SLIM: Functional Nanoscopy for Simultaneous Super‐Resolution Imaging and Quantitative Lifetime Mapping by Spatiotemporal Multiplexing of Spontaneous Emission

ABSTRACT Imaging nanoscale biological processes such as organelle interactions, protein aggregation, and metabolic regulation requires tools that concurrently resolve structural details and functional states. Stimulated emission depletion (STED)‐based super‐resolution can be integrated with fluorescence lifetime imaging (FLIM), but the depletion beam perturbs the fluorescence decay kinetics and compromises lifetime accuracy. Conventional FLIM, meanwhile, is fundamentally limited by optical diffraction. To overcome these limitations, we introduce SLIM, a functional nanoscopy technique based on spatiotemporal multiplexing of spontaneous emission. By combining a pulsed excitation laser with a temporally delayed STED depletion beam at an adjustable nanosecond interval, SLIM creates a temporal window within each acquisition cycle to separate spontaneous from stimulated emission, enabling the simultaneous acquisition of super‐resolved structural information and quantitative lifetime data. The technique delivers a 3‐ to 5‐fold improvement in structural resolution over diffraction‐limited FLIM, while preserving lifetime accuracy within 5% of confocal reference values, and operates at depletion powers one order of magnitude lower than conventional STED nanoscopy. We demonstrate SLIM's applicability across diverse biological specimens and its capability for long‐term live‐cell imaging of organelle dynamics. By merging structural imaging with quantitative functional mapping in a single modality, SLIM provides a practical and versatile platform for next‐generation biological discovery.