SignificanceFluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.AimWe aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.ApproachA single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.ResultsAn NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.ConclusionsFLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
SPAD array sensors support higher-throughput fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser-scanning to wide-field geometries. While a SPAD camera in epi-fluorescence geometry enables wide-field FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus culture medium masks cell autofluorescence and biases lifetime measurements. Here, we address this problem in a proof-of-concept implementation by integrating the SPAD camera in a light-sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling label-free wide-field FLIM of single-cell NAD(P)H autofluorescence.
The Flamingo is a modular, shareable light sheet microscope suited to a new model of scientific collaboration. Each microscope is customized for a given application, equipped to travel from lab to lab and providing widespread access to advanced microscopy. It is a compact selective plane illumination microscope (SPIM) that can be turned upright for multi-view imaging of hanging samples or turned on its side for samples in a dish. Rapid multi-color imaging is achieved via sCMOS cameras and several possible laser lines. With its selective sheet illumination, the Flamingo is well suited for fast and gentle imaging of developing organisms.
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