Single Photon, Time-Gated, Phasor-based Fluorescence Lifetime Imaging Through Highly Scattering Medium

Fluorescence lifetime imaging (FLI) is a powerful tool for in vitro and non-invasive in vivo biomolecular and cellular investigations. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to some extent, does not depend on excitation intensity and signal level. However, when used in vivo with visible wavelength emitting fluorophores, FLI is complicated by (i) light scattering as well as absorption by tissues, which significantly reduces fluorescence intensity, (ii) tissue autofluorescence (AF), which decreases the signal to noise ratio and (iii) broadening of the decay signal, which can result in incorrect lifetime estimation. Here, we report the use of a large-frame time-gated single-photon avalanche diode (SPAD) imager, SwissSPAD2, with a very short acquisition time (in the milliseconds range) and a wide-field microscopy format. We use the phasor approach to convert each pixels data into its local lifetime. The phasor transformation provides a simple and fast visual method for lifetime imaging and is particularly suitable for in vivo FLI which suffers from deformation of the fluorescence decay, and makes lifetime extraction by standard fitting challenging. We show, for single dyes, that the phasor cloud distribution (of pixels) increases with decay broadening due to scattering and decreasing fluorescence intensity. Yet, as long as the fluorescence signal is higher than the tissue-like phantom AF, a distinct lifetime can still be clearly identified with an appropriate background correction. Lastly, we demonstrate the detection of few hundred thousand A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering phantom layers, despite significant scattering and the presence of the phantom AF.