Single-photon avalanche diode (SPAD) arrays are solid-state detectors that offer imaging capabilities at the level of individual photons, with unparalleled photon counting and time-resolved performance. This fascinating technology has progressed at a very fast pace in the past 15 years, since its inception in standard CMOS technology in 2003. A host of architectures have been investigated, ranging from simpler implementations, based solely on off-chip data processing, to progressively “smarter” sensors including on-chip, or even pixel level, time-stamping and processing capabilities. As the technology has matured, a range of biophotonics applications have been explored, including (endoscopic) FLIM, (multibeam multiphoton) FLIM-FRET, SPIM-FCS, super-resolution microscopy, time-resolved Raman spectroscopy, NIROT and PET. We will review some representative sensors and their corresponding applications, including the most relevant challenges faced by chip designers and end-users. Finally, we will provide an outlook on the future of this fascinating technology.

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Summary form only given. Single photon detectors allow us to work with the weakest signals such as auto-fluorescent biological sources. In combination with time gated operation mode, an array of detectors can be used as Fluorescence Lifetime Imaging system with extremely high sensitivity.Here we present fluorescence lifetime imaging using the `SwissSPAD' [1] sensor, an extensive 512-by-128pixel array of time-gated single photon avalanche diodes (SPADs). By taking a series of gated measurements and changing the gate delay, fluorescent lifetimes can be retrieved for each individual pixels. Over 65 thousand independent SPAD pixels creates a detailed spatial map of fluorescence lifetimes in the field of view of the sensor. In this work, we are presenting two milestones of our research. Firstly, we present proof of principle imaging lifetime measurements of Rhodamine B and Fluorescein, with lifetimes of 1.68ns and 4ns ns respectively, excited using a 532 nm 10 picosecond laser. Secondly, we show that using 266 nm excitation, we can distinguish between free(0.5ns) and bound(2.2ns) NADH using unlabelled auto-fluorescence. Freeand bound-NADH are known to be markers for the cancer tissues [2].@en

sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.

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For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.@en

While CMOS single-photon avalanche diode (SPAD) technology has steadily advanced, improving noise, timing resolution, and sensitivity, spatial resolution has been increasing as well. The increase in the number of pixels has made a comprehensive analysis of nonuniformity and its effects meaningful, allowing a more accurate comparison of SPAD imagers with other high-end scientific imagers, such as electron multiplying charge-coupled device and scientific CMOS. A comprehensive nonuniformity analysis was conducted on a 512 × 128 pixel gated SPAD imager, where dark noise, afterpulsing, crosstalk, signal response, and shot noise were measured. This analysis has led to a variety of postprocessing algorithms to improve the linearity of the response as for example required by ground state depletion microscopy-based superresolution microscopy and other techniques. We derived a new correction formula for the count rate applicable to 1-b SPAD imagers, and we measured a significant improvement of photon detection efficiency using microlenses. These techniques were used to validate the suitability of the imager in fluorescence microscopy examples.@en