Re: quantitative confocal microscopy

Posted by Julian Smith III on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5349210.html

I'm running an Olympus Fluoview, but I think the answers are the same:
Yes--detector gain to adjust max signal--no saturated pixels where you
plan to measure.
Amp gain at 1
Offset at a few to no zero pixels in background (again, not where you
plan to measure).
Don't change the settings across specimens, if you plan to compare them
to each other.
Julian

Sandrine Pouvreau wrote:

> 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
>
>  

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

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