>1. Is it possible to accurately count the cellular events below the
>threshold one is using during acquisition?
>
The threshold is that level above which the trigger signal has to rise for
the instrument to define the beginning of an event; the electronics are
therefore essentially blind to what goes on below the threshold. If your
instrument allows you to set an acquisition gate, you can get the answer
you're looking for by lowering the threshold to include all of the events
you want to count, and restricting your analysis to events within the gate.
"Cellular events" is a tricky phrase; strictly speaking, unless you sorted
everything, you couldn't be sure which of the near-threshold events were
cells and which were noise, debris, etc.
It is now generally appreciated that good gating is essential for good flow
cytometry; threshold definition is probably at least as important. In this
context, it seems to me that a lot of manufacturers (myself included) might
improve their hardware design to permit multiparameter thresholding. The
Coulter Elite has this feature; some of my Cytomutts, the B-D FACSCalibur,
and the Bio-Rad have two-parameter thresholding, while the rest of the Mutts
and B-D's instruments and Cytomation's MoFlo have single-parameter
thresholding. As far as I know, nobody makes an instrument with two
threshold levels for the same parameter, which could be used to count
subthreshold events; this feature was incorporated in the Block instruments
20 years ago, primarily because they were originally designed as blood cell
counters.
-Howard