Wide-field Time-correlated Single Photon Counting Microscopy

Time-correlated single photon counting (TCSPC) is a sensitive, robust and precise technique to measure luminescence decays.[1] For fluorescence microscopy, it is often implemented with beam scanning and single point detectors, but we have implemented a camera-based wide-field TCSPC method. We employ a photon counting image intensifier in combination with a 1 MHz frame rate CMOS camera, thus combining an ultra-fast frame rate with single photon sensitivity.[2] This approach allows mapping the lifetime of europium-containing beads (570 μs), and decays of a few microseconds of ruthenium compound Ru(dpp) in living HeLa cells. Moreover, the invariant phosphor decay, or afterglow, of the image intensifier screen can be used for accurate timing of photon arrival well below the camera exposure time.[3] By taking ratios of the intensity of the photon events in two subsequent frames, decays of ruthenium and iridium-containing compounds with lifetimes of around 1 μs were measured with 18.5 μs frame exposure time (54 kHz camera frame rate), also in living HeLa cells. Fluorescence lifetime imaging (FLIM) microscopy with picosecond time resolution and widefield detection can be achieved using electronic read-out architectures, and an electron-bombarded CCD-based scheme has also been proposed.[4] These approaches are particularly appropriate for total internal reflection fluorescence, super-critical angle fluorescence or lightsheet microscopy, and the latest progress in this field will be discussed.