Autofluorescence lifetime flow cytometry with time-correlated single-photon counting

Autofluorescence lifetime imaging microscopy is sensitive to functional or treatment-induced metabolic changes in single cells. However, this label-free technique typically relies on laser-scanning microscopes that are not common for single-cell assessments in immunology. Here, we present a microfluidic lifetime-sensitive flow cytometer that measures time-domain decays of the metabolic co-enzyme NAD(P)H from single cells with picosecond temporal resolution using time-correlated single-photon counting (TCSPC) at a throughput of tens of cells per second. The system uses a picosecond-pulsed ultraviolet diode laser, alkali photomultiplier tubes, an FPGA-based time tagger, and provides real-time phasor-based classification (i.e., gating) of flowing cells for applications in immunology.