High-throughput time-correlated single photon countingElectronic supplementary information (ESI) available: IRF, reference fluorescence decay of Fl, fluorescence decay fitting procedures, and demonstration of all 6 equations in the paper and of the fitting function in Fig. 4. See DOI: 10.1039/c4lc00780h

We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high-throughput conditions. We discuss the fundamental limitations in the photon acquisition rate imposed by the single photon detection technique and show that it does not preclude accurate fluorescence lifetime (FLT) measurements at a droplet throughput exceeding 1 kHz with remarkable sensitivity. This work paves the way for the implementation of innovative biomolecular interaction assays relying on the FLT detection of nanosecond-lived fluorophores for high-throughput biotechnological applications, including high-throughput screening or cell sorting potentially allowed by droplet microfluidics or other fast sample handling facilities. Time-correlated single photon counting (TCSPC) was demonstrated in microfluidic droplets for high-throughput, time-resolved fluorescence (bio)sensing of nanosecond-lived fluorophores.