Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

Fast frame rate complementary metal–oxide–semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We show here how the image intensifier's P20 phosphor afterglow can be exploited for accurate timing of photon arrival well below the camera exposure time. By taking ratios of the intensity of the photon events in two subsequent frames, photon arrival times were determined with 300 ns precision with 18.5 μs frame exposure time (54 kHz camera frame rate). Decays of ruthenium and iridium-containing compounds with around 1 μs lifetimes were mapped with this technique, including in living HeLa cells, using excitation powers below 0.5 μW. Details of the implementation to calculate the arrival time from the photon event intensity ratio are discussed, and we speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could be possible.