Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

The “Central Dogma” of flow cytometry

The “Central Dogma” of flow cytometry Copyright 0 by the Society for Analytical Cytology Vol. 3, No. 6, 1983 Printed in U S A . LETTERS TO THE EDITOR The “Central Dogma” of Flow Cytometry Kerker et al. (1)in their analysis of the question, “Is the Central Dogma of Flow Cytometry True; That Fluorescence Intensity is Proportional to Cellular Dye Content?”, ignore the most pernicious and potentially largest failure of the “Central Dogma”. When the concentration of an inhomogeneous sample of chromophores (e.g., the dye molecules in a Feulgen-stained nucleus) is measured cytophotometrically as if it were a homogeneous solution in a cuvette with parallel walls which are perpendicular to the optic axis, an error, “distributional error”, results. This error varies directly with the optical density of the specimen and also with the magnitudes of inhomogeneities in optical path length and in concentration within the photometric field. This error which plagued many cytophotometric studies was analyzed in 1952 and methods for reducing or eliminating it were described (2). In that paper, it was fiist pointed out that the emission of fluorescent chromophore is linerly proportional to the amount of the chromophore within the photometric field, independent of distribution within that field, if, and only if the maximum optical density is very low. In 1957, this point was reiterated in the paper which introduced the fluorescent acriflavine-SO2 (Schiff) reaction (3). The photographs in that paper also illustrated that, at 436 nm, the optical density produced was comparable to that which is produced by the conventional Schiff reagent viewed in green light and therefore very likely to cause significant distributional error. Yet, when the first flow cytometric determinations of DNA histograms were published (7), (and this study used the acriflavine-SOz-Feulgen reaction), the authors made no mention of the potential error due to excessively high optical density at the exciting wavelength. The demonstration of a pretty good 4C to 2C ratio in this, and then many following papers, gave birth to the central dogma. It was repeatedly noted that the central dogma will fail badly (without necessarily impacting the 4C to 2C ratio, if the distributional errors of the measurements of the 4C and 2C cells were similar) when the optical densities of nuclei at the exciting wavelength are too high (4). Acriflavine-S02-Feulgen stained nuclei, after optimal Feulgen hydrolysis and optimal staining, typically exhibit optical densities above 0.3 at 488 nm. Therefore it was recommended that such preparations be either excited at off-peak wavelengths, where the peak optical densities are sufficiently low (e.g.,less than 0.05 (2) ), or, (with less confidence that the cure would not be worse than the disease) be “underhydrolized” or “under-stained”, intentionally (4). Recently, Tanke et al. (6) have also reviewed the impact of excessive optical density on fluorescence flow cytometric measurements therein r e c o n f i i n g its importance. As a pragmatic rule, in every study which aims to collect quantitative data on cellular composition by fluorescence flow cytometry, a sample of stained cells should always first be examined visually by bright-field microscopy at the excitation wavelength (or with a narrow wavelength band bracketing that wavelength). If the preparation is very refractile the refractive index of the medium surrounding, and if fiied then that permeating the cells, should be raised to match the cell index (5). If the stained structure (e.g., a nucleus) then is easily resolved because the contrast above background is appreciable the optical density is probably above 0.1, and the central dogma will fail for that sample. One must then consider resorting either to excitation at another wavelength, or understaining. Leonard Ornstein Department of Pathology Mount Sinai School of Medicine of the City University of New York 100th Street and Fifth Avenue New York, New York 10029 Literature Cited 1. Kerker M, Van Dilla MA, Brunsting A, Kratohvil JP, Hsu P, Wang DS, Gray JW, Langlois RG: Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content? Cytometry 3: 71, 1982 2. Ornstein L: Distributional error in microspectrophotometry. Lab Invest 1: 250,1952 3. Ornstein L (open discussions at a number of cytometry meetings) n 4. Omstein, L. Mautner, W, Davis BJ, Tamura R New horizons i fluorescence microscopy. J Mt Sinai Hosp 24: 1066, 1957 5. Pollister AW, Ornstein L The photometric chemical analysis of cells. In: Analytical Cytology, Mellors RC (ed.) McGraw-Hill, New York, 1959 6. Tanke HJ, van Oostveldt P, van Duijn P A parameter for the distribution of fluorophores in cells derived from measurements of inner fiiter effects and reabsorption phenomenon. Cytometry 2: 359, 1982 7. Van Dilla MA, Trujillo TT, Mullaney PF, Coulter J R Cell microfluorometry:a method for rapid fluorescence measurement. Science 163 1213, 1969 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cytometry Part A Wiley

The “Central Dogma” of flow cytometry

Orenstein, Leonard
Cytometry Part A , Volume 3 (6) – May 1, 1983
Download PDF
1 page

Loading next page...
 
/lp/wiley/the-central-dogma-of-flow-cytometry-ofka5nT5Ri

References (4)

Publisher
Wiley
Copyright
Copyright © 1983 Wiley Subscription Services, Inc., A Wiley Company
ISSN
1552-4922
eISSN
1552-4930
DOI
10.1002/cyto.990030614
pmid
6851795
Publisher site
See Article on Publisher Site

Abstract

Copyright 0 by the Society for Analytical Cytology Vol. 3, No. 6, 1983 Printed in U S A . LETTERS TO THE EDITOR The “Central Dogma” of Flow Cytometry Kerker et al. (1)in their analysis of the question, “Is the Central Dogma of Flow Cytometry True; That Fluorescence Intensity is Proportional to Cellular Dye Content?”, ignore the most pernicious and potentially largest failure of the “Central Dogma”. When the concentration of an inhomogeneous sample of chromophores (e.g., the dye molecules in a Feulgen-stained nucleus) is measured cytophotometrically as if it were a homogeneous solution in a cuvette with parallel walls which are perpendicular to the optic axis, an error, “distributional error”, results. This error varies directly with the optical density of the specimen and also with the magnitudes of inhomogeneities in optical path length and in concentration within the photometric field. This error which plagued many cytophotometric studies was analyzed in 1952 and methods for reducing or eliminating it were described (2). In that paper, it was fiist pointed out that the emission of fluorescent chromophore is linerly proportional to the amount of the chromophore within the photometric field, independent of distribution within that field, if, and only if the maximum optical density is very low. In 1957, this point was reiterated in the paper which introduced the fluorescent acriflavine-SO2 (Schiff) reaction (3). The photographs in that paper also illustrated that, at 436 nm, the optical density produced was comparable to that which is produced by the conventional Schiff reagent viewed in green light and therefore very likely to cause significant distributional error. Yet, when the first flow cytometric determinations of DNA histograms were published (7), (and this study used the acriflavine-SOz-Feulgen reaction), the authors made no mention of the potential error due to excessively high optical density at the exciting wavelength. The demonstration of a pretty good 4C to 2C ratio in this, and then many following papers, gave birth to the central dogma. It was repeatedly noted that the central dogma will fail badly (without necessarily impacting the 4C to 2C ratio, if the distributional errors of the measurements of the 4C and 2C cells were similar) when the optical densities of nuclei at the exciting wavelength are too high (4). Acriflavine-S02-Feulgen stained nuclei, after optimal Feulgen hydrolysis and optimal staining, typically exhibit optical densities above 0.3 at 488 nm. Therefore it was recommended that such preparations be either excited at off-peak wavelengths, where the peak optical densities are sufficiently low (e.g.,less than 0.05 (2) ), or, (with less confidence that the cure would not be worse than the disease) be “underhydrolized” or “under-stained”, intentionally (4). Recently, Tanke et al. (6) have also reviewed the impact of excessive optical density on fluorescence flow cytometric measurements therein r e c o n f i i n g its importance. As a pragmatic rule, in every study which aims to collect quantitative data on cellular composition by fluorescence flow cytometry, a sample of stained cells should always first be examined visually by bright-field microscopy at the excitation wavelength (or with a narrow wavelength band bracketing that wavelength). If the preparation is very refractile the refractive index of the medium surrounding, and if fiied then that permeating the cells, should be raised to match the cell index (5). If the stained structure (e.g., a nucleus) then is easily resolved because the contrast above background is appreciable the optical density is probably above 0.1, and the central dogma will fail for that sample. One must then consider resorting either to excitation at another wavelength, or understaining. Leonard Ornstein Department of Pathology Mount Sinai School of Medicine of the City University of New York 100th Street and Fifth Avenue New York, New York 10029 Literature Cited 1. Kerker M, Van Dilla MA, Brunsting A, Kratohvil JP, Hsu P, Wang DS, Gray JW, Langlois RG: Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content? Cytometry 3: 71, 1982 2. Ornstein L: Distributional error in microspectrophotometry. Lab Invest 1: 250,1952 3. Ornstein L (open discussions at a number of cytometry meetings) n 4. Omstein, L. Mautner, W, Davis BJ, Tamura R New horizons i fluorescence microscopy. J Mt Sinai Hosp 24: 1066, 1957 5. Pollister AW, Ornstein L The photometric chemical analysis of cells. In: Analytical Cytology, Mellors RC (ed.) McGraw-Hill, New York, 1959 6. Tanke HJ, van Oostveldt P, van Duijn P A parameter for the distribution of fluorophores in cells derived from measurements of inner fiiter effects and reabsorption phenomenon. Cytometry 2: 359, 1982 7. Van Dilla MA, Trujillo TT, Mullaney PF, Coulter J R Cell microfluorometry:a method for rapid fluorescence measurement. Science 163 1213, 1969

Journal

Cytometry Part A – Wiley

Published: May 1, 1983

There are no references for this article.