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Abstract

Single-molecule imaging in live cells now plays key roles in elucidating molecular dynamics and interactions. However, the imaging time resolution has remained limited, despite its critical importance for precisely capturing molecular events in cells, in contrast with major spatial resolution advances in fluorescence microscopy. To address this issue, we developed an ultrafast camera system that achieves the highest time resolutions reported to date for single fluorescent-molecule imaging and tracking (SFM-IT), reaching fluorophore photophysics-limited (photon-limited) frame times of 33 and 100 μs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore identified. Using this system, we directly detected the hop diffusion of membrane molecules in the plasma membrane, confirming its compartmentalization. Thus, ultrafast SFM-IT has helped to elucidate the principles governing the plasma membrane organization and molecular dynamics. Building on this platform, we further established ultrafast, live-cell, two-color single-molecule localization microscopy (SMLM). This method reduced the data acquisition time by ≈30-fold relative to standard methods, while simultaneously enabling much larger view-fields, with localization precisions of 29 and 19 nm for PALM and dSTORM, respectively. Combining ultrafast SMLM with ultrafast SFM-IT revealed the mesoscopic dynamical organization of focal adhesions (FAs), termed an archipelago architecture, showing that FAs consist of ≈30-nm-diameter FA-protein islands loosely clustered into ≈300-nm-diameter functional units embedded in the compartmentalized fluid membrane (74 nm inside vs. 110 nm outside the FA). Ultrafast SFM-IT and ultrafast SMLM techniques open previously inaccessible spatiotemporal regimes for biophysical cell biology research.