Direct microsecond wide-field single-molecule tracking and super-resolution mapping via CCD vertical shift

Megan A. Steves and Ke Xu ()
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Megan A. Steves: Berkeley, Department of Chemistry, University of California
Ke Xu: Berkeley, Department of Chemistry, University of California

Nature Communications, 2025, vol. 16, issue 1, 1-10

Abstract: Abstract Wide-field single-molecule tracking is often limited by the ~10 ms camera frame time. We introduce SpeedyTrack, which directly enables microsecond wide-field single-molecule tracking/imaging on common setups. Harnessing the native sub-microsecond vertical charge shifting capability of EM-CCDs, SpeedyTrack staggers wide-field single-molecule images along the CCD chip at ~10-row spacings between consecutive timepoints, effectively projecting the time domain to the spatial domain. Wide-field tracking is achieved for molecules diffusing at up to 1000 µm2/s at 50 µs temporal resolutions for >30 timepoints. Concurrent Förster resonance energy transfer measurements further elucidate molecular states. By implementing temporally patterned vertical shifting, VS-SpeedyTrack next deconvolves the spatial and temporal information to map trajectories at the super-resolution level, resolving the diffusion mode of a fluorescent protein in live cells with nanoscale resolution. Without modifications to existing optics or electronics, SpeedyTrack provides a facile solution to the microsecond tracking of single molecules and their super-resolution mapping in the wide field.

Date: 2025
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DOI: 10.1038/s41467-025-65529-x

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