Open AccessExperimental and model investigations of bleaching and saturation of fluorescence in flow cytometry
Author(s) -
Doornbos Richard M. P.,
de Grooth Bart G.,
Greve Jan
Publication year - 1997
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19971101)29:3<204::aid-cyto3>3.0.co;2-b
Subject(s) - fluorescence , flow cytometry , population , kinetics , fluorescein isothiocyanate , phycoerythrin , allophycocyanin , chemistry , autofluorescence , analytical chemistry (journal) , reaction rate constant , cytometry , fluorescein , biophysics , photochemistry , chromatography , microbiology and biotechnology , biology , optics , physics , phycocyanin , cyanobacteria , demography , genetics , quantum mechanics , sociology , bacteria
We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein‐isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate k b1 = 6 · 10 3 s −1 , triplet state population rate k 12 = 2 · 10 5 s −1 , and depopulation rate k 20 = 5 · 10 4 s −1 . The fluorescence kinetics of FITC‐ and PE‐labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water. Cytometry 29:204–214, 1997. © 1997 Wiley‐Liss, Inc.