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

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  • Megan A. Steves

    (Berkeley, Department of Chemistry, University of California)

  • Ke Xu

    (Berkeley, Department of Chemistry, University of California)

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.

Suggested Citation

  • Megan A. Steves & Ke Xu, 2025. "Direct microsecond wide-field single-molecule tracking and super-resolution mapping via CCD vertical shift," Nature Communications, Nature, vol. 16(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-65529-x
    DOI: 10.1038/s41467-025-65529-x
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