Cytometric sorting based on the fluorescence lifetime of spectrally overlapping signals

Flow cytometry is a well-established and powerful high- throughput fluorescence measurement tool that also allows for the sorting and enrichment of subpopulations of cells expressing unique fluorescence signatures. Owing to the reliance on intensity-only signals, flow cytometry sorters cannot easily discriminate between fluorophores that spectrally overlap. In this paper we demonstrate a new method of cell sorting using a fluorescence lifetime-dependent methodology. This approach, referred to herein as phase-filtered cell sorting (PFCS), permits sorting based on the average fluorescence lifetime of a fluorophore by separating fluorescence signals from species that emit differing average fluorescence lifetimes. Using lifetime-dependent hardware, cells and microspheres labeled with fluorophores were sorted with purities up to 90%. PFCS is a practical approach for separating populations of cells that are stained with spectrally overlapping fluorophores or that have interfering autofluorescence signals.