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why don't we get a linear line of events on a pulse width/forward scatter (linear scale) bivariate, but
rather a hyperbolic curve ?
Take for example mouse bone marrow cells. This curve tells us that 'junk' produces very little (and a
small range of) light scatter but varies a lot in pulse width value. The majority of the cells, however,
(usually larger than junk) show a wide variety in forward scatter (lymphocytes, blasts, granulocytes)
and a very small range in pulse width value. In other words, cells with different forward light scatter
characteristics may have the same pulse width value. This may be explained by the use of a laser beam
diameter that is wider than the diameter of the largest cell.
You'll also find events above the curve: particles with the same forward light scatter characteristics
but with much (approx.2X) higher pulse width values than the events within the curve. Reasonable to
assume that these events are the doublets passing through the laser one after the other (but 'stuck' to
each other).
In a pulse width/forward light scatter bivariate I expect to see a difference between the detection of
two small cells that are stuck together and a large cell that is more than twice the size of a small cell:
The doublet will show up at the same forward light scatter value as the small cells but the pulse width
value will be higher. The large single cell will also have a high pulse width value but will have a high
forward light scatter value. Not ?
And now I've found really big single cells above the curve ...
Any suggestions ?
Ruud
P.S.
What is the difference between 'pulse width' and 'time of flight' ?
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web