LeicaSP2_ UserGuide

8/15/2019 LeicaSP2_ UserGuide

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The Leica Confocal Microscope SystemB46 Weill Hall (revised6/5/13)

Startup Turn ON 3 red buttons on far right:1. PC ON

2. Scanner ON3. Fan ON

Turn Laser Power Level to lowest setting (Min)

Turn argon laser KEY to Start briefly, and then back to ONTurn Green and Red HeNe Lasers ON with keys if neededTurn on microscope—must be on to start softwareTurn on Mercury lamp

If microscope says SET? Press and hold upper stop button until “0” appears

Login to PC with your netid and netid passwordClick on Leica icon to start softwareWhen 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 softwareIf 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

ShutdownIf another user within 1 hour Save imagesExit Leica softwareCopy files to Imaging Lab ShareLog off WindowsWipe oil or water off objectives

ShutdownIf another user >2 hours

As above plus:Mercury lamp offHeNe lasers offLaser power to lowest setting

Total ShutdownIf no appt within 3-4 hours

Argon laser to minTurn off all lasers with keysSave imagesExit Leica softwareCopy files to Imaging Lab ShareLogoff Windows

Shut down Windows (or not)Mercury lamp off (must cool before restarting)Scope and brightfield lamp offScanner and PC off (2 red buttons)

Wipe oil or water off objectivesCover microscope**Leave fan on for 10 min to coolFan off

REMEMBER: after hours, always shuteverything off if the next person is notthere. When in doubt shut down.


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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 cubesPosition 1 DAPI or CFP fluorescencePosition 2 Green FluorescencePosition 3 Red FluorescencePosition 4 Scan or brightfieldWollaston Prism—use BF for confocal

Mercury Lamp Light Path Control--upper right1st position is field stop, 4th is aperture stop, keep both up2nd is empty position3rd is Hg shutter, marked with orange. up=closed, down=open

Transmitted LightHalogen lamp switch on lower right, next to on/off switchON for eyepiecesOFF for scanning

Transmitted Light Detector (TLD) (in front of on/off switch)ON for scanning =INOFF 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 RheostatOn Base: Lower polarizer moved out gives a better bright field image

Objectives - See Specs at endFocus Motor2 button group in back – upper to move stage up, lower to move stage downSingle button above focus knob – adjusts coarseness of focus knob1 = fine, 2 = medium, 3 = coarseTurn focus knob away to raise stage

Finding Specimen PlaneFor oil or water immersion, raise stage with motor until close, then use knob to focusmanually until fluid joins objective. Continue up with focus knob while looking througheyepieces. 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 StopPress and hold upper button on front to delete previous stop, releasePress and hold again to set to zero at your focal planeDo not leave in SET? When exiting Leica software

Stage Rotation—Galvo stage onlyLoosen thumbscrews in front and rotate center, tighten screws


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Basic Procedure

See Startup system page 1Choose your objectiveChoose your fluorescence filter (usually 2 or 3) for viewing in the microscope.If no light, see troubleshooting, below.

Focus on sampleWhen ready to scan, switch filter to position 4Pull out knob on upper left of microscope (laser interlock)

Software Always click on Beam IconDouble click on desired settingCheck 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 objectivesCheck Objective icon and make sure the one you are using is checked. If not, see ‘Thingsto Remember,’ p.5. Hit Continuous to start scanning

Adjust gains and focusWhen you have a nice image, set Line Averaging and use Single Scan to collect an imageTo save it, click on Save icon

This asks for the name of the experiment folderTo return to viewing with microscope

Change filter to 2 or 3Push 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 microscopefocus 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.)


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Problem: No light thru eyepieces with bright field (transmitted light)Turn on lamp, switch is next to scope on/off switchTurn 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 scopeSet filter wheel to 4Push in laser interlock knob

Problem: Green Image not greenIf you start with a config that does not have green color, then switch to a green-redconfig, 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 blackSet filter wheel to 4Turn up gain for PMT in use (check settings for control panel, bottom of screen)Increase laser power

Adjust focusDid 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 onIf scope says SET? Press and hold upper stop button (front base of scope, hold

until it says 0) to set a stop temporarilyClick on initializeIf system freezes, you will have to restart the software, see below

Problem: stage won’t go up far enoughPress 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 keyIs 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, restartthe program

Problem: Red image weak Adjust 543nm laser to 100%--this laser is not very powerfulIncrease 488nm laser powerIncrease Gain, try Accumulate

Problem: Stage makes loud, rapping noiseIf you are pushing down on the stage or stage is too high, lower the stage with motorIf 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 torestore your data.


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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 onIs 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 thescanner (middle red button) off and on again, wait 30 sec and restart the software. If thisdoes not work, the problem will be that the system does not know which beam splitterposition is active. In the Beam window, click on the spot where the green line crosses theyellow light path arrows. Then try each position until you get the image you expect. Threewill work for green (488), two for red (543), only one for far red (633). This may soundunscientific 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 areunsure.

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 youwant the data it found. Say YES.

*When software not responding, exit by:

Ctrl-Alt-DelTask ManagerHighlight Leica softwareEnd TaskRepeat if necessary, you should return to Windows

Things to Remember:

LasersTurn 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 softwareLaser 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 fieldFor 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 softwareto activate TLD. You will need the Smart Mode on the Control Panel to adjust the TLD.


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When scanning, the bright field lamp must be off. Whenever using fluorescence, the brightfield lamp must be off.

Polarizer on upper right (just below laser interlock) should be out. If you see a little knobwhere 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 inone 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 numbersDrag desired objective to desired position. Close window.

Remember to put everything back when you are finishedIf you forget to change the objective back and you have already exited the software, leave anote for the next person.

After Hours

If you have a problem after hours, reread these pages. If you still have a problem, feel freeto call Carol at home: 275-9090 or cell 379-1544.


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Control PanelThis 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 nextto them the actual function each knob controls. Just above on the right are tiny icons to loadand save new configurations. The most common ones are ‘red-green’ and ‘smart.’ Smartgain 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 generallygood. A new configuration can be saved by clicking on the tiny disk icon. Right click on thename in the list to save it as the default.

Software Acquire OptionsBeam icon – always click on this and choose a settingChoose setting from Leica list

Adjust to your needs:

Laser power and lines neededEmission ranges and PMTs neededDichroic beam splitter —must match laser linesLUTS (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 whereBit—8 bits or 12 bits. Use 12 bits for quantitative work or if you have low signal.Expan—use 6 alwaysMode—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 x256) for faster scanning.Speed—400 MHz, (400 lines/s) 200MHz is slow but gives a cleaner image. Faster scansrequire a slight increase in gain and a zoom factor is automatically set.800MHz=1.7x 1000MHz= 3.0x 1400 MHz=6.0xScan -> 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 degreesZoom—set a zoomZ In—zoom to region drawn on imagePan arrows—move image when zoomed

Continuous Scan—to start scanning—Creates a Preview that cannot be savedSingle 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.


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View Options -- Next to the displayed imageQLUTThis calls up the glow scale with upper and lower saturation limits displayed. The glow scaleis a non-linear color range that highlights bright objects. Saturated pixels will be blue andpixels that are too black will be green. Proper settings for gain and offset will show just afew blue and green pixels (none if you want to quantify). However, these are only guidelinesand your image may require different settings. This button has 3 settings: glow scale, black

and white, and original colors.

LUTs=Look-up tablesThis opens the list of Look-up tables so you can change the color of the image without re-acquiring it. If you save again, the image will be saved with this color.

Zoom, Z-INUse 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=OverlayClick 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 lightimage. 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 iconOrig – to get out of x-sec

Z-Series AcquisitionSmall 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 theZ knob clockwise makes the yellow line go up. This is the stage is moving up, and you aremoving deeper into your specimen.

Sect 123—to choose # of sections or step sizeYou must stop scanning to set thisChoose Others to set step size, then click on

calculate # sections -- to leave step size unchangedor calculate step size -- to leave height unchanged

cubic voxels is recommended for highest resolution and 3D rotationsget 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


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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) tobottom, the depth size in the .txt file or the physical length of Z in properties, will be anegative value. For bottom to top, these values will be positive.

Sequential Scanning

Between LinesThis is the easiest to use but the only things that can change are which laser is on andwhich channel is active. You cannot change emission collection ranges or beamsplitterfilters.1) Set up a program f or 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 isactive. 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 firstchannel (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 doone green line then one red line sequentially. It is slower than simultaneous scanning butstill very fast. Work as usual. You can change gains without resaving the program.

Between FramesThis allows you to vary all of the parameters checked in the list. The checked parameterswill use the saved settings in the separate green or red program. If you want to change thegain 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 activeMode=xy The buttons will highlightExample: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 thenumber 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.ResultsQuantify 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 Leicato create an emission scan like the ones supplied by Leica.


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Optimizing Image Quality

Increasing SignalCheck focus—choose brightest planeTurn up gain—this increases noise, line or frame average to reduce noise

Turn up laser power —this can increase photobleaching, watch for itWiden your PMT regions to collect more wavelengths—to avoid laser lines, you may have todo sequential imagingIncrease the pinhole (use Airy Unit scale)—this can reduce your Z-resolution, compare topinhole = 1 Airy. Image may be slightly fuzzy.

Accumulate—use with line averaging, lower the offset to darken your backgroundCollect 12-bit images and adjust LUT (contrast stretch)Use the LUT called P4 to collect dim images

Increasing signal to noise ratioKeep Gain below ~700; Offset ~ 0Use 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 moreaveraging is needed.Scan Speed at 200 will also decrease noise

Increasing ResolutionFor maximum resolution, use the maximum zoom on the table posted.Use objective lens with higher Numerical Aperture (NA)—This usually means an oil- orwater-immersion lens and higher magChange 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. SeeSoftware defaults.Collect fewer lines, eg 512 x 256Use bi-directional scanning, adjust Phase so image looks normal

Types of OverlaysDefault=Fast BitwiseOn 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, theFast 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 trybrightening by adjusting the Wollaston prism or increasing the gain. To brighten withoutsaturating 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 colorslows down scanning considerably. You can adjust the overlay later in Leica Lite.


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Process Menu3D Visualization and AnimationsProjections –use Maximum option

Or try color-coded projectionProjections 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 goodEditing—use for Image Separation (eg separate red and green Z-series)

Use to remove planesUse for file merging (make 2 separate images into one file for overlay)

Quantify MenuTo 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 yourexpt. Folder where it says ‘File name’. You image will go into this folder. This folder nameCANNOT 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 imagescan 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 thegreen and red channels, you will have 2 .tif files. If you save a Z-series, each plane andchannel will be a separate .tif file. If you save in color, they will open in color.

File NamingImages will have VERY long names,

eg. foldername_imagename_plane00_channel00.tifDo 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 tellsthe Leica software all of the hardware settings under which all the images in the expt. folderwere taken

Image Properties and ApplyTo view the conditions under which an image was acquired, right click on the file andchoose Properties. The Apply button will apply the properties listed to the system. You canalso get this information from the .txt file, which is in the expt folder.

New FolderTo make a new folder, click on the New icon. Do NOT try to name it with rename! First putan image in it and hit the Save icon. Now you can name the folder. Check the path beforesaving so it does not become a subdirectory of the preceding experiment folder.


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Saving the Overlay ImageRight 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 animage with a scale bar. Saving all vs selection saves the entire screen.

Saving Overlay of Z-SeriesClick on gallery to see entire series. Right click, Send to—Experiment—raw. This saves aseries of .tif images just like the red or green series.Saving as .AVIRight 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 clickon the scale bar button at the bottom. Change size and hit Apply. Move if desired. Clickon Auto-rescale to keep the same value at all zooms. Must save as Snapshot.

Transferring your files to the Imaging Lab ShareDrag files from your Leica folder (D:\All User Images Here) to your folder on the Imaging LabShare (Z:). Both the Leica hard drive and the server have limited space. Please downloadyour 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 imagesNote 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 simpleprocessing, like projections. You can get it from the Imaging lab share. Look under LeicaLite. Use the Installation folder to install. It does not work with Vista or Windows 7. You caninstall a free XP simulator from Microsoft. See your IT person. This works great and is setup on our workstation in the outer room.Projections, go to View—Fix Max—send to expt to save each channelYou can project a time series this way.Overlays, go to viewTo change the colors of your image with Leica Lite, double-click on the color bars at the rightof the image. This will bring up the LUT options and you can change the color of the imageand 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 imageScale Bar —you can put a scale bar on your image but you cannot adjust it.

LAS AF LiteThis works with Windows 7 and you can open and view .lei files with it. But you cannot domuch else. You can get this from the Imaging Lab share also.


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Leica SoftwareWe have a full version of the Leica software on the MetaMorph computer in MIF. Does allthe 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 signupon 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 reliableinstructions yet.


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Transmitted Light ( Brightfield )

IntroductionYou 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 IlluminationFocus 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 sectioningqualities and is very good for viewing surfaces. The effect looks similar to a scanning EMimage, 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 DICFirst follow procedures above for Kohler IlluminationNeed 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 placeWollaston Prism (below filter wheel)—set for objective: C=20x D=63x/100x E=40xCondenser --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. Thefull range of the screw is such that the image is darkest in the center (extinction) andbrighter 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 ScopeHalogen lamp onTransmitted Light Detector (TLD) offScanning Halogen lamp offTLD onTo increase contrast, lower offset to -10 to -20 and increase gain


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PhotoshopThe most valuable feature in Photoshop is:Image--Adjustments--Levels

choose RGB or individual coloruse the histogram to adjust white level, black level, and gamma

Save with a new name. This does a contrast stretch and is permanent.

Coloring ImagesYour Leica images should open in colorTo make a Red/Black or Green/Black image from greyscale.

Open imageImage--Mode—choose RGB ColorImage--Adjust--LevelsChoose colors not wantedDrag left triangle under histogram all the way to the right to eliminate this colorRepeat 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 ColorImage--Adjust--Hue/SaturationCheck ColorizeSaturation at 100% is probably best

Adjust Hue to ~120 for green, red is the defaultLower Lightness to color white areas and darken backgroundSave image

Specifications for Leica Confocal SP23 Laser system

4-line argon laser, lines at 458nm, 473nm, 488nm, 514nm.

458 for CFP, 514 for YFP, 488 for all green dyesGreen Helium Neon laser at 543nm for all orange / red dyes eg. rhodamine, Cy3Red Helium Neon laser at 633nm for far red dyes eg. Cy5, TOPRO, DRAQ5

4 fluorescent detectors1 transmitted light detector5 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 250umcan 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 100um4 58 63x oil HCX PL APO 63x / 1.32 oil WD 70um5 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 12mmIn 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 MicroscopeUV 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


Emission BP 527/30 512 - 542 nm

Red Excitation BP 510-560 510-560 nm


Emission LP 590 590 + nm

CFP Excitation 436 / 20 426 - 456 nm


Emission 480 /40 460 - 500 nm

Green-Red Excitation BP 450-490 450-490 nm

(in drawer) Dichroic 510

Emission LP515 515 + nm

Documents

LeicaSP2_ UserGuide