Phasor-based widefield FLIM using a gated 512×512 single-photon SPAD imager

摘要

Single-photon avalanche diode (SPAD) imagers can perform fast time-resolved imaging in a compact form factor, byexploiting the processing capability and speed of integrated CMOS electronics. Developments in SPAD imagers haverecently made them compatible with widefield microscopy, thanks to array formats approaching one megapixel andsensitivity and noise levels approaching those of established technologies. In this paper, phasor-based FLIM isdemonstrated with a gated binary 512×512 SPAD imager, which can operate with a gate length as short as 5.75 ns, aminimum gate step of 17.9 ps, and up to 98 kfps readout rate (1-bit frames). Lifetimes of ATTO 550 and Rhodamine 6G(R6G) solutions were measured across a 472×256 sub-array using phasor analysis, acquiring data by shifting a 13.1 nsgate window across the 50 ns laser period. The measurement accuracy obtained when employing such a scheme based onlong, overlapping gates was validated by comparison with TCSPC measurements and fitting analysis results based on astandard Levenberg-Marquardt algorithm (>90% accuracy for the lifetime of R6G and ATTO 550). This demonstratesthe ability of the proposed method to measure short lifetimes without minimum gate length requirements. The FLIMframe rate of the overall system can be increased up to a few fps for phasor-based widefield FLIM (moving closer toreal-time operation) by FPGA-based parallel computation with continuous acquisition at the full speed of 98 kfps.