Re: quantitative confocal microscopy

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5349227.html

Hi Sandrine, your question is sure to draw answers as it has been asked several times before. Quantitative fluorescence microscopy is not trivial, but can be somewhat reliable if you try to carefully control as many experimental parameters as possible. Other people on this list server can give you more details, but as a quick reference, try reading Jim Pawley's "39 steps":
http://labs.pbrc.edu/cellbiology/documents/39steps.pdf

As for detector gain and offset settings, Alan Hibbs' book has a nice section that outlines a good method for determining the best settings:
http://www.amazon.com/Confocal-Microscopy-Biologists-Alan-Hibbs/dp/0306484684

All the best,

John Oreopoulos



On 2010-07-28, at 9:05 AM, 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