Fig. 2: Fast wide-field tracking of freely diffusing molecules through SpeedyTrack.

a–f SpeedyTrack data of Cy3B-labeled carbonic anhydrase diffusing in PBS, with an exposure time of 300 µs and a vertical shift time of 7.5 µs for 15 rows at each timepoint. a A portion of one CCD frame, showing multiple streaks from single molecules entering/exiting the focal plane at different locations and timepoints. b Zoom-in of one streak, with colored boxes indicating different time points. c Single-molecule trajectory reconstructed from the localizations in (b) by back-shifting 15 rows between consecutive timepoints, colored as the boxes in (b). d Additional example trajectories. e Distribution of durations for single-molecule trajectories collected over 4000 SpeedyTrack frames. f Squares: MSD at different time lags calculated from the trajectories. Error bars: standard errors. Line: Linear fit to the first 4 data points, yielding a diffusion coefficient of 90 ± 3 µm²/s (95% confidence interval) from 5573 tracks. Inset: distribution of apparent diffusion coefficients estimated for individual trajectories longer than 12 timesteps. g–j SpeedyTrack data of Cy3B free dye. g A portion of one SpeedyTrack frame showing two single-molecule streaks for Cy3B diffusing in PBS, with exposure and vertical-shift times of 50 and 7.5 µs at each timepoint. h Examples of reconstructed Cy3B trajectories in PBS and acetone at 57.5 and 50 µs timesteps, respectively. The arrow points to the trajectory reconstructed from the long streak in (g). i Data points: MSD vs. time lag for Cy3B in PBS (black, 1860 tracks) and acetone (red, 1924 tracks). Error bars: standard errors. Lines: Linear fits to the first 4 data points, yielding diffusion coefficients of 378 ± 9 and 1022 ± 30;µm²/s (95% confidence intervals), respectively. j Circles: SpeedyTrack-measured diffusion coefficients of Cy3B in solvents and mixtures of varying viscosities. Each data point is obtained from the MSD-time lag fit to one experiment of 10³ − 10⁴ tracks (Supplemental Table 1). Error bars: 95% confidence intervals. Dashed curve: Trend predicted by scaling the PBS value by the medium viscosity. Inset: Results in methanol-water mixtures of varying methanol mole fractions, compared to the expected trend based on the known mixture viscosity.