(306e) High Throughput Fluorescence Decay Measurements Using Time-Resolved Flow Cytometry

Flow cytometry is a fluorescence-dependent measurement approach that enables the screening of single cells at a high-throughput level.  Current flow cytometry assays involve tagging cells with several differently colored fluorophores so that through spectral analysis, cell surface features or intracellular targets can be discriminated and quantified.  Yet owing to the severe spectral overlap that occurs between the emission of organic fluorophores, complex cytometry assays are becoming increasingly limited.   In this contribution a high-throughput fluorescence lifetime-dependent flow cytometry system is characterized and evaluated in order to provide a new cytometric parameter that is not dependent on fluorescence intensity output.  Here we present new time-resolved approaches for flow cytometry analysis that involve a multi-frequency methodology.  By utilizing the idea that a laser excitation source is modulated as a square wave, where multiple odd harmonics are included in the signal, we can extract multiple phase shifts and correlate those to multiple fluorescence lifetimes that are present in each cell.   Additionally, we test this novel approach on a range of applications including measurement of fluorescence lifetime changes of green fluorescent protein.  Specifically, enhanced green fluorescence protein (EGFP) molecules are fused to a protein (human LC3 type B) that changes location within cells during the process of autophagy.  The localization of the LC3 protein into punctae occurs during autophagy.  Thus tagging EGFP to LC3 will indirectly cause the fused EGFP to group in to the punctae.  Our fluorescence lifetimes are shown to be sensitive to the presence of diffuse EGFP-LC3 in the cytosol and to the organized EGFP-LC3 localization into punctate autophagosomes during autophagy induction. We verify this with low-throughput imaging and compare to less-quantitative standard fluorescence intensity-only flow cytometry.  In general this work prefaces a new rapid compound-screening assay for effects on protein location.  If autophagy or other biologically relevant phenomena can be quantitatively detected then time-resolved flow cytometry can become a powerful sorting and analysis tool.