Jim - While your light scatter sizing statement is true, cells of all
refractive indices, fixed or unfixed can be measured very accurately by
pulse-width time-of-flight techniques using polystyrene or other size
standards. If performed properly with pulse height normalization, the
measurement is refractive index independent. We have also sized objects as
small as 0.3 microns diameter with a 15 micron laser beam using a high
resolution biased amplifier to subtract the laser beam width out of the
time-of-flight measurement. In addition, nuclear sizes can be measured by
fluorescence time-of-flight. And you can even measure N/C ratios of cells
using two such measurements. I and others have been doing this routinely for
more than 20 years. For example:
Rudolph, N.S., Ohlsson-Wilhelm, B.M., Leary, J.F., Rowley, P.T.:
"Single-Cell Analysis of the Relationship Between Transferrin Receptor,
Proliferation and Cell Cycle Phase in K562 Cells" Cytometry 6: 151-158
(1985).
There seems to be a lot of confusion on light scatter and time-of-flight
sizing. Would there be any interest in my writing a short technical
review/overview for Cytometry? I've been doing light scatter and
time-of-flight sizing since 1974 and have tried teaching these concepts. A
quick and simple overview of light scatter and time-of-flight sizing is also
on my Molecular Cytometry Course website (lecture 6) at
http://stem.utmb.edu/98pth6311 . Let me know if you find it helpful and
clear.
Best regards, Jim Leary
James F. Leary, Ph.D.
Chief, Molecular Cytometry Unit
4.216 Mary Moody Northen Building - Route 0435
Professor of Internal Medicine, Pathology,
Microbiology & Immunology, Biophysics,
Human Biological Chemistry & Genetics.
and Biomedical Engineering
University of Texas Medical Branch
Galveston, Texas 77555-0435
Tel: 409-747-0547; Fax: 409-772-4227
Email: jleary@utmb.edu
-----Original Message-----
From: James Weaver 301-594-5879 FAX 301-594-3037
[mailto:WEAVER@CDER.FDA.GOV]
Sent: Thursday, October 12, 2000 12:55 PM
To: Cytometry Mailing List
Subject: Re: Cell Volume Revisited
Sensitivity: Confidential
Despite what is written in various places, flow cytometry can only be used
for size measurement on homogenous particles such as plastic beads. It does
not work for particles with complex optics such as cells. With beads,
increasing size leads to increased light scatter. With cells the situation
is quite different, a treatment that resulted in a 30% change in cell volume
caused a light scatter decrease in a lymphoid cell line and no change in
light scatter from NIH3T3 cells.
Coulter volume measurements are regarded as the gold standard of cell volume
measurements. I have done cell volume measurements on nucleated cells in the
past with no problems. If you are not seeing changes in cell volume, than
either the treatment is not causing a change or the instrument needs repair.
* Jim Weaver
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* Division of Applied Pharmacology Research *
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