Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

Fluorescence lifetime imaging (FLIM) is widely used for functional and multiplexed bioimaging, but is limited in speed, volumetric sampling of multicellular specimen, and live cell compatibility. To overcome these limitations, we have combined single objective light-sheet microscopy with pulsed excitation and time-resolved detection on a SPAD array detector. We report excellent quantitative agreement with confocal FLIM at 10-100-fold shorter acquisition times, down to 100 ms per image. We demonstrate lifetime-based multiplexing in 3D and time-lapse FLIM of mechanosensitive tension probes on living embryonic organoids, providing a powerful tool for functional imaging of dynamic multicellular systems.