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

We introduce spatially-encoded dynamics tracking (SpeedyTrack), a strategy to enable direct microsecond wide-field single-molecule tracking/imaging on common microscopy setups. Capitalizing on the native sub-microsecond vertical charge shifting capability of popular electron-multiplying charge-coupled devices (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 thus achieved for freely diffusing molecules at down to 50 μs temporal resolutions for >30 timepoints, permitting trajectory analysis to quantify diffusion coefficients up to 1,000 μm ² /s. Concurrent acquisition of single-molecule diffusion trajectories and Förster resonance energy transfer (FRET) time traces further elucidates conformational dynamics and binding states for diffusing molecules. Moreover, with a temporally patterned vertical shifting scheme, we deconvolve the spatial and temporal information to map long, fast single-molecule trajectories at the super-resolution level, thus resolving the diffusion mode of a fluorescent protein in live cells with nanoscale resolution. While these demonstrated capabilities substantially outperform existing approaches, SpeedyTrack further stands out for its simplicity by directly working off the built-in functionalities of EM-CCDs without the need to modify existing optics or electronics. We thus provide a facile solution to the microsecond tracking/imaging of single molecules and their super-resolution mapping in the wide field.