Direct microsecond wide-field single-molecule tracking and super-resolution mapping via CCD vertical shift.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 41290642.
- Also identified by DOI 10.1038/s41467-025-65529-x and PMC identifier 12647137.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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 µm<sup>2</sup>/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.