Fig. 1: Schematics: SpeedyTrack leverages the native fast vertical shifting of EM-CCD to record microsecond single-molecule dynamics in the wide field.

a CCD timing diagram of SpeedyTrack. Many rounds of exposure-vertical shift are made before the final readout. b SpeedyTrack recording of single-molecule trajectories. After each exposure, the entire frame is shifted down by δ ~ 10 rows, just enough to separate the single-molecule images at each timepoint. A trail (streak) of images (colored by timepoint) is thus generated for each molecule along the vertical direction of the CCD chip. After a series of exposures, the chip-stored image is read out collectively, which contains multiple single-molecule streaks projecting discrete timepoints in the vertical direction. c Single-molecule trajectories reconstructed from the three streaks depicted in (b) by deducting the preset vertical shifts between timepoints. d The SpeedyTrack exposure-shift scheme is tailored for single-molecule/single-particle imaging: for continuous structures, ~10-row shifts in the wide field lead to unresolvable signal overlap.