Dear list readers,

I have just started playing around with photon counting on the
BioRad MRC-600 confocal and I have a couple of questing concerning
its use with living cells. Specifically, I am interested in its
application to quantitative confocal ratio imaging to determine
intracellular ion concentrations in living material.

If you can obtain a reasonable fluorescence signal with gain settings
of 6.5 to 8.0, slow scan speed and low signal enhance in analogue
mode and are, therefore, able to use Kalman averaging (over 3-5)
frames is there any real advantage in using photon counting mode
accumulating over a similar number of frames?

Should ensuring sufficient fluorescence signal to allow image
collection in analogue mode (with Kalman filtering) take priority
over using photon counting when using live (growing/active)
material i.e. should photon counting only be used as a last resort if
insufficient signal can be achieved?

Does a pixel intensity of say, 8, for an image taken in photon
counting mode correspond to 8 photons detected? If that image is
contrast streched (to a suitable degree) is it roughly equivalent to
the image of the same fluorescent sample taken with a single direct
scan in analogue mode once a "dark signal" image has been
subtracted?

Thanks, Richard