High-efficiency digitally scanned light-sheet fluorescence lifetime microscopy (DSLM-FLIM)

Recently-developed kilopixel single-photon avalanche diode (SPAD) arrays with in-pixel timing hold great promise for fluorescence lifetime imaging microscopy of dynamic samples, thanks to their widefield single-photon time-of-arrival imaging capabilities. However digitally-scanned light-sheet microscope (DSLM) and two-photon microscope systems present significant technical barriers which have to date prevented full and efficient use of the capabilities of SPAD arrays. Because the 12.4 kHz frame-rate of our array camera is faster than achievable DSLM scan rates, most pixels would be sitting idle most of the time. We present a new optical design based around astigmatic imaging optics, enabling rapid and efficient acquisition of fluorescence lifetime imaging data. We demonstrate our system with both one- and two-photon excitation sources, validate performance with lifetime reference beads, and demonstrate separation of similar fluorescence emission spectra in biological samples via lifetime contrast.