Workflow for light sheet-based Fluorescence Lifetime Imaging Microscopy (FLIM). (A) A pulsed laser (blue) is used to excite fluorescent molecules within the sample (green). These fluorescent molecules have a characteristic relaxation time back to their ground state. (B) To measure the lifetime of these fluorescent molecules, the detector is briefly exposed (white box) at a known delay from the excitation pulse (gate delay). This is repeated at continually-increasing gate delays. (C) The total intensity is integrated for each gate delay. An exponential decay curve is fit to the data from which one can extract the lifetime. (D) This process (A-C) is then parallelized via light sheet microscopy and an array detector.

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Instrument Summary

Volumetric fluorescence intensity–based microscopy has transformed the life sciences, but it faces several intrinsic limitations. Multiplexing is constrained by the ability to spectrally separate fluorophores, making simultaneous imaging of multiple signals difficult. Autofluorescent tissues can obscure labeled structures when emission spectra overlap, and quantitative measurements of biosensors in large or optically complex samples are often distorted by scattering and optical aberrations.

Fluorescence Lifetime Imaging Microscopy (FLIM) overcomes many of these challenges by measuring the fluorescence decay rate rather than signal intensity (1). Fluorophores with overlapping emission spectra can be distinguished based on their lifetimes, enabling expanded multiplexing. Autofluorescence, which typically exhibits distinct lifetime signatures, can be effectively separated from true signal. Because lifetime measurements are largely independent of fluorophore concentration and excitation intensity, FLIM provides more robust and quantitative readouts in thick, heterogeneous specimens (2,3).

Despite these advantages, most FLIM implementations rely on point-scanning confocal systems and small fields of view, making fast volumetric imaging impractical for large living samples. To address this, we developed a volumetric FLIM platform that combines a next-generation SPAD (Single-Photon Avalanche Diode) array detector with static light-sheet illumination to perform time-domain lifetime imaging. This approach merges the speed and low phototoxicity of light-sheet microscopy with the quantitative power of FLIM, enabling rapid, gentle, and truly volumetric lifetime imaging in living systems ranging from organoids to developing zebrafish.

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Suggested Reading

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1. Torrado, B., et al. Fluorescence lifetime imaging microscopy. Nature Reviews (2024)

2. Farrants, H. A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.  Nat. Methods. (2024).

3. Dunsing-Eichenauer, V., et al. Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy Communications Biology (2025).