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BP: Fachverband Biologische Physik

BP 17: Bioimaging

BP 17.8: Talk

Wednesday, March 20, 2024, 11:45–12:00, H 2032

Video-rate volumetric fluorescence lifetime imaging of living multicellular systems using single-objective lightsheet microscopy — •Valentin Dunsing-Eichenauer¹, Johan Hummert², Claire Chardès¹, Felix Koberling², Ivan Michel Antolovic³, Léo Guignard¹, and Pierre-François Lenne¹ — ¹IBDM & CENTURI, Aix-Marseille University/ CNRS, Marseille, France — ²PicoQuant GmbH, Berlin, Germany — ³Pi Imaging Technology SA, EPFL Innovation Park, Lausanne, Switzerland

Fluorescence lifetime imaging microscopy (FLIM) is a widely used technique for functional and multiplexed bioimaging. It is commonly performed on confocal laser scanning microscopes equipped with time correlated single photon counting hardware. However, high excitation powers or long acquisition times are needed to obtain sufficient photon statistics, preventing applications on sensitive living specimen such as embryos or organoids. To overcome these limitations, we have combined single objective lightsheet microscopy with pulsed excitation and time-resolved detection on a 512x512 pixel gated SPAD array detector. We report excellent quantitative agreement with confocal FLIM at 100-1000-fold shorter acquisition times, down to 150 ms per image. We further demonstrate 3D FLIM on live embryonic organoids, lifetime unmixing of two spectrally overlapping fluorophore species, and time-lapse 3D FLIM of mechanosensitive tension probes. Our approach facilitates volumetric FLIM at unprecedented speed and throughput, providing a powerful tool for functional imaging of dynamic multicellular systems.