Hi everyone,

I would like to quantify both sizes and intensities of fluorescently
labelled bacteria (combined DNA/RNA staining) by confocal microscopy,
which is difficult on a conventional epifluorescence microscope
because of lamp aging and uneven illumination. So far I have no
experience at all in confocal microscopy but some background in 2d
image analysis.

My institution has just acquired a Zeiss LSM 510 equipped with a
Coherent Innova Enterprise UV laser, which I thought of using for
DNA staining with DAPI or Hoechst 33342. I immediately ran into
a very annoying problem, which I suspect might be a consequence of
being a total CLSM newbie:

The images I get with either the 63x Apochromat or the 100x
Apochromat are totally "unevenly illuminated", very bright in the
central part and dim towards the margins. This is most pronounced
when I select the largest scanning fields. It actually does not seem
to depend on the lens, a colleague had similar troubles
with the 10x Neofluar. I don't even understand theoretically what the
reason could be. The pinhole adjustment? I have been optimizing
pinholes for hours according to the instructions of the Zeiss
technicians, using this little mirror and linescan mode...The
collimator position?

At another excitation wavelength (543nm for RNA staining with
labeled oligonucleotide probes) I seem to not encounter this problem,
but I have not examined it really thoroughly with fluorescent beads
yet.

Can anybody give me some advice?

Jakob Pernthaler
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Jakob Pernthaler
Max Planck Institute for Marine Microbiology
Celsiusstrasse 1
D-28359 Bremen
Germany

email: [log in to unmask]
Phone: +49-(0)421/2028-940
Fax:   +49-(0)421/2028-580
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