Zeiss LSM510 Meta Confocal Microscope User Guide

Zeiss 510 Meta w/ ZEN Confocal Microscope Handbook
(Revised 01/15/2010)
Startup
HBO/Hg lamp ON - below table
Little box beside the keyboard:
System/PC ON
Components ON
Computer tower ON
Login with your user name and password to Biotech domain
Double-click start ZEN
Start System (initialize hardware e.g. lasers and microscope
Image Processing is just for after-acquisition processing and analysis)
Start Lasers:
Click Laser under Setup Manager
Select the lasers you need:
Green fluorescence: Argon laser (458, 477, 488, 514 nm)(30mW)
Standby first, when status changes to ready- Click ON
output starts at 25%
increase laser power to 45% = ~6.1 Amps is good working level
To see laser status, click the Laser Properties on bottom of laser window
Red Fluorescence: DPSS 561 nm 15 mW
Far Red fluorescence: HeNe 633 nm 5.0 mW
Blue or UV fluorescence: Diode 405 nm 30mW
Standby first, when ready-ON
Minimize laser window
Microscope
In upper right corner
Ocular (eye) to use microscope
Light Path icon in setup manager to setup scope with software
choose objective
choose filter
Choose lamp - Shutters on lower right near front of microscope
Top is Fluorescence (FL) or bottom is brightfield (HAL)
Manual microscope set up:
Behind focus knob are 2 sets of buttons:
Lower buttons - Filter changes
Upper buttons - Obj changes (be careful not to get oil on dry lenses)
The button pointing toward the back moves the nosepiece
clockwise
Note: it is safer to change objectives manually
Screen at top of scope gives settings

Fluorescence light adjustment
no ND filters
Near the back of the scope on the right are adjusters:
Field stop slide up and down
Aperture stop in back
Focusing
Manual focus as usual
To set the focal plane as Zero
Press Zero button on right of scope
The 2 other buttons are Work (top) and Load (bottom)
When in focus, press and hold Work button to set
Load will then drop nosepiece and Work will return to Work position
Do not change focus in Load position or a new Work is set
If nosepiece won’t move up, you have to delete the Work position
Press top button and hold while manual focusing until in focus, then
release and new Work is set
To avoid confusion, don’t use these 2 buttons, just manually return to the
Zero position using the output on the little screen
Brightfield – Transmitted light
Switch lamps with buttons on right near front of scope
Halogen lamp rheostat in front of scope at bottom
Set Kohler Illumination
Adj condenser aperture for desired contrast
motor on top left of condenser

Confocal Mode
LSM icon to switch to confocal scan
Changes lamps, light path
Imaging Setup icon under the setup manager
Single Track for simultaneous acquisition, click the pro button at the
upper right corner of imaging setup window, you can see the single track
configs on the bottom.
Multi Track configs are on the top in Configuration, the button next to
it is "Apply".
You can assign colors to the channels here.
If you want to see the brightfield/DIC channel, check Ch D under
Setup Manger / Light Path / Acquisition
If you want to manually set up the lasers / filters / mirrors / dichriocs,
also go to Setup Manger / Light Path / Acquisition
Mode
Channel Mode for normal scanning
For Transmitted Light must use NONE for filter below stage
After adjusting, Store (button next to apply) with a new name
You can minimize this window
Acquisition Mode icon under Online Acquisition
Frame size in pixels, 512 x 512
Speed, use 6-9
Bits, 8 bits = 256 grey levels, 12 bits = 4096 grey levels
For Scan direction, click pro at the upper right corner
Averaging
Line or Frame (line is not faster)
Mean (averages) or Sum (accumulates)
Scan Area (at the bottom of the Acquisition Mode window)
Zoom – (use crop tool on image window) Must Stop scanning.
Under 1.0x you cannot rotate the scan
Rotation, X- or Y- move
To get rid of the Zoom/Rotation, Reset All
Channels tab
SET BEFORE SCAN for EACH channel every time you switch
objectives:
Pinhole = 1 Airy
make all channels have = section thickness for Z-series or
colocalization
start from the largest section (longest wavelength, or red)
Gain - start at ~500
Offset is OK
Amplifer Gain - keep at 1 unless very low signal
Laser (AOTF) keep optimal low %

Confocal Scanning
Find -the system will try to find a preliminary image for you
Fast -to continuous scan at fastest speed and w/o averaging - for focusing
and gain/offset adjustment
Single -to collect one scan - with averaging. Do this before saving.
Continuous -to continuous scan with selected scan speed (with averaging)
Stop
ALWAYS STOP SCANNING
New to open a new image window
Note: While fast scanning has a short dwell time, the repeated scans can
Photobleach as much or more than slow scan!
To focus while scanning, use microscope focus knob or use Focus icon under
Online Acquisition
Note: the nosespiece drifts very badly for first few hours due to temp
changes, move knob towards you to correct (this is down!)
Viewing Images
Image Window (in the middle of ZEN window)
Display
2D-Overlay image
Split-different channels
Range Indicator - click the channels under the image window to switch
between ranger indicator and colored images
Red - saturated pixels
Blue - pixels that are too black (background, or 0s in pixel value)
Adjust gain and laser power to minimize saturation
Adjust offset to minimize black pixels
Crop
Zoom
Overlay – adds icons to window
Scale bar is here. Click on icon and draw on image.
Saving images
Set speed and averaging and use single to collect an image you want to
save
Use save icon to the right of image window or File / Save or Save as to your
folders on the DATA Drive E:
Images is saved in .lsm format (preferred, you can manipulate later
with ZEN LE or ImageJ) or tiff/jpeg format as you want.

Z-Stack
Z-Stack icon under Multidimensional Acquisition
Mark first/last
While Fast scanning use focus knob on scope
Mark first, mark last
Can do this manually or use Stage icon
Z-series ALWAYS collects with nosepiece moving UP. If you set up your Z
series to start deep in your sample and move down to the coverslip, it
won’t. It will go to the end (coverslip) and move up to the start.
Focus values get larger as nosepiece moves down.
Use optimal interval to set up section thickness or number of slices
Change interval / number of slices as desired
Before start, check Z-Stack under Multidimensional Acquisition /
Information On Experiment
Start to start Z-series
Viewing Z-Stack
Image Window
Z Position icon makes scroll bar below image window
Gallery is overlay only, check Show Text under Gallery below image window
shows the depth from first section
3D allows you view the Z-stack in 3D
Auto Brightness Correction
When images get dimmer as you go deeper you can brighten them as you
collect them
Choose first slice, adjust settings, Set A
Choose Last slice: re-adjust settings to match (?), Set B
The settings that you change will adjust linearly through this series
Gain, Offset, Amp Gain, Laser Power
Time Series
Cycles - choose how many cycles you want to acquire
Interval – set time interval between cycles
See Actual scan time and dwell time under the Acquisition Mode
You can set more than one interval and change in the middle of the
series
Start – Manual or choose a clock time based on computer clock
Stop - Manual, set number of time points
Before start, check Time Lapse under Multidimensional Acquisition /
Information On Experiment
Start to start time series
You can save these settings for future use.

Focus, Stage and Tile Scan
Focus - set / change focus (Z) with software
Choose step size
Up arrow moves nosepiece up
Stage - move / change positions on stages in X-Y directions
Finding and marking sites of interest
Move to first position
Zero – defines this position as 0,0
Mark position
Set distance to move
Arrows go in opposite direction from image on screen
Or move to a new location and mark
Go to zero
Goes to each position and scans
To combine with Time, see Multitime series
Tile Scan (aka stitching)
Move to first position - this is center (?)
Choose #X and #Y – will move based on pixel resolution (eg 512 x 512)
Moves up and to the right first (?)
Before start, check Tile Scan under Multidimensional Acquisition /
Information On Experiment
Start to start tile scan
Bleaching (under Multidimensional Acquisition)
Draw ROIs with Regions (under Online Acquisition)
Setup bleaching / FRAP experiment
Before start, check Bleaching under Multidimensional Acquisition /
Information On Experiment
Start to start bleaching
You could combine all or any of the Z-stack, Time series, Bleaching, and Tile Scan.
You could always Reuse your imaging settings by open a previous .lsm file and click
the "reuse" button under the image window. All settings are saved for reuse on a
.lsm file except the objective, please match the objective properly before reuse your
settings.
Shutdown
Lasers OFF
Hg lamp OFF
Copy files to removable disk
File – Exit
Shut down Windows
Wait until fan for argon laser shuts off automatically
then – Components OFF and System OFF

Zeiss Axiovert 200 Microscope Specs
Filters:
1 blank = LSM???
2 Blue LP FS 02 ex 365/100 (300-400) bs 395 em 420 LP
3 Green FS 10 ex 470/40 (450-490) bs 510 em 540/50 (515-565)
4 Red FS 15 ex 546/12 (534-558) bs 580 em 590 LP
5 DIC
Objectives:
Mag NA Desc coverslip WD (mm)
5x 0.15 C Plan-NEOFLUAR 0.17 13.6
10x 0.3 C Plan-NEOFLUAR 0.17 5.6
20x 0.8 Plan-APOCHROMAT 0.17 0.55
40x 1.3 oil EC Plan-NEOFLUAR 0.17 0.20
63x 1.4 oil Plan-APOCHROMAT 0.17 0.19
40x 1.2 W C-APOCHROMAT c slip adj 0.28
DIC for all except 5x and 10x
Section thickness in um
Wavelength
Obj 488nm 561nm 633mn
5x/0.15 47.2 um 52.7 um 60.1 um
10x/0.3 11.7 13.1 14.9
20x/0.8 1.6 1.7 2.0
40x/1.3 0.9 1.0 1.1
63x/1.4 0.8 0.8 0.9
40x/1.2W 0.9 1.0 1.1
“Optimal” step size is ½ section thickness.

Filter choices
NFT-T
None
DAPI
FITC
RHOD/TEXAS RED
Analyzer module D
Main beam splitter HFT
NT80/20 (for reflected light)
HFT 405/488/561/633/KP725
HFT 405/488/561
HFT 405/514/633
HFT 458/514
HFT 458/561
HFT 458
HFT 488
Beam Splitter NFT-1
None
Mirror
NFT 490
NFT 515
NFT 565
NFT 635 VIS
NFT KP 560
Plate
Beam Splitter NFT-2
Mirror
NFT 490
NFT 515
NFT 565
Em Filter Chan 3
LP 505
LP530
LP 575
LP 560
BP 505-530
BP 505-550
BP 530-600
BP 575-615 IR
Em Filter Chan 2
LP 420
LP 475
LP 505
BP 420-280
BP 470-500
BP 475-525
BP 505-550
Change Pos
Beam Splitter NFT-3
None
Plate
None
Mirror
Em Filter Chan 4
LP 420
LP 475
LP 505
LP 530
LP 575
LP 560
BP 505-550
BP 575-615 IR
Notes
Plate transmits 100%
Mirror deflects 100%
KP = short pass
LP = long pass
BP = band pass
IR = blocks IR light
Suitable for IR excited dyes

Set-up for First-time users of the Zeiss LSM510 software
(System #209385)
Login to Windows with your Domain username and password
Find the New User Initialization files (Usually on the D drive)
Initialize_New_User_Singletracks
Initialize_New_User_Multitracks
Initialize_New_User_Macros (if desired)
Initialize
From the LSM 510 software:
Click Options, Settings
Program Start:
Check “don’t show logo”
Save:
Check the third bullet - “At “Create Database” automatically
create a subdirectory with same name as specified database
and create dataset image files in that subdirectory.”
Check “Save prompt at Closing Modified Windows” and “Warning
before overwriting existing recordsets”
Temporary Files:
“Use RAM” is fine, unless you are doing time lapse, then you may
want to set a temporary location, e.g. your server directory
Recording/Reuse:
UNcheck all
Time series: Check “Time Interval”
Image Status Display:
Check all selections under “Status display in image window” and “Image
status bar”
Don’t check “Show status display upon opening of new image display”