>The probability information is the point of my comment. I use this
>"probability"
>in sorting, where one can determine the most probable regions for sorting
>G1, S,
>and G2M. In this context, cells defined as falling within these regions
>by the DNA
>fitting algorithm are certainly specific to that compartment . . . unless you
>suggest there is no real relationship between DNA content and DNA-fluorochrome
>signal intensity . . .
By which model are "cells defined as falling within regions by the DNA
fitting algorithm"?
I wasn't knocking modelling (nor the model-makers), but was pointing out
that it doesn't allow you to identify which cell, in an area of overlap,
belongs to which compartment.
>It's not the fault of the modeling, yet the nature of the measurement that
>forces
>these apparent overlaps. In a perfect world, perfect resolution may
>result in the
>ability to define a single channel for G1, a specific channel for
>transition to
>S-phase, channels that designate the interface of G2 and Mitosis . . .
>but, our
>flow cytometers, labeling techniques, operators, cells, dyes, all conspire to
>dither these measurements so that what we actually measure requires
>accommodation
>through use of this broadened S-phase polynomial function. Logic predicts
>that
>there will be S-phase cells having DNA content close to G1, and at the
>other end,
>close to G2 and Mitosis (keeping in mind here that we're actually measuring PI
>content, of course). The "overlap" allows for this.
The reason for deconvolution is to create a perfect world where those
assignments can be made. The conspiring spread functions are modelled and
how well the model's output matches the real histogram is a measure of
perfection. But the nature of modelling still doesn't allow you to
identify a cell.
Jill wanted exact boundaries that she could gate on, I don't see how
modelling could give them. It identify a point where there is a
satisfactory tradeoff between contamination from an unwanted compartment
and loss of the required one, but would it improve the analysis of her
horseshoes where she already has an indicator of S-phase?
You mention sorting, and I agree that deconvolution would be able to give
you an estimate of contaminants, but even if you deconvolved in real-time
it wouldn't improve the quality of your sort decisions (in that it wouldn't
be able to tell you if a cell that was in the G0/G1 mean channel was
actually a G0/G1 cell or an S one).
Ray
Ray Hicks
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