From: Bob Leif
To: cyto-inbox
The latest in the series of papers from Professor Deutsch's group is D.
Fixler, "Monitoring the fluorescence intensity and anisotropy decay of
individual cells within a population" in Optical Diagnostics of Living Cells
IV, Eds. D. L. Farkas and R. C. Leif, Proc. SPIE Vol. 4260, pp 210-218
(2001). This book and the description of SPIE BiOS including next year's,
Advanced Techniques in Analytical Cytology session are available at
www.spie.org
-----Original Message-----
From: Howard Shapiro [mailto:hms@shapirolab.com]
Sent: Wednesday, June 20, 2001 8:16 PM
To: cyto-inbox
Subject: Re: Fluorescence polarization
Maris Handley wrote-
>Someone recently brought me an article entitled: Fluorescence polarization
>assay by flow
>cytometry. The authors are J.M. Rolland, K. Dimitropoulos, G.R. Hocking,
>and R.C. Naim.
>The article is from 1985. My first question is whether anyone is
>routinely looking at
>fluorescence polarization in 2001? If not, is this because there is a
>newer method,
>or better technology for looking at subtle cellular changes?
>I would be interested in talking to anyone who has experience with these
>kinds of
>measurements.
See pages 327-9 of the 3rd Edition of Practical Flow Cytometry for
additional background information.
The fluorescence polarization assay in question was based on observations
made by Boris and Lea Cercek, working in Manchester, England in the
mid-1970's, that the polarization of fluorescein fluorescence in
lymphocytes (produced by intracellular enzymatic hydrolysis of fluorescein
diacetate, also known as FDA) changed within a relatively short time after
mitogenic stimulation by phytohemagglutinin (PHA). The measurements,
described as showing changes in the "structuredness of cytoplasmic matrix",
or SCM, were originally done on lymphocyte suspensions in a
spectrofluorometer. The Cerceks also reported that lymphocytes from
patients with cancer exhibited a diminished polarization response to PHA,
but, unlike patients without cancer, showed a polarization response to
proteins derived from cancer cells, and proposed this as a test for
cancer. While Rolland et al and others attempted to implement the assay
using flow cytometry, with varying degrees of success, the most precise
single cell fluorescence polarization measurement system is the Cellscan,
developed by Prof. Mordechai (Motti) Deutsch and his colleagues at Bar Ilan
University in Israel. They have published numerous papers in recent years
(see bibliography below), and now believe that the polarization changes
reflect changes in the cytoskeleton and/or intracellular hydration state.
Motti Deutsch has criticized the notion of measuring polarization in flow,
because the constant observation time means fewer photons are counted for
cells with weaker fluorescence, making measurements less
precise. Computation of the polarization value requires taking a ratio,
and the change in this ratio is relatively small, meaning that any source
of imprecision decreases the likelihood of obtaining significant
results. The Cellscan illuminates the cell with very low intensity light
(minimizing bleaching) for as long as is necessary to count 10,000 photons
in each of two polarizations for each of two fluorescence wavelengths,
providing a very precise polarization measurement. I don't know of recent
data bearing on whether commercial flow cytometers are up to the job.
An overall problem with the polarization assay is that the Cellscan
apparatus, while precise for polarization measurements, has not been
equipped with sufficient multiparameter measurement capability to allow
determination of other cellular characteristics, such as phenotype,
activation antigen expression, RNA or DNA content, cytoplasmic calcium
concentration, or membrane potential, which also reflect lymphocyte
activation. The differences in lymphocyte activation patterns between
patients with and without cancer that are reported to be detected by the
polarization assay should presumably be detectable using more widely
accepted indicators of lymphocyte activation, but the necessary comparison
experiments remain to be done. I personally think this would be
worthwhile, because some of the results I have seen from both the Cellscan
and flow cytometric studies suggest that pursuing this line of inquiry
could yield some interesting new information relevant to tumor immunology.
Older references cited in the book; the newer papers are:
Deutsch M, Weinreb A: An apparatus for high-precision repetitive sequential
optical measurement of living cells. Cytometry 1994; 16:214-26
Ron IG, Deutsch M, Tirosh R, Weinreb A, Eisenthal A, Chaitchik S:
Fluorescence polarisation changes in lymphocyte cytoplasm as a diagnostic
test for breast carcinoma. Eur J Cancer 1995; 31A:917-20
Eisenthal A, Marder O, Dotan D, Baron S, Lifschitz-Mercer B, Chaitchik S,
Tirosh R, Weinreb A, Deutsch M Decrease of intracellular fluorescein
fluorescence polarization (IFFP) in human peripheral blood lymphocytes
undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A
(ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Biol Cell 1996;
86:145-50
Marder O, Shoval S, Eisenthal A, Fireman E, Skornick Y, Lifschitz-Mercer B,
Tirosh R, Weinreb A, Deutsch M: Effect of interleukin-1 alpha,
interleukin-1 beta and tumor necrosis factor-alpha on the intracellular
fluorescein fluorescence polarization of human lung fibroblasts.
Pathobiology 1996; 64:123-30
Deutsch M, Ron I, Weinreb A, Tirosh R, Chaitchik S: Lymphocyte fluorescence
polarization measurements with the cellscan system: application to the SCM
cancer test. Cytometry 1996; 23:159-65
Eisenthal A, Marder O, Lifschitz-Mercer B, Skornick Y, Tirosh R, Weinreb A,
Deutsch M Inhibition of mitogen-induced changes in intracellular
fluorescein fluorescence polarization of human peripheral blood lymphocytes
by colchicine, vinblastine and cytochalasin B. Cell Struct Funct 1996;
21:159-66
Zurgil N, Deutsch M, Tirosh R, Brodie C: Indication that intracellular
fluorescence polarization of T lymphocytes is cell cycle dependent. Cell
Struct Funct 1996; 21:271-6
Rahmani H, Deutsch M, Ron I, Gerbat S, Tirosh R, Weinreb A, Chaitchik S,
Lalchuk S: Adaptation of the cellscan technique for the SCM test in breast
cancer. Eur J Cancer 1996;32A:1758-65; Comment in: Eur J Cancer. 1997;
33:1333-5
Merimsky O, Kaplan B, Deutsch M, Tirosh R, Weinreb A, Chaitchik S:
Detection of melanoma by monitoring the intracellular fluorescein
fluorescence polarization changes in lymphocytes. Cancer Detect Prev 1997;
21:167-77
Eisenthal A, Marder O, Lifschitz-Mercer B, Skornick Y, Fixler D, Avtalyon
R, Tirosh R, Deutsch M: Influenza A virus affects the response of human
peripheral blood mononuclear cells to phytohaemagglutinin A by altering the
cytoskeleton. Pathobiology 1997;65:69-74
Sunray M, Deutsch M, Kaufman M, Tirosh R, Weinreb A, Rachmani H: Cell
activation influences cell staining kinetics Spectrochim Acta A Mol Biomol
Spectrosc 1997;53A:1645-53
Cohen-Kashi M, Deutsch M, Tirosh R, Rachmani H, Weinreb A:
Carboxyfluorescein as a fluorescent probe for cytoplasmic effects of
lymphocyte stimulation.Spectrochim Acta A Mol Biomol Spectrosc 1997;
53A:1655-61
Eisenthal A, Marder O, Lifschitz-Mercer B, Skornick Y, Tirosh R, Irlin Y,
Avtalion R, Deutsch M: Infection of K562 cells with influenza A virus
increases their susceptibility to natural killer lysis. Pathobiology 1997;
65:331-40
Fixler D, Tirosh R, Eisenthal A, Marder O, Irlin Y, Lalchuk S, Deutsch M:
Monitoring of effector and target cell stimulation during conjugation by
fluorescence polarization. Biol Cell 1997; 89:443-52
Gelman-Zhornitsky E, Deutsch M, Tirosh R, Yishay Y, Weinreb A, Shapiro HM:
2, 7'- bis-(carboxyethyl)-5-(6)-carboxyfluorescein (BCECF) as a probe for
intracellular fluorescence polarization measurements. J Biomed
Optics. 1997; 2:186-194
Avtalion N, Avtalion R, Tirosh R, Sheinberg A, Weinreb A, Avinoach I,
Deutsch M: Preparation of a diagnostic antigen of human melanoma based on
lymphocyte activation as measured by intracellular fluorescein fluorescence
polarization. Cancer Detect Prev 1999; 23:64-71
Sunray M, Kaufman M, Zurgil N, Deutsch M: The trace and subgrouping of
lymphocyte activation by dynamic fluorescence intensity and polarization
measurements. Biochem Biophys Res Commun 1999; 261:712-9
Zurgil N, Levy Y, Deutsch M, Gilburd B, George J, Harats D, Kaufman M,
Shoenfeld Y: Reactivity of peripheral blood lymphocytes to oxidized
low-density lipoprotein: a novel system to estimate atherosclerosis
employing the Cellscan. Clin Cardiol 1999; 22:526-32
Zurgil N, Kaufman M, Solodiev I, Deutsch M: Determination of cellular thiol
levels in individual viable lymphocytes by means of fluorescence intensity
and polarization. J Immunol Methods 1999; 229:23-34
Zurgil N, Schiffer Z, Shafran Y, Kaufman M, Deutsch M: Fluorescein
fluorescence hyperpolarization as an early kinetic measure of the apoptotic
process. Biochem Biophys Res Commun 2000; 268:155-63
Deutsch M, Zurgil N, Kaufman M, Berke G: Fluorescence polarization as an
early measure of T-lymphocyte stimulation.Methods Mol Biol 2000; 134:221-42
eutsch M, Kaufman M, Shapiro H, Zurgil N: Analysis of enzyme kinetics in
individual living cells utilizing fluorescence intensity and polarization
measurements. Cytometry 2000;39:36-44
-Howard
.
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