Leica SP2 Confocal Microscope User Guide

The Leica Confocal Microscope System
B46 Weill Hall (revised6/5/13)
Startup Turn ON 3 red buttons on far right:
1. PC ON
2. Scanner ON
3. Fan ON
Turn Laser Power Level to lowest setting (Min)
Turn argon laser KEY to Start briefly, and then back to ON
Turn Green and Red HeNe Lasers ON with keys if needed
Turn on microscope—must be on to start software
Turn on Mercury lamp
If microscope says SET? Press and hold upper stop button until “0” appears
Login to PC with your netid and netid password
Click on Leica icon to start software
When software is loaded, Always click on Beam Icon to set lasers, channels, etc.
Adjust Power Level of Argon Laser to mark (~11:00)
Note: Z-galvo stage moves down, then up ~80 um as software loads.
Always lower stage before starting software
If you get a request to initialize microscope, do it
See Troubleshooting pages 3-4 if you have a problem
Read this handout if you forget how to do something
Shutdown
If another user within 1 hour
Save images
Exit Leica software
Copy files to Imaging Lab Share
Log off Windows
Wipe oil or water off objectives
Shutdown
If another user >2 hours
As above plus:
Mercury lamp off
HeNe lasers off
Laser power to lowest setting
Total Shutdown
If no appt within 3-4 hours
Argon laser to min
Turn off all lasers with keys
Save images
Exit Leica software
Copy files to Imaging Lab Share
Logoff Windows
Shut down Windows (or not)
Mercury lamp off (must cool before restarting)
Scope and brightfield lamp off
Scanner and PC off (2 red buttons)
Wipe oil or water off objectives
Cover microscope
**Leave fan on for 10 min to cool
Fan off
REMEMBER: after hours, always shut
everything off if the next person is not
there. When in doubt, shut down.
1

Microscope (DMRE-7)
Light path control / Laser Interlock—upper left
out for scan --- in for eyepieces -- do not use middle position
(Polarizer, just below, should be out completely from microscope)
Fluorescence filter cubes
Position 1 DAPI or CFP fluorescence
Position 2 Green Fluorescence
Position 3 Red Fluorescence
Position 4 Scan or brightfield
Wollaston Prism—use BF for confocal
Mercury Lamp Light Path Control--upper right
1 st position is field stop, 4 th is aperture stop, keep both up
2 nd is empty position
3 rd is Hg shutter, marked with orange. up=closed, down=open
Transmitted Light
Halogen lamp switch on lower right, next to on/off switch
ON for eyepieces
OFF for scanning
Transmitted Light Detector (TLD) (in front of on/off switch)
ON for scanning =IN
OFF for eyepieces =OUT (push in to pop it out)
On left of base:
Back controls: Field stop (lower) and Condenser Aperture (upper)
Front controls: Neutral density filter and Rheostat
On Base: Lower polarizer moved out gives a better bright field image
Objectives - See Specs at end
Focus Motor
2 button group in back – upper to move stage up, lower to move stage down
Single button above focus knob – adjusts coarseness of focus knob
1 = fine, 2 = medium, 3 = coarse
Turn focus knob away to raise stage
Finding Specimen Plane
For oil or water immersion, raise stage with motor until close, then use knob to focus
manually until fluid joins objective. Continue up with focus knob while looking through
eyepieces. Wiggle stage as you move up.
DO NOT USE MOTOR WHILE LOOKING THROUGH EYEPIECES!!!
For dry lenses, get close, then move down with focus knob while looking thru eyepieces.
Stage Stop
Press and hold upper button on front to delete previous stop, release
Press and hold again to set to zero at your focal plane
Do not leave in SET? When exiting Leica software
Stage Rotation—Galvo stage only
Loosen thumbscrews in front and rotate center, tighten screws
2

Basic Procedure
See Startup system page 1
Choose your objective
Choose your fluorescence filter (usually 2 or 3) for viewing in the microscope.
If no light, see troubleshooting, below.
Focus on sample
When ready to scan, switch filter to position 4
Pull out knob on upper left of microscope (laser interlock)
Software
Always click on Beam Icon
Double click on desired setting
Check control panel settings at bottom of screen (for the gain-black level knobs)
Check that pinhole is at Airy 1, especially if you have changed objectives
Check Objective icon and make sure the one you are using is checked. If not, see ‘Things
to Remember,’ p.5.
Hit Continuous to start scanning
Adjust gains and focus
When you have a nice image, set Line Averaging and use Single Scan to collect an image
To save it, click on Save icon
This asks for the name of the experiment folder
To return to viewing with microscope
Change filter to 2 or 3
Push in knob on upper left
Troubleshooting
Problem: Focus knob on Control Panel not working.
This needs to be set to z-wide (until further notice). There is a separate instruction sheet
with images of the software describing how to fix this. You can focus with the microscope
focus knob, but if you want to do a Z-series, you will have to follow the other directions.
Problem: No light thru eyepieces with fluorescence:
Set filter wheel to 2 (green) or 3 (red)
Push in laser interlock knob (upper left side of microscope)
Open shutter in fluorescence light path
(upper right side of scope, see orange tape)
Turn on mercury lamp (white box on table labeled Mercury lamp)
Focus specimen in microscope
Problem: Light thru eyepieces with fluorescence seems very dim:
Check settings on upper right of microscope, the fluorescence light path
Two levers slide up and down. Up is fully open for max light.
Check for polarizer just below laser interlock knob on upper left.
If there is a polarizer here (it has a knob and an arrow on it, take it out.)
3

Problem: No light thru eyepieces with bright field (transmitted light)
Turn on lamp, switch is next to scope on/off switch
Turn off TLD, in front of on/off switch, push in and push out to pop it out
Adj brightness with wheel on lower right side of scope
Set filter wheel to 4
Push in laser interlock knob
Problem: Green Image not green
If you start with a config that does not have green color, then switch to a green-red
config, the green image will not be green. You can make it any other color except green.
The only solution is to restart the software and be sure to open the green-red config first.
Problem: Image on screen all black
Set filter wheel to 4
Turn up gain for PMT in use (check settings for control panel, bottom of screen)
Increase laser power
Adjust focus
Did you select a setting with the Beam Icon? Do that first.
Check that lasers are on-Yellow light at each laser key should be on
Problem: software can’t initialize microscope
Turn scope on
If scope says SET? Press and hold upper stop button (front base of scope, hold
until it says 0) to set a stop temporarily
Click on initialize
If system freezes, you will have to restart the software, see below
Problem: stage won’t go up far enough
Press and hold upper stop button (front of scope) until it says del, then SET?
—when in focus, press and hold again to set position = zero
Problem: Image on screen not as bright as it should be
Are the proper lasers on? Yellow light should be on at key
Is the Pinhole set at Airy 1?
Is the sample very bright thru the eyepieces?
Try exiting the program, turn scanner (middle red button) off and on, wait 30 sec, restart
the program
Problem: Red image weak
Adjust 543nm laser to 100%--this laser is not very powerful
Increase 488nm laser power
Increase Gain, try Accumulate
Problem: Stage makes loud, rapping noise
If you are pushing down on the stage or stage is too high, lower the stage with motor
If this is not the case or the noise does not stop, turn off the Scanner-middle red button.
Then turn it back on, wait 30 sec, and restart the Leica software. You will be able to
restore your data.
4

Common Error Messages:
Error: Laser Interlock: pull out knob (rod) on upper left of scope
Error: Can’t initialize microscope or lost microscope initialization or other microscope error:
Is the microscope on
Is the stage is in SET? If it is, press and hold upper button on front of scope to set a
focal plane.
Go to Tools-Microscope, hit Initialize (our scope is the default, DMRE-7)
After software is loaded you can reset the stage reset in your desired focal plane.
Error: Long message about Beam Splitter Motor.
Say OK and let it continue. This is not a fatal error. You can try to exit the software, turn the
scanner (middle red button) off and on again, wait 30 sec and restart the software. If this
does not work, the problem will be that the system does not know which beam splitter
position is active. In the Beam window, click on the spot where the green line crosses the
yellow light path arrows. Then try each position until you get the image you expect. Three
will work for green (488), two for red (543), only one for far red (633). This may sound
unscientific but actually it is perfectly legitimate. You do need to have a well-labeled
sample to figure this out. Please ask Carol or Becky or Johanna in B35 for help if you are
unsure.
Other Errors or System Freezes:
Any kind of scanning error, device error, system freezes or won’t stop scanning:
Exit software* and turn scanner off (middle red button). Then turn scanner on,
wait 30 sec and restart Leica software. You don’t have to reboot the computer. When
in doubt, turning the scanner off and on again and restarting the software is a good idea.
You will NOT lose your images. When the Leica software restarts, it will ask you if you
want the data it found. Say YES.
*When software not responding, exit by:
Ctrl-Alt-Del
Task Manager
Highlight Leica software
End Task
Repeat if necessary, you should return to Windows
Things to Remember:
Lasers
Turn on the lasers you need to use (don’t assume they are on—check!)
Adjust laser power knob to marked position (~10:00) (this is for green only)
Do not turn up laser power beyond top mark unless 488 laser line is at 100% in software
Laser power adjustment knob does not affect the Green He Ne (543nm) or the Red He Ne
(633nm) lasers. It only affects 458, 476, 488, 514 laser lines.
Bright field
For transmitted light thru the eyepieces, lamp must be on but Transmitted Light Detector
(TLD) must be off. Turn off TLD by pushing in and pulling out so the button pops out.
To collect a transmitted light image, lamp must be off and TLD on. Click on box in software
to activate TLD. You will need the Smart Mode on the Control Panel to adjust the TLD.
5

When scanning, the bright field lamp must be off. Whenever using fluorescence, the bright
field lamp must be off.
Polarizer on upper right (just below laser interlock) should be out. If you see a little knob
where there usually is not one, pull it out completely.
Objective changes
To remove an objective, use 2 hands. When it is finally loose it drops quickly. Then put it in
one of the objective boxes. Screw it securely into the cap and then screw on the covering.
Put it somewhere where you won’t bump it. These lenses are extremely expensive.
To screw in a new objective, also use 2 hands. Then tell the software what you did:
Tools-Objective
Sort by number- see list on right of scope for numbers
Drag desired objective to desired position. Close window.
Remember to put everything back when you are finished
If you forget to change the objective back and you have already exited the software, leave a
note for the next person.
After Hours
If you have a problem after hours, reread these pages. If you still have a problem, feel free
to call Carol at home: 275-9090 or cell 379-1544.
6

Control Panel
This is the set of knobs (dials) next to the keyboard used to control gain, offset, zoom, focus,
etc. At the bottom of the screen are tiny diamonds representing this control panel and next
to them the actual function each knob controls. Just above on the right are tiny icons to load
and save new configurations. The most common ones are ‘red-green’ and ‘smart.’ Smart
gain and offset will control the highlighted channel and is useful if you have more than 2
channels. Red-Green sets PMT2 and PMT3 gains and offsets.
Each knob can be configured by right-clicking on its function at the bottom of the screen.
The sensitivity of the knob rotation should be kept fine. The second option is generally
good. A new configuration can be saved by clicking on the tiny disk icon. Right click on the
name in the list to save it as the default.
Software
Acquire Options
Beam icon – always click on this and choose a setting
Choose setting from Leica list
Adjust to your needs:
Laser power and lines needed
Emission ranges and PMTs needed
Dichroic beam splitter—must match laser lines
LUTS (Look-up tables provide colors)
Save with new name. You can customize what is included in this setting by going to Tools
— Settings — Instr. Parameter Settings.
Other Defaults (adjust as desired)
Obj—should have the correct objective—use this to see what obj is where
Bit—8 bits or 12 bits. Use 12 bits for quantitative work or if you have low signal.
Expan—use 6 always
Mode—xyz mostly. xyt for time course, xzy for cross-sections
Format—512 x 512, go to 1024 x 1024 for higher resolution. Use narrow window (512 x
256) for faster scanning.
Speed—400 MHz, (400 lines/s) 200MHz is slow but gives a cleaner image. Faster scans
require a slight increase in gain and a zoom factor is automatically set.
800MHz=1.7x 1000MHz= 3.0x 1400 MHz=6.0x
Scan -> single direction (for bidirectional must set phase while acquiring)
Pinh—pinhole, 1 Airy disk (AU) recommended. Use AE scale when adjusting.
Field—rotate field being scanned. –2 to 90 degrees
Zoom—set a zoom
Z In—zoom to region drawn on image
Pan arrows—move image when zoomed
Continuous Scan—to start scanning—Creates a Preview that cannot be saved
Single Scan—to do one scan or averaging—Creates an Image file that can be saved
Li A—line average does each line as it scans—This is faster than Frame average
Aver—Frame Ave goes frame by frame
Accumulate—Integrates (adds) images together to increase signal. These images
can be line averaged but not frame averaged. Very good for low signals.
7

View Options -- Next to the displayed image
QLUT
This calls up the glow scale with upper and lower saturation limits displayed. The glow scale
is a non-linear color range that highlights bright objects. Saturated pixels will be blue and
pixels that are too black will be green. Proper settings for gain and offset will show just a
few blue and green pixels (none if you want to quantify). However, these are only guidelines
and your image may require different settings. This button has 3 settings: glow scale, black
and white, and original colors.
LUTs=Look-up tables
This opens the list of Look-up tables so you can change the color of the image without reacquiring
it. If you save again, the image will be saved with this color.
Zoom, Z-IN
Use Z-in to draw a box around the area you want to zoom in on.
First click highlights the channel, second click draws box.
OVL=Overlay
Click on the OVL button next to image for a view of the overlay. To get an overlay image
without the grey transmitted light image, unclick the channel containing the transmitted light
image. To save the overlay image, see Saving images
Gallery - To see all the planes in a Z-series or Time series. Unclick to undo.
X-sec - Use with a Z-series to get a cross section wherever the lines are on the image.
This may be in the View menu next to the acquire menu just above the Beam icon
Orig – to get out of x-sec
Z-Series Acquisition
Small series button—–to design Z-series with nice graphic
**To get the most accurate starting plane, set the end plane first.
Use Z position (=focus) knob to find end plane, Click on End.
Note: Clockwise moves deeper into your specimen
Use Z position knob to find first plane, Click on Begin.
When you look at the graphic of the box when setting your begin and end points, moving the
Z knob clockwise makes the yellow line go up. This is the stage is moving up, and you are
moving deeper into your specimen.
Sect 123—to choose # of sections or step size
You must stop scanning to set this
Choose Others to set step size, then click on
calculate # sections -- to leave step size unchanged
or calculate step size -- to leave height unchanged
cubic voxels is recommended for highest resolution and 3D rotations
get voxel size from list on screen
Intensity compensation
Designed to increase signal as you go deeper into your sample. Go thru your focus and
when the image gets dim, brighten it with gain or laser power and add this location.
Can use this to go to top or bottom—go to top before starting
8

Large Series Box —click to begin collecting Z-series, will use frame or line averaging if set
If you forget how you collected your series and want to know later on: For top (cover slip) to
bottom, the depth size in the .txt file or the physical length of Z in properties, will be a
negative value. For bottom to top, these values will be positive.
Sequential Scanning
Between Lines
This is the easiest to use but the only things that can change are which laser is on and
which channel is active. You cannot change emission collection ranges or beamsplitter
filters.
1) Set up a program for each color separately and save each, eg ‘green’ and ‘red’.
Remember, the only differences between them will be which laser is on and which PMT is
active. You can change gains anytime. Changes in laser power must be resaved.
2) Click on Sequential in the beam window, between lines is the default. Activate the first
channel (green), click Add. Activate the second channel (red), click Add. You can do as
many as you like and can collect a transmitted light image with any color.
3) Keep the sequential window open and start scanning. A continuous scan will actually do
one green line then one red line sequentially. It is slower than simultaneous scanning but
still very fast. Work as usual. You can change gains without resaving the program.
Between Frames
This allows you to vary all of the parameters checked in the list. The checked parameters
will use the saved settings in the separate green or red program. If you want to change the
gain between samples without resaving each time, uncheck gain/offset.
Set up separate programs as above and Add each.
Keep the sequential window open use the Series button to collect. If a Z-series is set up,
including the number of sections, it will be active also.
Lambda Scan
Only one laser line and one channel can be active
Mode=xy The buttons will highlight
Example:
488nm excitation
Set your channel range to be 500-510 and hit beg
Set your channel range to be 690-700 and hit end
Set steps to be 20. This will collect every 10 nm between 500 and 700. If you set the
number to 40, this will collect every 10 nm with 5nm overlap, giving better resolution.
Start collecting with Series button. If frame or line averaging are set they will be used.
Results
Quantify menu, choose Profile in Z.
Choose an area of interest, you may want to keep it very small and specific.
Click on Graph to see results. You can export the data or it can be entered back into Leica
to create an emission scan like the ones supplied by Leica.
9

Optimizing Image Quality
Increasing Signal
Check focus—choose brightest plane
Turn up gain—this increases noise, line or frame average to reduce noise
Turn up laser power—this can increase photobleaching, watch for it
Widen your PMT regions to collect more wavelengths—to avoid laser lines, you may have to
do sequential imaging
Increase the pinhole (use Airy Unit scale)—this can reduce your Z-resolution, compare to
pinhole = 1 Airy. Image may be slightly fuzzy.
Accumulate—use with line averaging, lower the offset to darken your background
Collect 12-bit images and adjust LUT (contrast stretch)
Use the LUT called P4 to collect dim images
Increasing signal to noise ratio
Keep Gain below ~700; Offset ~ 0
Use QLUT in the view menu to determine best gain and offset settings (see above)
For quantification, avoid all blue (saturated) and green (too black) pixels
Always average (frame or line average) to decrease noise. The higher the gain, the more
averaging is needed.
Scan Speed at 200 will also decrease noise
Increasing Resolution
For maximum resolution, use the maximum zoom on the table posted.
Use objective lens with higher Numerical Aperture (NA)—This usually means an oil- or
water-immersion lens and higher mag
Change format to 1024 x 1024 or higher to maintain large field
Increasing Speed
Use a faster scan speed. 400 Hz is 400 lines/sec. Faster speeds require zooming. See
Software defaults.
Collect fewer lines, eg 512 x 256
Use bi-directional scanning, adjust Phase so image looks normal
Types of Overlays
Default=Fast Bitwise
On the far left is a small icon that says ‘Display’. If you have an overlay image in the view,
there will also be an icon that says ‘Overlay’. Click on it. For RGB fluorescence images, the
Fast bitwise, which is the default, works fine.
Yellow,cyan or any color other than RGB and for greyscale (brightfield) images:
the fast bitwise default is very bad. You should switch to True Color with or without dynamic
adjustment, ie scaling. The grey will not be as bright as the original, for DIC you can try
brightening by adjusting the Wollaston prism or increasing the gain. To brighten without
saturating the fluorescence, adjust the gamma and black level in Photoshop (see below).
The result will be more or less the same.
Save your overlays with the proper setting and switch back to Fast for scanning. True color
slows down scanning considerably. You can adjust the overlay later in Leica Lite.
10

Process Menu
3D Visualization and Animations
Projections–use Maximum option
Or try color-coded projection
Projections with animations—to do 3D rotations
Click on rotation, choose angle (eg. half is +/- 90 degrees)
Choose # frames (eg 10 with half is 18 degree jumps)
Essentially makes a projection at each angle and you ‘play’ them for the 3D effect
Enhancement—use to adjust Brightness, Contrast and Gamma—not very good
Editing—use for Image Separation (eg separate red and green Z-series)
Use to remove planes
Use for file merging (make 2 separate images into one file for overlay)
Quantify Menu
To see a histogram of a line or a profile through a series or across an image. Etc.
Saving Files and File Management
All images are saved in an Experiment folder.
You cannot name or save an Expt folder until you acquire an image using single scan.
After acquiring an image, click on the Save icon
Select the D:\ALL USER IMAGES HERE folder and your personal folder. Then name your
expt. Folder where it says ‘File name’. You image will go into this folder. This folder name
CANNOT be easily changed. Keep it short. Don’t let the experiment folder get too big.
300-500MB should be max.
Always save (use icon) every image after collecting. If the software crashes, your images
can be recovered but they go into a new folder and it gets confusing.
Right click on image name to Rename or Delete images.
Images are saved as .tif files. Each channel is saved as a separate file. If you collect the
green and red channels, you will have 2 .tif files. If you save a Z-series, each plane and
channel will be a separate .tif file. If you save in color, they will open in color.
File Naming
Images will have VERY long names,
eg. foldername_imagename_plane00_channel00.tif
Do not rename or delete anything outside of the Leica software. This will confuse the
.lei file and you will never be able to open that folder in the Leica software. The .lei file tells
the Leica software all of the hardware settings under which all the images in the expt. folder
were taken
Image Properties and Apply
To view the conditions under which an image was acquired, right click on the file and
choose Properties. The Apply button will apply the properties listed to the system. You can
also get this information from the .txt file, which is in the expt folder.
New Folder
To make a new folder, click on the New icon. Do NOT try to name it with rename! First put
an image in it and hit the Save icon. Now you can name the folder. Check the path before
saving so it does not become a subdirectory of the preceding experiment folder.
11

Saving the Overlay Image
Right click on the overlay image, choose Send to—Experiment—Selection Raw is best.
The Snapshot option saves it as a bitmap image. Use snapshot to save a Gallery or an
image with a scale bar. Saving all vs selection saves the entire screen.
Saving Overlay of Z-Series
Click on gallery to see entire series. Right click, Send to—Experiment—raw. This saves a
series of .tif images just like the red or green series.
Saving as .AVI
Right click on series name. Choose Export, name it and save as type AVI.
Scale Bar
On the left side of the screen, click on display icon and check the scale bar box. Then click
on the scale bar button at the bottom. Change size and hit Apply. Move if desired. Click
on Auto-rescale to keep the same value at all zooms. Must save as Snapshot.
Transferring your files to the Imaging Lab Share
Drag files from your Leica folder (D:\All User Images Here) to your folder on the Imaging Lab
Share (Z:). Both the Leica hard drive and the server have limited space. Please download
your files promptly and delete the files from both places when you have them stored safely.
After 1 month, your files may be deleted.
Retrieving files from the imaging share—see Carol for instructions
Software for viewing your images
Note that all the images are .tif and can be opened almost anywhere if 8 bit.
Leica Lite (for Win XP)
This is a free version of the Leica software for viewing your images and doing simple
processing, like projections. You can get it from the Imaging lab share. Look under Leica
Lite. Use the Installation folder to install. It does not work with Vista or Windows 7. You can
install a free XP simulator from Microsoft. See your IT person. This works great and is set
up on our workstation in the outer room.
Projections, go to View—Fix Max—send to expt to save each channel
You can project a time series this way.
Overlays, go to view
To change the colors of your image with Leica Lite, double-click on the color bars at the right
of the image. This will bring up the LUT options and you can change the color of the image
and resave.
Image Editing—you can do separations, merges, convert to 8 bit.
To change the color, click on the color bar on the right side of the image
Scale Bar—you can put a scale bar on your image but you cannot adjust it.
LAS AF Lite
This works with Windows 7 and you can open and view .lei files with it. But you cannot do
much else. You can get this from the Imaging Lab share also.
12

Leica Software
We have a full version of the Leica software on the MetaMorph computer in MIF. Does all
the 3D reconstructions, etc.and is good for putting on scale bars.
It is free to use anytime the Metamorph system is not being used. You may want to signup
on the Metamorph calendar.
See Carol for a login so you don’t get charged for the time.
Image J (now FIJI)
This is a free program that should be able to open the .lei files but I don’t have reliable
instructions yet.
13

Transmitted Light ( Brightfield )
Introduction
You can collect a transmitted light image at the same time as the fluorescent image
to aid in localization of your fluorescence. The bright field image can be overlaid (merged)
with the fluorescent image(s).
**Note: Adjusting the microscope for an optimal bright field image is more complicated than
fluorescence. It is highly recommended that you ask for help the first few times.
Kohler Illumination
Focus on your specimen in the microscope.
Close the field stop diaphragm on left side of microscope base. Lower back wheel
Adjust the height of the condenser until the aperture is sharply focused. Silver knob on left.
Center the aperture with the centering screws.
Open the field stop until the light just fills the field and aperture is no longer visible.
Open the condenser diaphragm for desired contrast. Upper back wheel. Wide open is best.
Remove the polarizer at the base of the scope if you have annoying video lines.
Differential Interference Contrast (DIC) -- Nomarski
DIC is a method of enhancing the contrast of a bright field image. It has optical sectioning
qualities and is very good for viewing surfaces. The effect looks similar to a scanning EM
image, with a black ‘shadow’ on one side and a white ‘shadow’ on the other side. The
‘shadows’ are formed by differences in refractive index or by differences in height.
Microscope setup for DIC
First follow procedures above for Kohler Illumination
Need upper polarizer in for eyepieces-- out when scanning
Find in upper drawer and put in slot below laser interlock knob, removing plug first.
Rotate lower polarizer into place
Wollaston Prism (below filter wheel)—set for objective: C=20x D=63x/100x E=40x
Condenser --Set for objective: H-bright field 10/20 100 oil 40/63
The condenser diaphragm should be open.
Wollaston Prism
The Wollaston Prism has a thumbscrew, which can be adjusted for desired contrast. The
full range of the screw is such that the image is darkest in the center (extinction) and
brighter near the ends. The difference between the two sides is the direction on which the
‘shadows’ fall. The optimum setting is usually somewhat off center on either side.
Note that this prism can cause a slight doubling of your image at very high magnification.
Switching between the ‘scope and scanning
Scope
Halogen lamp on
Transmitted Light Detector (TLD) off
Scanning
Halogen lamp off
TLD on
To increase contrast, lower offset to -10 to -20 and increase gain
14

Photoshop
The most valuable feature in Photoshop is:
Image--Adjustments--Levels
choose RGB or individual color
use the histogram to adjust white level, black level, and gamma
Save with a new name. This does a contrast stretch and is permanent.
Coloring Images
Your Leica images should open in color
To make a Red/Black or Green/Black image from greyscale.
Open image
Image--Mode—choose RGB Color
Image--Adjust--Levels
Choose colors not wanted
Drag left triangle under histogram all the way to the right to eliminate this color
Repeat with other color not wanted. You will be left with the desired color.
Adjust as above.
Save image (You can get rid of the color by changing Mode back to Grayscale)
Saturated pixels will turn white with this method. To avoid this, try the following method:
Image--Mode--RGB Color
Image--Adjust--Hue/Saturation
Check Colorize
Saturation at 100% is probably best
Adjust Hue to ~120 for green, red is the default
Lower Lightness to color white areas and darken background
Save image
Specifications for Leica Confocal SP2
3 Laser system
4-line argon laser, lines at 458nm, 473nm, 488nm, 514nm.
458 for CFP, 514 for YFP, 488 for all green dyes
Green Helium Neon laser at 543nm for all orange / red dyes eg. rhodamine, Cy3
Red Helium Neon laser at 633nm for far red dyes eg. Cy5, TOPRO, DRAQ5
4 fluorescent detectors
1 transmitted light detector
5 channel simultaneous collection
Confocal Beamsplitter Filters
Beam Splitter Laser Lines Range 1 Range 2 Range 3
RSP 500 458, 476, 488 500-600
488/543 488, 543 500-530 555-700
488/543/633 488, 543, 633 500-535 555-620 650-750
458/514 458, 514 475-500 530-660
RT 30/70 any reflects 30% transmits 70%
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Microscope Leica Upright DMRE-7
Objectives (new arrangement 9/1/08)
Position ID# Description
1 35 10x dry HC PL APO 10x / 0.30 WD 12mm
2 30 20x Imm HC PL APO 20x / 0.70 Corr WD 250um
can be used with oil or water
Adjust collar for oil or water and cover slip
3 49 40x oil HCX PL APO 40x / 1.25 oil WD 100um
4 58 63x oil HCX PL APO 63x / 1.32 oil WD 70um
5 61 100x oil HCX PL APO 100x / 1.40 oil WD 90um
All oil lenses: keep aperture open
6 56 63x water HCX PL APO 63x / 1.20 Corr WD 220um
Adjust collar to .17 for #1.5 cover slip
7 45 40x dipping HCX APO L 40x / 0.8 W WD 3.3mm
In Drawer 32 5x dry HC PL FLUOTAR 5x / 0.15 WD 12mm
In Drawer 52 40x dry HCX PL APO CS L 40x / 0.85 Dry, WD 240um
Adjust collar to .17 for #1.5 cover slip
Filters on Microscope
UV Excitation BP 360/40 340 – 380 nm
(position 1) Dichroic 400
Emission BP470/40 450 – 490 nm
GFP (green) Excitation BP 480/40 460 - 500 nm
(position 2) Dichroic 505
Emission BP 527/30 512 - 542 nm
Red Excitation BP 510-560 510-560 nm
(position 3) Dichroic 580
Emission LP 590 590 + nm
CFP Excitation 436 / 20 426 - 456 nm
(position 1) Dichroic 455
Emission 480 /40 460 - 500 nm
Green-Red Excitation BP 450-490 450-490 nm
(in drawer) Dichroic 510
Emission LP515 515 + nm
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