Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

Spectrally resolved fluorescence lifetime imaging(1-3) and spatial multiplexing(1,4,5) have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (similar to 40 ps temporal resolution) using fewer than N-2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Frster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.