Dear Adaleta,

your question is too vague formulated. Please give us more infos.

1/ What a kind of fluorochrome and cells (fixed or not) (e.g. in vivo or in vitro staining) have you used? For fixed cells /fixation method.

What a fluorescence method are you using (e.g. antibodies, bacteria.....)

2/ How have you mounted your cells on smears? What a kind of oil are you using?

3/ for a maximum fluorescence intensity, please give us infos about your filter cube and excitation filter used for your research, dichromatic mirror and suppression filter.

4/ Give us infos about your short and long pass filters.

5/ Are you using (Leica) Hg 100W and Xenon 75 W ?

6/ Give us infos about your high performance objectives e.g. Leica'planapochromat or PL Fluotar ...

Thank you in advance.

JRG

Dear Adaleta, not seeing any fluorescence may indicate any of the following possible reasons:

(1) Your target protein is too low = Try to increase exposure time during fluorochrome excitation, or use the Auto-exposure setting. If the target it too low, the time may be longer than 1 min.

(2) Antibodies are not compatible (i.e. primary and secondary did not bind to each other) = Check your antibody specifications.

(3) Mercury lamp has depleted. The fluorescence microscope in my faculty is Nikon, and only allows 200 hours of fluorescence. = Check the hours on the lamp.

As noted above, there are several things that could be causing problems for you. Here are some simple things to check as part of your trouble-shooting.

1) Fluorescence lamp. It seems unlikely to me that the mercury bulb would be the problem if it has not been used. I would try starting the bulb and letting it warm up for a few minutes. Most fluorescence microscope lamp housings for mercury lamps that I am familiar with have slits for ventilation to help keep the lamp cool. If the lamp housing has ventilation slits for cooling, try holding a sheet of paper next to the slits. If the lamp is working, you should see intense white light hitting the paper. Do not look directly into the lamp housing! Alternatively, hold your hand near the lamp housing. If the lamp is working you should feel the heat produced by the lamp. (Don’t touch the housing directly).

If you do not see any light or feel it getting warm, the lamp isn’t working.

2) Assuming the Fluorescence lamp works, is the shutter working properly? The manual should indicate what type of shutter(s) the microscope has. If the fluorescence shutter doesn’t open properly, then no fluorescence-Stimulating light will hit the specimen. Some microscopes have manual shutters that the user has to open and close, some have automated shutters controlled electrinically or via software. Check to be sure the shutters are working properly.

3) Is the image being sent to the port that the camera is mounted on. This can be tested using the white light illumination. Turn it on, and test whether the camera can detect the image (you may need to adjust the intensity). Many microscopes have a selector that sends the image to eye pieces or to the camera. Check to be sure it is in the proper position. Some microscopes have selectors that split the image between the eyepieces and the camera, which reduces the fluorescence image intensity reaching the camera and can compromise the ability of the camera to detect the image.

4) How sensitive is the camera? Fluorescence images are not especially bright and a camera that is not sufficiently sensitive may not detect the image. Check the camera settings to be sure that it is set properly for fluorescence microscopy. As a way to check this, you could place a specimen known to be well labeled on the microscope and see whether the camera detects the image. Many cameras permit adjustment of gain and binning to improve sensitivity.

5) Do your Fluorescence filters match your fluorescent tags? If the excitation and emission characteristics of your fluorescent tag(s) do not match the excitation and emission characteristics of the filters, you won‘t get a good fluorescent signal. Also, some microscopes are configured so that the excitation and emission filters are not housed within the same cube, but are actually separate from one another. In this case, if the two filters you need don’t get paired up properly, you won’t get a fluorescent image.

6). Do you know that your labeling worked properly? Can you put your labeled specimen on a different microscope and see your labeling?

7). Are all the proper software drivers for your hardware installed in your image capture software? If this isn’t properly configured you won’t be able to grab the images.

Alot of this detailed information should be in the microscope, camera, and software manuals. Filter information may be specified on the purchase documents. A useful resource for matching fluorescent tags and your filters is the Molecular Probes Spectra Viewer (Just type those words into Google and the link will come up. There are several other comparable websites out there, too).

Good luck.

Dave Sherry

Thank You very much for detailed help. We have checked the specimens on other microscope and they are OK. I think, problem is not with camera, because the same bad picture I also see on oculars, and camera can show only these bad segnals on 100x magnification. The shutter is manual and seems OK. I have to contact servisers, there is no other possible way to resolve problem . Thank you

Adaleta