ConspectusDiffusion underlies vital physicochemical and biological processes and provides a valuable window into molecular states and interactions. However, it remains a challenge to map molecular diffusion at subcellular and submicrometer scales. Whereas single-particle tracking of fluorescent molecules provides a path to quantify motion at the nanoscale, its typical pursuit of long trajectories limits wide-field mapping to the slow diffusion of bound molecules.Single-molecule displacement/diffusivity mapping (SMdM) rises to the challenge. Rather than following each fluorescent molecule longitudinally as it randomly visits potentially heterogeneous environments, SMdM flips the question to ask, for every location (e.g., a 100 × 100 nm² spatial bin) in a wide field, how different single molecules of identical nature move locally. This location-centered strategy is naturally effective for spatial mapping of diffusivity. Moreover, by focusing on local motion, each molecule only needs to be detected for its transient displacement within a fixed short time window to achieve local statistics. This task is fulfilled for fast-diffusing molecules using a tandem excitation scheme in which a pair of closely timed stroboscopic excitation pulses are applied across two tandem frames, so that wide-field single-molecule images are recorded at a pulse-defined ≲1 ms separation unlimited by the camera frame rate. With fitting models robust against mismatched molecules and diffusion anisotropy, SMdM thus successfully achieves super-resolution D mapping for fluorescently labeled molecules of contrasting sizes and properties in diverse cellular and in vitro systems.For intracellular protein diffusion, SMdM uncovers nanoscale diffusion heterogeneities in the mammalian cytoplasm and nucleus and further elucidates their origins from the macromolecular crowding effects of cytoskeletal and chromatin ultrastructures, respectively, through correlated single-molecule localization microscopy (SMLM). Across diverse compartments of the mammalian cell, including the cytoplasm, the nucleus, the endoplasmic reticulum (ER) lumen, and the mitochondrial matrix, SMdM further unveils a striking charge effect, in which the diffusion of positively charged proteins is biasedly impeded. For cellular membranes, the integration of SMdM with fluorogenic probes enables diffusivity fine-mapping, which, in combination with spectrally resolved SMLM (SR-SMLM), elucidates nanoscale diffusional heterogeneities of different origins. For biomolecular condensates, another synergy of SMdM and SR-SMLM uncovers the gradual formation of diffusion-suppressed, hydrophobic amyloid nanoaggregates at the surface of FUS (fused in sarcoma) protein condensates during aging. Beyond spatial mapping, the mass accumulation of single-molecule displacements in SMdM further affords a valuable means to quantify D with exceptional precision. This advantage is harnessed to show no enhanced diffusion of enzymes in reactions, to uncover ubiquitous net charge-driven protein-protein interactions in solution, and to show with strategically manipulated cytoplasmic extracts that molecular interaction in the crowded cell is defined by an overwhelmingly negatively charged macromolecular environment with dense meshworks, echoing our parallel results in the mammalian cell.Together, by uniquely enabling super-resolution mapping and high-precision quantification of molecular diffusion across diverse systems, SMdM opens a new door to reveal fascinating spatiotemporal heterogeneities in living cells and beyond.

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