bioRxiv2021
The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy (FFS). However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark-noise, photon-detection efficiency, after-pulsing probability, and optical-cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present a novel SPAD array detector with an active cooling system, which substantially reduces the dark-noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15{degrees}C significantly improves the signal-to-noise ratio due to a 10-fold decrease in the dark-count rate compared to room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing GFP-tagged proteins. For FFS, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements whose dark-noise is substantially higher than the median value. Overall, this detector represents a further step towards the integration of SPAD array detectors in any FLSM system. SIGNIFICANCESingle-photon avalanche diode (SPAD) array detectors are revolutionizing fluorescence laser-scanning microscopy (FLSM). Thanks to their single-photon timing and sensitivity ability and their imaging faculty, a SPAD array detector transforms any FLSM into a super-resolution microscope, and opens a whole range of possibilities for the study of sample dynamics by means of fluorescence fluctuation spectroscopy (FFS). However, dark-noise can be a severe problem for both imaging and FFS. For imaging, the signal overcomes noise only for a relatively high illumination intensity, which can be detrimental for live-cell experiments. For FFS, the noise leads to artifacts in the correlation curves, potentially leading to wrong conclusions about the sample. We show that lowering the temperature of the detector to -15{degrees}C solves both problems