Measurement of modulated autofluorescence signals in flow cytometry

Abstract

In flow cytometry autofluorescence from cells often interferes with dim exogenous emission and presents signal-overlap problems during complex multi-color, multi-intensity, and/or low-density antigen experiments. An alternative to intensity measurements in flow is to capture the fluorescence lifetime, or the average time a fluorophore spends in an excited state before decay. The lifetime can discriminate between two emission signals independently of intensity and fluorophore concentration. Thus, in order to distinguish between overlapping autofluorescence signals and other dim emissions, we present phase-sensitive flow cytometry (PSFC) experiments and investigate the average autofluorescence lifetime of a variety of cell types. We demonstrate lifetime fluctuations with cell cycle, vitality, and differing excitation wavelengths. Establishing a catalog of intrinsic lifetimes motivates future PSFC filtering studies in which the intrinsic lifetime is used to subtract out background autofluorescence noise. Since it is the case that in any given cellular condition, a varying level of intrinsic fluorophores are present, determining the average decay times in a high-throughput manner is ideal for separating cytometry signals on a cell-to-cell basis. © 2009 Springer Berlin Heidelberg.