Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy

Highlights: • Optical crosstalk in SPAD arrays introduces correlation between pixels. • Fluorescence Correlation Spectroscopy analysis can be affected by crosstalk. • We describe a method to compute crosstalk from photon timestamps. • Optical crosstalk is negligible in the tested in 48-pixel SPAD arrays. • High-throughput single-molecule setup is not affected by optical crosstalk. Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability $\le 1.110^{-3}$) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.