Cataloging autofluorescence lifetimes of intracellular proteins for flow cytometry

Flow cytometry is a high-throughput cellular analysis technique in which fluorescence from dye-tagged cells is measured.  A flow cytometer allows for in vitro molecular screening for drug discovery.  Currently, many cytometry screening protocols utilize microspheres or cells that are multiplexed.  The cell or particle harbors multiple molecular targets, all of which can be bound to a fluorescent dye of a different color. Unfortunately these polychromatic assays have been coupled with the issue of fluorescence spillover, which makes distinguishing between different fluorophores difficult.   In our research time-resolved measurements are investigated in an attempt to avoid problems with spectral overlap.  We have developed techniques in cytometry to measure the fluorescence lifetime, or average time the fluorescent dye spends in its excited state.  In this contribution, we demonstrate one lifetime-dependent application---the separation of endogenous cellular autofluorescence from external dye fluorescence.  Experiments were performed to characterize autofluorescent species and lifetimes thereof with Chinese Hamster Ovary Cells (CHO-K1).   We found that endogenous proteins in CHO-K1 cells express two dominant lifetimes that vary slightly depending on growth phase of the cell, its viability, and the solvent fixative. Because autofluorescence issues arise for eukaryotic cells of all types, the future of this work will be to extend measurements across other cell lines while delving into the measurement of multiple lifetimes for the purpose of discriminating individual proteins, amino acids, or other cellular constituents that fluoresce.