Autofluorescence lifetime flow cytometry with time‐correlated single photon counting

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm