Analyzing blinking effects in super resolution localization microscopy with single-photon SPAD imagers

Abstract

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.