Wide-field time-gated phasor analysis of visible fluorescence through highly scattering medium

Fluorescence sensing is an invaluable research tool in life science and biomedical imaging. Despite its major use and advantages, it mainly remains a tool for in vitro studies since exist fluorophores mainly absorb and emit light in the visible region, in which light penetration through the tissue is very low. Here, we use fluorescence lifetime as an intrinsic characteristic of any fluorescent dye, which to some extent, does not depend on excitation intensity and signal level. We use a large time-gated single-photon avalanche diode (SPAD) array detector, SwissSPAD2, for wide-field fluorescence acquisition. We use the phasor approach [1,2], which provides a simple and fast visual method for lifetime imaging, to convert each pixel’s data into a quantity which can be mapped to the local lifetime. We show, for single dyes, that the phasor dispersion increases with decay broadening due to scattering and decreasing fluorescence intensity (shot noise effect). Yet, as long as the fluorescence intensity is higher than the tissue-like phantom autofluorescence, a distinct lifetime can still be clearly identified with the appropriate background correction.