In vitro and in vivo NIR fluorescence lifetime imaging with a time-gated SPAD camera

Abstract

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events <i>in vivo</i>. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for <i>in vivo</i> macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report <i>in vivo</i> NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled <i>in vitro</i> experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for <i>in vivo</i> studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for <i>in vivo</i> preclinical applications in the NIR window.