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Abstract:
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 mus frame
exposure time (54 kHz camera frame rate). Decays of ruthenium and
iridium-containing compounds with around 1 mus lifetimes were mapped
with this technique, including in living HeLa cells, using excitation
powers below 0.5 muW. 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.
