High-efficiency digitally scanned light-sheet fluorescence lifetime microscopy (DSLM-FLIM)
Nutt, K. J.; Olesker, D.; McGhee, E.; Hungerford, G.; Leburn, C. G.; Taylor, J. M.
Show abstract
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.
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