Excited-state annihilation reduces power dependence of single-molecule FRET experiments.

Single-molecule Förster resonance energy transfer (FRET) experiments are an important method for probing biomolecular structure and dynamics. The results from such experiments appear to be surprisingly independent of the excitation power used, in contradiction to the simple photophysical mechanism usually invoked for FRET. Here we show that excited-state annihilation processes are an essential cause of this behavior.

High-throughput time-correlated single photon counting.

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.

Out-of-equilibrium biomolecular interactions monitored by picosecond fluorescence in microfluidic droplets.

We developed a new experimental approach combining Time-Resolved Fluorescence (TRF) spectroscopy and Droplet Microfluidics (DμF) to investigate the relaxation dynamics of structurally heterogeneous biomolecular systems. Here DμF was used to produce with minimal material consumption an out-of-equilibrium, fluorescently labeled biomolecular complex by rapid mixing within the droplets. TRF detection was implemented with a streak camera to monitor the time evolution of the structural heterogeneity of the complex along its relaxation towards equilibrium while it propagates inside the microfluidic channel.