FAQs - Frequently Asked Questions: The Answers

1. Can see specimen through oculars but no image is seen on the computer display panel while scanning.
   Check for the presence of scattered laser light from specimen on the stage.   If none ensure that scan head shutter rod is pulled out.  Give it a firm tug.   Check that laser power is not at zero or too low.  Check sensitivity (PMT voltage).  Check focus (see 2).  Open pinhole to at least 1.0
2. Sudden loss of focus in the conventional microscope or when initially scanning.
   The stage position of the microscope is really quite stable. Sudden loss of focus is almost always due to one of two things: 1) an arm brushing the focus knob when reaching over to the sliders or fluorescent filters and 2) establishing initial focus on the sample while the galvo stage is in the parked position. The galvo stage is in this lowered position when the TCS program is not loaded or the scan electronics are off. Upon starting of the TCS program, the stage rises by about 85 m m (one-half of its total throw of 170 m m) to its operational and holding position. Focus is lost, of course. Starting from the original focus position, the new focus position can usually be found quite simply by raising the lenses 85 m m, as indicated by the LCD. See also Smooth Starts.
3. Focusing is at higher level than previous positions.
   This can happen when slide is being hinged up by the objective pushing on the slide.  One slide of the slide remains on the stage while the spring clips allow the other edge to lift up off the stage.  A wedge profile of oil forms between the objective and the slide creating a gap equal to the working distance of the objective.  Typically the objective is about 400 mm higher than the upper limit, if the limit has been set correctly.  This situation is a disaster for z-series sections, and can be a bigger disaster if another objective is swung into place, causing the nose of the objective slide to scrape into the edge of the slide.   Solution: move objective close to slide and find focal plane while increasing distance between the slide and the objective.  Typically z-position on microscope should not be more than 200 mm from the correctly set upper limit.
4. Knurled Knob - Iris control on 20X, 40X and 100X oil immersion lenses should be fully open.
   The iris control diaphragm on the 40X and 100X oil immersion lenses need to be fully open (rotated clockwise when viewing objective from above).  Having this diaphragm closed down reduces the numerical aperture of the objective and reduces image resolution and increases depth of focus.  The only reason to deliberately reduce the numerical aperture is to acquire z-series sections through thick tissue.
5. False co-localization with multi-channel labeling
   Injudicious use of laser power and detector sensitivity. This is a complicated issue, but excessive laser power can cause out of channel band excitation of fluorophore.   Direct fluorescence energy coupling is another mechanism of fluorescence channel cross talk. See m.c. for more information.
6. With z-series use "save as".
   Save selected strategy with z-series images is un-intuitive and complicated.   Unless the user has tried, tested and thoroughly analysed data gathered using the "save selected" method, it is far safer to use "save as" and sort out image channels and planes after the image has been saved to a file.
7. Missing channels when viewing saved images.
   This is a problem with multi-channel fluorescent images or Z-series images. Most image viewing programs cannot handle Mutli-paged image files. However ThumbsPlus (for PC’s) or WangImage (for PC’s) can view such files. Multi-paged image files may be split into individual images for each channel using ThumbsPlus or Tiffsplit.exe. (see M.C. if you would like these programs).
8. Mouse speed
   It looks like one can click pretty fast with the mouse without causing problems, but until we are sure, it is probably wiser to pace oneself. We know for sure of one instance in which the computer hung up for this reason.
9. Scan speed/AOTF interaction
   Adjusting scan speed then moving directly to the AOTF to adjust laser power will cause the TCS program to lock up. Nothing to do but exit the program (CTRL-SHIFT-ESC, followed by two "END TASK" commands from the dialog boxes) and start over. All settings except those controlled by "METHOD" (filter choice) will have to be re-entered.
10. Sudden loss of image
    Zoom suddenly leaps to 32. The cause of this is not identified for sure, but seems to be associated with changing scan speeds. The sign: you set up a beautiful image, then turn SCAN SERIES on to do the final scan and the image has disappeared. In fact, it is still there, it is just now so big you can't see anything. This can be a bummer because even one scan at zoom 32 may photo-bleach your sample to smithereens (depending on characteristics of the sample; some survive). Best protection is to keep an eye on zoom on the right hand side of the screen. New view: Looks like you turn off SERIES SCAN, then change SCAN SPEED, the zoom jumps. We're still working on it.  Accidental handling of the focus button on the panel box. For very thin samples, a small change in focus can completely eliminate the image. Can happen if one grabs focus when one wants to grab zoom, for example.
11. Solid colors on screens
    At the start of a scan, the screens for the three channels show solid green, solid red, and solid blue. This may exist for only one scan, then correct itself. More usually, it persists, and the only solution is to exit the program and start over. Sometimes associated with a leap of zoom. If you are using the GLOW-OVER/UNDER FEATURE, the screens appear uneven yellow-white.
12. Inactive panelbox positions
    Certain panel box positions say they are programmed for something, but are inactive when you start up or change METHOD selection. The solution is to hit the INIT PANEL button on the left end of the drawing of the panelbox. The drawing is called up starting in the left hand corner of the screen: SETTINGS-PREFERENCES-PANELBOX. Wish they were all this easy.
13. Can not focus on specimen
    1. Specimen is on top of slide. Needs to be on bottom since this is an inverted microscope. Or the galvanometer stage is at the limits of its travel.  If the z-position display on the right hand side of the right monitor is close to -/+ 82 um then use the z-position know to set the z-position display to about 0 and then refocus the sample at the microscope while viewing with the eye-pieces.
    2. Focus successfully with 10X objective and image may appear fuzzy but can not focus with higher power objectives or focal plain is much higher.  There is a pool of oil on the concave lens of the 10x objective.  Gently clean it with a layer of lens tissue pushed into the lens with the cotton end of a q-tip.
14. Unexpectedly high background level
    1. Mercury lamp is spilling light into microscope.  Close mercury lamp port rod. and/or 2. Transmitted light lamp is on.  Make sure it is off by turning the lamp power wheel and checking on the LCD display on the microscope that lamp voltage is zero.
15. Can not save after scanning is completed
    If scanning has completed and the 'File->save ...etc' pull down menu is grayed out check the series scan button.  If it is still depressed (grayed) press it carefully once (and only once).  'File->save ...etc' menus should be active.   This will happen if another program is clicked on while acquiring the image(s).   Thanks to Bill Eckert for pointing this problem out.
16. Saving very slow after acquiring a large z-series stack/time-series with multiple channels and 1024 by 1024 pixel format.
    If this happens the file size is close to or exceeds the amount of free memory available on the \\Leica_tcs computer.  Either be very patient with the save, which may take many minutes, or cancel the save and acquire with fewer slices or time points or decrease the number of channels or reduce the image pixel size.
17. While x-z scanning objects seem elongated in the vertical direction.
    This is frequently due to a restriction of the vertical movement of the stage by, for example, 1. the condenser being too close to the slide or 2. the galvanometer stage being at the limit of its travel.  The fix for 1. is to remove the restriction by focusing the condenser away from the slide and for 2. is by moving the galvanometer stage closer to its middle position (use z-pos knob and open and look at z-pos display on gray panel to the right of the display box(es) and refocusing with the fine focus control on the microscope).
18. Microscope does not change objectives or does not focus
    The microscope's embedded computer has crashed.  Just turn the power on the scope off for about 10 seconds and turn back on.  Note that the upper limit will be remembered and does not need to be re-found.
19. A slight offset image is seen when zoomed 2 or more fold with the 40x or 100x objectives. 
    The Walliston prism is probably in place.  This prism will split the light path into two polarized beams, slightly offset from each other.  This is used to create a Normaski image, but when not setup for Nomarski a double image can be seems.  Make sure that the Walliston prism dial is on "H".  If a letter is flashing on the LCD display of the microscope this is a sure sign that a Walliston prism is inappropriately in place.

Last updated 07/31/03

					© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC.  All rights reserved.
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