Re: quantitative confocal microscopy--photon-counting

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5350421.html

Hi Martin

The primary problem is speed (IMHO). The counters start to show pile up
at ~1MHz. To get (say) 10 photons per pixel this implies a pixel scan
rate <100 KHz which for a 1MHz image means >10s /frame. Even at this low
rate the image is still very poor (10 phot in brightest pixel).
Basically the low speed, loss of linearity at high photon count rates
and extra complexity doesn't make it worthwhile. On the other hand,
analogue is fast and linear and is only slightly lower signal to noise.

Cheers Mark

> This is a bit of a digression for this thread, but I wonder if anyone
> can tell me why photon-counting isn't available on more confocals.  It
> seems as if it would be the method of choice for quantitative
> experiments--assuming that you're avoiding pile-ups, the counts for a
> given wavelength will be directly proportion to the fluorescence.  But
> perhaps I'm missing something.
>
> Thanks--
>
> Martin