Time-domain single photon-excited autofluorescence lifetime is sensitive to activation state of T cells

A single photon-excited confocal whole-cell autofluorescence lifetime system is described that uses fast field-programmable gate array (FPGA)-based time tagging electronics to implement time-correlated single photon counting (TCSPC) with simultaneous near-IR brightfield imaging. This system resolves variations in the fluorescence decay of the metabolic coenzyme NAD(P)H that provides high accuracy and precision for classifying activated and quiescent primary human T cells (ROC AUC = 0.98). This performance is similar to that obtained using commercial two-photon fluorescence lifetime imaging microscopy (FLIM). The small footprint, low cost, and high acquisition speed make this system attractive for monitoring cell biomanufacturing.