Dear Calman,
A question we all come to at some stage; I'm glad that you raised it.
A question that I don't remember seeing discussed:
Probably not raised because it is a contentious issue.
How fast (events per second) can a FACScan or FACSCalibur run?
When I have tried it on a FACScan, it peaked at 10,000 events/s. This
implies that its dead time is around 100 microseconds.
By the time you get to that rate though, the Scan will be missing more than
half the total events, and you will be getting many measurement errors due
to undetected coincidences. These occur when two cells overlap in the flow
path as they are illuminated, and the machine registers a signal which is a
reduced sum of the signals each would separately generate.
To minimise these errors, you need to run at a rate where the incidence of
these is lower, and/or you need to set gates in s/w that will accept only
singlet events. In the MoFlo, the pulse width enables you to do this
exclusion/acceptance. You would need to talk to BD about that in their
machines. It is harder to do in conventional signal space, since one effect
of the undetected coincidences is to smear the normal lymphocyte scatter
gate areas in both directions; you are placed on the edge of uncertainty!!
We are looking at rare events (0.01-0.03% of total cells) and want to
speed up the acquisition.
One to three events per ten thousand is probably beyond the Scan when you
run it at a rate which will yield a valid statistical sample, ie ca 10,000
events. This will take one-three hours; the time relationship is linear, so
1000 will take 6-18 minutes, and the error then 3%, not including the
coincidence errors.
Your solution is to reduce the machine's dead-time and run in peak mode
(refer to BD for a retro-fit, of whatever their performance is claimed to
be). There is a better alternative: rent or buy a MoFlo, either desktop or
MLS system; it comes as an analyser or a sorter. The dead-times are
respectively 15 and 5 microseconds, so you can run at 20,000 and 60,000
cells/s without much trouble from the sources I described.
Take it up with me if you wish.
Cheers, Bob
_______________________
Calman Prussin
Laboratory of Allergic Diseases
NIAID/ National Institutes of Health