, feedback on possible failures,and instructions to be carried out.NextWhen the user must carry out some instruction (as given in the Status field), the Next button becomesenabled. Press the Next button to continue (after having carried out the instructions) or the Focus orTune button to cancel further execution.FlapoutThe flapout button leads to the Settings and Calibrate tabs of the AutoAdjust Control Panel.

<strong>Tecnai</strong> on-line help Options 156Options Software version 211.3 AutoAdjust Calibrate (Expert) Control PanelThe AutoAdjust Calibrate Control Panel provides the calibrationprocedures necessary for proper operation of the AutoAdjustfunctions.CalibratePressing the calibrate button starts the calibration procedure selected in the drop-down list, if the buttonis gray. If a procedure is already running (the button is yellow), the procedure is canceled. Calibrationsmust be done in the proper sequence and if a procedure is selected that is not yet possible (becausecalibrations higher up in the hierarchy have not been done), the list will jump back to the lowermostprocedure possible.Calibration selectionThe drop-down list allows selection of one of the calibration procedures.Calibration statusThe Status field shows information about the currently active procedure, feedback on possible failures,and instructions to be carried out.NextWhen the user must carry out some instruction (as given in the Status field), the Next button becomesenabled. Press the Next button to continue (after having carried out the instructions) or the Calibratebutton to cancel further execution.11.3.1 CalibrationsCalibrations have a set hierarchy (some calibrations can only be done once others have been done).They are specific to a number of conditions :• CCD camera used in combination with the microscope lens series (Normal or EFTEM)• High tension• Microscope mode (LM, Microprobe, Nanoprobe, Lorentz)• For the microscope modes, only the Nanoprobe mode does not require full calibrations. For thatmode only the Focus/Stigmator calibration must be done separately (the rest of the calibrations if thesame as in the Microprobe mode).Calibrations can only be done by TEM Experts (private calibrations for these users) and Supervisor,Service or Factory (dependent on availability, the se calibrations are what basic microscope Users get.Calibration is done on the basis of a standard specimen, a cross-grating. This specimen has squares onit with a spacing of 463 nm.

<strong>Tecnai</strong> on-line help Options 157Options Software version 2The following calibration procedures are used :• Magnification calibration• Image-shift calibration• Stage calibration• Stigmator alignment• Focus/stigmator calibrationMagnification calibrationThe software will acquire an image of the cross-grating and analyse that to find the basic spacing. Theanalysis is done using an auto-correlation (at high magnifications) or an FFT (at low magnifications) inwhich the two basic vectors will be outlined by circles.An image of a cross-grating, showing the 463 nm squares.The result of the magnification calibration. The autocorrelationof the cross-grating image (zoomed in 2x) shows the centralpeak pointed at by the line (which has been added manually forclarity). The software adds the two circles around the two basevectors of the pattern. It is important that these circles arelocated at the correct positions and not e.g. at the diagonals ofthe squares. Additional circles are placed at the multiples of thebase vectors used to show how many base vectors were usedtogether for the analysis.Image-shift calibrationThe image-shift calibration procedure performs a calibration of the image shift on the CCD. Thiscalibration must be done for all magnifications in the required range (up to magnifications of ~150kx).Normally the whole range is calibrated in one procedure, but it also possible to (re)calibrate the imageshift for an individual magnification (e.g. when it appears that the calibration in the complete procedurewent wrong for one magnification, that magnification can be redone later).

<strong>Tecnai</strong> on-line help Options 158Options Software version 2Stage calibrationThe stage calibration calibrates the stage x and y shifts. This calibration is needed for consistencychecks of the eucentric height procedure.Stigmator alignmentThe stigmator alignment ensures that image shifts caused by changes of the stigmator (objective in HMimage and Nanoprobe; diffraction in LM image) are minimised. This procedure performs the same as thestigmator alignment procedures themselves (in the Alignment Control Panel) except in AutoAdjust thealignment is done automatically. It should be noted that the focus/stigmator calibration is dependent onthis alignment and a change in the alignment will interfere with proper operation of the AutoAdjustfunctions.Focus/Stigmator calibrationThe focus/stigmator calibration procedure performs the calibration of beam tilt, focus/stigmator andimage shift. Once this calibration has been done, the focus/procedure can be run for any magnificationthat is smaller than ~150kx (above the beam-tilt induced image-shift method doesn't work anymorebecause random fluctuations of image position start to play an important role and interfere too much withthe focus/astigmatism measurement).

<strong>Tecnai</strong> on-line help Options 159Options Software version 212 TrueImage Focus Series AcquisitionThe Automated Experiments Control Panel with TrueImageFocus Series Acquisition active.12.1 IntroductionThe TrueImage suite of software provides the tools for the PAL-MAL focus series reconstruction ofHRTEM images. The software consists of two parts, one for the acquisition and the other for thereconstruction. The acquisition on the <strong>Tecnai</strong> microscope is covered by the Focus series acquisitionautomated experiment. This software is the only reliable way for acquiring HRTEM through-focus serieswherein the focus steps are kept the same throughout the series. In order to do so, the focus seriesacquisition software controls the objective-lens current at the level of the DACs (Digital-to-AnalogConverters that control the actual current through the objective lens). Using manual control via the Focusknob is not sufficiently accurate for this purpose because the optics software focus step size is not linkeddirectly to the control of DACs.The objective-lens current is set by two (coarse, fine) or three (coarse, fine, superfine) DACs which arestacked on top of each other, the number depending on the type of microscope and objective lens.During focus series acquisition the resetting of the finest DAC (as happens when the DAC runs out ofrange) must be avoided because the compensation of the reset by the coarser DAC is not accurateenough. The software ensures there is enough range on the finest DAC for recording the whole focusseries. In order to do so, it may reset the finest DAC before the series is recorded. When that happens,the software gives a message, the user must refocus (to be sure the starting focus is still correct), andpress the Next button to continue.Because of the DAC control, the step available for the focus is always in integer values of the DAC step,and not a value in physical units. The software indicates the physical focus step associated with thesmallest DAC step. The step you select is then a multiple of that value (e.g. if you want steps of 5 nmand the smallest step is 1.6 nm, you choose as step size 3 which the closest to 5/1.6). Anotherconsequence is the possibility of a small mismatch between the indicated focus on the microscope andthe actual focus in the series (the former is controlled by the focus knob while the latter is solelydependent on the DAC settings). Typical step sizes normally used for focus series acquisition range from~2 nm (300kV U-TWIN) to ~6nm (200kV S-TWIN).

<strong>Tecnai</strong> on-line help Options 160Options Software version 212.2 Binary file formatsYou can save the image of the focus series in different binary file formats. However, only the extendedheaderbinary stores all the information that helps to simplify TrueImage reconstruction. The binary fileformats are :• Binary : only data are stored in the file. Further information about data type (2- or 4-byte integer,single- or double-precision floats) and image dimensions is put into a separate .log file with same filename.• Extended-header :the binary data are preceded by an extended header that contains the data type,image dimensions, and several types of information stored specifically for Focus seriesreconstruction (see extended-header definition below). Note that depending on the particularsoftware that writes the file, not all values are necessarily defined.12.2.1 Data type1 - unsigned 1-byte integer2 - unsigned 2-byte integer3 - unsigned 4-byte integer4 - signed 1-byte integer5 - signed 2-byte integer6 - signed 4-byte integer7 - 4-byte (single-precision) floating point8 - 8-byte (double-precision) floating point12.2.2 Extended-header format2-byte integer : data type of the image (see above)4-byte integer : image width in pixels4-byte integer : image height in pixels4-byte integer : total size of the header in bytes (including preceding values). In effect the offset tothe data.double : Microscope type 0 = T10 and T12, 1 = T20, 2 = T30double : Gun LaB6 = 0, FEG = 1double : Lens type HC = -2, BioTWIN = -1, TWIN = 0, STWIN = 1, UTWIN = 2double : D number of microscopedouble : High tension (kV)double : Focus spreaddouble : MTFdouble : Starting defocus (nm)double : Focus step (nm)double : DAC settingdouble : Focus value (nm)double : Pixel size (nm)double : Spherical aberration (mm)double : Semi-convergence (mrad)double : Info limit (nm-1)double : Number of imagesdouble : Image number in seriesdouble : Coma xdouble : Coma ydouble : Astigmatism 2 Xdouble : Astigmatism 2 Y

<strong>Tecnai</strong> on-line help Options 161Options Software version 2doubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoublethree doublesdoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoubledoublefurther doubles: Astigmatism 3 X: Astigmatism 3 Y: Camera type serial number: Camera position (WA, MSC, GIF): Magnification: Camera length: STEM magnification: Lens series: Lorentz: Spot size: Microscope mode: Objective lens value: Currently undefined: Image shift X (m): Image shift Y (m): Image shift X (pixels): Image shift Y (pixels): Stage X (m): Stage Y (m): Stage Z (m): Stage A (rad): Stage B (rad): Image minimum level: Image maximum level: Image real (0) or reciprocal space: undefinedMany of the values stored in the extended header are used by the TrueImage reconstruction software asa first guess to optimize the reconstructed wave function.

<strong>Tecnai</strong> on-line help Options 162Options Software version 212.3 MRC file formatThe MRC file is a file with extension .mrc. It contains a primary header and an extended header asdescribed below, followed by the data. The file format is derived from the MRC file format which wasoriginated at the Medical Research Council in Cambridge, England.• The MRC format can contain a series of images in a single file. The format consists of:• A primary header. A 1024 byte section.• An extended header. Additional information about the individual images.• Image data.12.3.1 The primary header.Data type Name Byte Commentpositioninteger (4-byte) nx 0 number of columsinteger ny 4 number of rowsinteger nz 8 number of sections (i.e. images)integer mode 12 data type, 1 = pixel value stored as short integerinteger nxstart 16 lower bound of columsinteger nystart 20 lower bound of rowsinteger nzstart 24 lower bound of sectionsinteger mx 28 cell size in Angstroms (pixel spacing=xlen/mx)integer my 32integer mz 364-byte floating point xlen 404-byte floating point ylen 444-byte floating point zlen 484-byte floating point alpha 52 cell angles in degrees4-byte floating point beta 564-byte floating point gamma 60integer mapc 64 mapping colums, rows, sections on axis(x=1,y=2,z=3)integer mapr 68integer maps 724-byte floating point amin 764-byte floating point amax 804-byte floating point amean 842-byte integer ispg 88 space group number (0 for images)2-byte integer nsymbt90 number of bytesused for storingsymmetry operators4-byte integer next 92 number of bytes in extended header. This is animportant number. It defines the offset to the firstimage2-byte integer dvid 96 creator idchar (byte) extra[30] 98 extra 30 bytes data (not used)2-byte integer numintegers 128 number of bytes per section in extended header2-byte integer numfloats 130 number of floats per section in extended header2-byte integer sub 1322-byte integer zfac 134

<strong>Tecnai</strong> on-line help Options 163Options Software version 24-byte floating point min2 1364-byte floating point max2 140 extra 28 bytes data4-byte floating point min3 1444-byte floating point max3 1484-byte floating point min4 1524-byte floating point max4 1562-byte integer idtype 1602-byte integer lens 1622-byte integer nd1 164 divide by 100 to get float value2-byte integer nd2 1662-byte integer vd1 1682-byte integer vd2 1704-byte floating point tiltangles[9] 172 used to rotate model to match rotated image4-byte floating point zorg 208 origin of image; used to auto translate model4-byte floating point xorg 212 to match a new image that has been translated4-byte floating point yorg 2164-byte integer nlabl 220 number of text labels with useful data (0-10)char data[10][80] 224 10 text labels with 80 characters12.3.2 The extended headerTotal number of extended headers (one for each image) is 1024.Please note: Some values have rather odd numbering. That is because prior usage determined thatcertain values were fixed too low and the addition of more types dictated the necessity for numbersbelow, while 0 is generally avoided. The 0 is typically an undefined number.Name Byte position Commenta tilt 0 Alpha tilt in degreesb tilt 4 Beta tilt in degreesx stage 8 Stage position in umy stage 12z stage 16x shift 20 Image shift position in umy shift 24defocus 28 Defocus in micrometersexposure time 32mean intensity 36tilt axis angle 40pixel size 44 Image pixel size in nanometersmagnification 48Microscope type 52 <strong>Tecnai</strong> 10 = -2, <strong>Tecnai</strong> 12 = -1,<strong>Tecnai</strong> 20 = 1, <strong>Tecnai</strong> 30 = 2, forTitan add 10 to HT equivalentGun type 56 W = -2, Lab6 = -1, FEG = 1Lens type 60 HC = -3, BioTWIN = -2, TWIN = -1, STWIN = 1, UTWIN = 2,XTWIN = 3D number of microscope 64High tension (kV) 68Focus spread 72

<strong>Tecnai</strong> on-line help Options 164Options Software version 2MTF 76Starting Df (nm) 80Focus step (nm) 84DAC setting 88Spherical aberration 92Semi-convergence 96Info limit (nm-1) 100Number of images 104Image number in series 108Coma 1 112Coma 2 116Astigmatism 2 1 120Astigmatism 2 2 124Astigmatism 3 1 128Astigmatism 3 2 132Camera type number 136Camera position 140 WA = 1, MSC = 2, GIF = 3Unused 144Unused 148

<strong>Tecnai</strong> on-line help Options 165Options Software version 212.4 The Automated Experiments control panelThe Automated Experiments Control Panel.ActiveWhen the Active button is pressed the Automated Experiment currently selected is switched on (thebutton becomes yellow) or off. DigitalMicrograph must be running before the Focus Series Acquisitionexperiment can be activated.Euc HeightNot available in Focus series acquisition.Auto FocusNot available in Focus series acquisitionStartWhen the Start button is pressed, the function currently selected is started. The button remains yellowduring execution of the function selected. When the button is pressed again while it is yellow, furtherexecution of the current function is canceled.NextDuring some procedures, the user must set some conditions (specimen area, illumination, etc.) on themicroscope, then press the Next button for the procedure to continue.Define FileWhen the Define File button is pressed, the software brings up the standard Save File dialog in whichyou select a folder and file name for the focus series data. The images are saved in separate files, withthe file name followed by an underscore ( _ ) and then a three-digit number (001, 002, etc.; for exampletest_001.ebn). If you collect another series without changing the file name, the second series is savedunder the file name followed by an underscore, then a serial number (e.g. 02) followed again by theunderscore and then the three-digit number (for example test_02_001.ebn).ExperimentThe Experiment drop-down list contains a list of all experiments available on the system. Select theexperiment required and press Active to get started.FunctionFor Focus series acquisition the only function currently available is Acquire series.

<strong>Tecnai</strong> on-line help Options 166Options Software version 2InstructionsThe instructions field will display instructions on how to proceed or a status description of the functioncurrently running or finished.Flap-out buttonThe flap-out button leads to the Settings tabs of the Automated Experiments Control Panel.12.5 TrueImage Focus Series Acquisition SettingsThe Automated Experiments Settings Control Panel.Each Automated Experiment will typically have a number of associated functions, each of which willhave its own settings. The settings are controlled on the Settings tab of the Automated ExperimentsControl Panel.Settings listFrom the drop-down list, a function can be selected, whose settings will then be listed in the propertyeditor underneath. The settings list for tomography contains the following:• Acquisition parameters• Reconstruction parametersSettingsThe settings are displayed in a so-called property editor. This is a two-column grid, listing the setting onthe left-hand side and its value on the right-hand side. The settings in the property editor are sortedalphabetically. You can change the settings by clicking on the value. Sometimes you have to type in anumber or other setting. In the case where a limited selection is possible (for example with the booleanoperators true or false) a small down arrow on the right-side allows selection of the one of thepossibilities. Setting changes are updated as soon as you move to another setting value (e.g. bypressing the down or up arrow). If the new setting is not allowed, it will be switched back to a propervalue.Tip: To change the settings for the function selected back to their default values, click on the propertyeditor with the right-hand mouse button and select Reset to defaults in the popup menu. All settings ofthe current set will be restored to their software default values.Note: The property editor does not allow having some properties as read-only, but the Focus seriesacquisition has several such values that are either used for information display or which definition isrestricted to some users. In practice this means that the value can be changed, but it will jump back to itsoriginal value.

<strong>Tecnai</strong> on-line help Options 167Options Software version 212.6 SettingsBelow is given a description of the groups of settings used for Focus series acquisition and theirmeaning.When you change values, the software will do check on suitable values and if necessary, adjust thevalues. Note that you can change the values even when no experiment is currently active, but you willnot see any update if the values you entered were incorrect. The update in the user interface is onlydone when the experiment is active.12.6.1 Acquisition parametersThese settings are stored and recalled for each individual user.Check Drift BeforeData PathExposure TimeFilenameFile TypeFocus IncrementFocus StepImage BinningDrift can spoil the reconstruction of a focus series because there is not enoughoverlap area between the first and last images. When the Check Drift Beforeoption is switched on (true), the software will check the drift before acquiring theseries, by recording two images with about 30 seconds between them andmeasuring the shift. When the drift exceeds a certain value, dependent on anestimate of the time needed to acquire the focus series, a warning is given andyou can cancel further acquisition. Otherwise the value of the drift measured isgiven.The path where the data will be stored. Define this path through the Define Filefunction on the main control panel.The exposure time (in seconds) used for the acquisition of the focus seriesimages.The filename under which the data will be stored. Define this name through theDefine File function on the main control panel. If the name has already beenused in the same session, the software will automatically use the same namebut with an additional serial number (02, 03, ..) to avoid overwriting thepreviously acquired focus series.Images from the tomography tilt series can be saved as native server format(either DigitalMicrograph or TIA), as one of a series of binary formats, or both. Inthe latter case each image is saved in binary and native server format. Note thatfor TrueImage reconstruction the file format must be one of the binary formats,with the extended binary advised.The true minimum focus step on the microscope, corresponding to a single stepof the finest DAC available. This value is as currently set as calibration on themicroscope. This value is read-only (you can change it, but it will jump back to itsprevious value).The number of focus increments you want to use as step between images.The binning value used for the acquisition of the focus series.

<strong>Tecnai</strong> on-line help Options 168Options Software version 2Image HeightImage ServerThe height of the image in pixels for the acquisition of the tilt series. This numberis adjusted for the binning used, so that at binning 1 a value of 1024 pixelsbecomes 512 at binning 2, etc. The only allowed values are 1024 or 2048. For a2048x2048 camera both 2048 and 1024 are allowed. The latter can be binning 1(center area of CCD) or binning 2.A choice of DigitalMicrograph (DM) or Tem Imaging and Analysis (TIA).Image WidthLast Image AtNo Of ImagesStart At CurrentFocusThe width of the image in pixels for the acquisition of the tilt series. This numberis adjusted for the binning used, so that at binning 1 a value of 1024 pixelsbecomes 512 at binning 2, etc. The only allowed values are 1024 or 2048. For a2048x2048 camera both 2048 and 1024 are allowed. The latter can be binning 1(center area of CCD) or binning 2.A parameter used together with Start At Current Focus. If the latter is false, theseries acquisition will not start at the focus you have currently set, but instead itwill aim to have the last image of the series recorded at the value specified(usually this would be the Lichte or Alpha Null focus). In order to estimate whereat which focus to start acquisition, the software assumes that the focus you havecurrently set is at the Minimum Contrast setting (~0.4 * Scherzer focus). Thereason for using Minimum Contrast rather than any other value (such as 0defocus) is that Minimum Contrast focus can be recognized reasonably well,whereas any other defocus cannot.The number of images recorded automatically in the series.This parameter defines whether you want to start the series at the focus as youhave it currently set or whether the software should aim at recording the lastimage at the value specified for the last image (see Last Image At, above).12.6.2 Reconstruction parametersThe reconstruction parameters are values that are stored in the extended-header binary file. Thesevalues are used as input for the TrueImage reconstruction software. Some of the values are systemsettings and as such can only be set by the special microscope users Supervisor, Service or Factory.Astigmatism 2 and3 X and YComa X and YFocus SpreadReconstructionLimitLichte focusThe values for two-fold and three-fold astigmatism. For future use.The values for coma. For future use.The values for the focus spread of the microscope. For future use. Value canonly be set by Supervisor, Service or Factory.The effective reconstruction limit to be used for focus-series reconstruction. Thisvalue is one of the factors determining the Lichte focus. It is in nanometer units.A value calculated by the software from the value for the reconstruction limit andhigh tension on the microscope. Generally the focus series is aimed at ending atthe Lichte focus value. Reference: H. Lichte (1993) Ultramicroscopy 51, 15.

<strong>Tecnai</strong> on-line help Options 169Options Software version 2Alpha Null focusA value calculated by the software from the value for the reconstruction limit andhigh tension on the microscope. The focus series can be aimed at ending at theAlpha Null focus value. Reference: M.A. O'Keefe (2001) 59th Ann. Proc. MSA916.MTFSemi ConvergenceSphericalAberrationA value giving a single-number representation of the Modulation TransferFunction of the CCD camera. Value can only be set by Supervisor, Service orFactory.The measure of the incidence angle, relevant for the spatial-coherenceenvelope. For future use.The value of the spherical aberration of the microscope's objective lens.