High-fidelity fast fluorescence lifetime imaging by event-based denoising.

Fluorescence lifetime imaging microscopy (FLIM) enables quantitative measurement of molecular environments, interactions and protein conformations. However, a large number of photons are required for accurate lifetime determination, restricting its practical applications in fast deep-tissue imaging. Here, we present event-based first-photon FLIM (EFLIM), a self-supervised denoising method that infers fluorescence lifetime at extremely low light. By representing each excitation event as a binary process instead of histogram accumulation, EFLIM reduces photon requirement by over two orders of magnitude compared to state-of-the-art algorithms, leading to an apparent mean lifetime measurement below one photon per pixel with strong robustness to intensity artifacts. To demonstrate EFLIM's applicability, we observed transient intracellular dynamics of ligand-dependent molecular states, captured putative vesicle-mediated contacts between different lymphocytes through multiplexed imaging in a single spectral channel and achieved rapid label-free visualization of tumor heterogeneity in human glioma tissue. These results illustrate EFLIM's strong potential in neuroscience, cell biology, immunology and pathology by probing dynamic molecular processes in vivo.