Kees Jalink wrote:
> We observed that the light intensity delivered to the lens
> in our fibre-coupled, AOTF-equipped instruments fluctuated enormously
> with time... We have brought this to the attention of the manufacturer,
> Leica, and they have been very cooperative.

We have also seen fluctuations of similar magnitude in one out of 3
Zeiss 510 units. What caught our attention was fluctuations in intensity
during collection of a single image; i.e. faster fluctuations than you
mention. We will go back and test for longer term fluctuations now.

Kees, can you give us some idea of why the fiber coupling would produce
cyclical variations in transmittance, and explain how the alignment
procedures were changed to minimize the problem?


>     The big question of course is: how to work around it.

Two suggestions....

In the Zeiss LSM510 systems, there is a monitor diode option which lets
you collect an "image" of laser intensity simultaneously with your image
scan. The diode will collect info from all the lasers on during the
scan, so it is not an ideal system to collect info about the variation
in individual laser intensity under such conditions. However, it can be
quite good for appraising variation when only a single laser line is
used. For multi-line work it would work best with the multi-tracking
feature where you use the AOTF to switch between laser lines at the end
of each scan line.

The second suggestion is more "equipment independent", and is already
mentioned by Kees. Use emission ratioing whenever possible. The second
fluorescence measured must be in response to the same exciting line. You
may have to introduce a second dye into your experimental images (beads,
dye in solution, etc) as a reference. Please be aware you must control
for differential dye loss and/or photobleaching between two probes, but
it may still be a marked improvement in image reliability.


Chip Montrose

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