Ratiometric analysis calculations?

From: Darren Hickerson (dhickerson@brody.med.ecu.edu)
Date: Fri Sep 26 1997 - 13:03:04 EST


I have successfully been doing ratiometric Ca++ measurements by flow
for about two years for our own purposes.  However, data analysis has
always been a problem, since I have not been able to find a clear
description of what people are really dividing to get their ratio
numbers, and if I do the math myself (divide means from X & Y), I get
very different numbers compared to software which  automatically
calculates a ratio.

When a paper says "linear", I can't tell if they mean a true linear
scale, or what BD calls a linear scale (in its "preferences" box on
Lysys) which is actually a "log" scale.  On a true linear scale, the
fluorescent events are too spread out.  However, I have often seen
people convert the "log" (linear) scale to a "channel" scale, which
superimposes a false linear axis over the true "log" values during
data analysis.  How can that be a true representation of the
fluorescence values, especially when calculating ratios?

Please respond to the list, or preferably, directly to me.  Any help
with your experiences will be very, very much appreciated.  Please
include references to literature which deals with this, if available.
 Also, if this or a similar topic has been discussed on this list in
the past, please give approximate dates & message titles & I'll look
it up on the net.  Many thanks.

Darren Hickerson, ECU School of Medicine



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