quantitative confocal microscopy

Posted by POUVREAU SANDRINE on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185.html

Hello.
I had this discussion with several colleagues (biologist like me), and did some
research on my own, but I figurate the best would be to submit the question
to this list. Here’s the point: we are doing quantitative measurement with
confocal microscopy (calcium measurement) using a Zeiss LSM exciter. There
are 3 parameters of the PMT that can be configured: detector gain, amplifier
offset, amplifier gain. The only parameter I adjust to improve the signal is the
detector gain. I keep the amplifier gain at 1 as I read in several papers that
increasing it will not improve the signal over noise ratio (they also say that it
is bad for several reasons that I can not summarize here). Is that correct? I
put the offset usually at zero. I saw that a change in offset can affect the
calcium signal. In any case, I keep the same offset in a series of records that
I whish to compare. Any comment on this? My guess is that the same should
be done with the amplifier gain.
Ah, a last point: the offset should be tuned so no pixel in the background has
the value of zero right?
Does anybody have any clarification for these questions? Thanks for your help.

Regards
Sandrine