Microsecond wide-field TCSPC microscopy based on an ultra-fast CMOS camera

Ultra-fast frame rate CMOS cameras, combined with a photon counting image intensifier, can be used for microsecond resolution wide-field time-correlated single photon counting (TCSPC) microscopy. A sequence of frames is recorded after an excitation pulse, and the number and location of photons in each frame is determined. This process is repeated until enough photons are recorded for a photon arrival time histogram in the pixels of the image. This approach combines low, nanowatt excitation power with single-photon detection sensitivity and arrival timing in many pixels simultaneously, short acquisition times in the order of seconds and allows lifetime mapping with a time resolution of ∼1 microsecond. Moreover, we also show that the phosphor decay can be exploited to time the photon arrival well below the exposure time of the camera. This approach yields better time resolution and larger images than direct imaging of photon events. We show that both methods are ideal for lifetime imaging of transition metal compounds in living cells within a few seconds.