K. Suhling

Klaus Suhling, Jakub Nedbal, Liisa M. Hirvonen
Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise and robust technique to detect photon arrival times in fluorescence spectroscopy and microscopy. In confocal or multiphoton excitation fluorescence microscopy, it is often implemented with beam scanning and single point detectors. However, we have implemented a camera-based wide-field TCSPC method, where the position and the arrival time of the photons are recorded simultaneously. This has some advantages for certain types of microscopy. 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. Compatibility of this method with live-cell imaging was demonstrated by imaging europium-containing beads with a lifetime of 570 μs in living HeLa cells, as well as decays of ruthenium compound Ru(dpp) with lifetimes from around 1 to 5 μs. Moreover, the invariant phosphor decay of the image intensifier screen can be used 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, 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), including in living HeLa cells. These approaches bring together advantageous features for time-resolved live cell imaging such as low excitation intensity, single photon sensitivity, ultra-fast camera frame rates and short acquisition times. We also report on nanosecond fluorescence lifetime imaging (FLIM) microscopy based on a 40 mm diameter crossed delay line anode detector with picosecond time resolution, where the readout is performed by three standard TCSPC boards. We apply this wide-field TCSPC detector to FLIM of cells labelled with membrane dyes imaged with a TIRF microscope. Moreover, we demonstrate FLIM lightsheet microscopy with this detector.