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