EXPERIMENTAL AND MODEL INVESTIGATIONS OF BLEACHING AND SATURATION OF FLUORESCENCE IN FLOW-CYTOMETRY

We investigated the fluorescence emission from three fluorophores comm only used for labeling cells in flow cytometry. We have demonstrated t hat the fluorescence emission from cells labeled with fluorescein-isot hiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is co nsiderably saturated and bleached in standard flow cytometric conditio ns. Therefore, for optimization of fluorescence detection in a flow cy tometer, it is important to know the emission kinetics in detail We ma de a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and trip let state occupation. The validity of the model was experimentally tes ted with a set of averaged fluorescence pulses, measured in a large ra nge of intensities and illumination times. The fluorescence of APC cou ld be com pletely described by the model and produced the following ra te constants: photodestruction rate k(b1) = 6.10(3) s(-1), triplet sta te 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 cell s could not be fitted with only three parameters over the entire range , indicating that other optical processes are involved. We used the mo del to determine the sensitivity of our flow cytometer and to calculat e the optimum conditions for the detection of APC. The results show th at in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering b y water. (C) 1997 Wiley-Liss, Inc.