Extended Data Fig. 5: Frame rate determines SMT dynamic range.
(a) State array analysis plotted as a function of frame rate comparing DMSO and 1 μM KI-696 treated Keap1-HaloTag U2OS cells. The number of FOV replicates per frame rate was as follows: n = 88 (100 Hz), n = 88 (200 Hz), n = 132 (400 Hz), n = 198 (800 Hz), and n = 264 (1250 Hz). Lines are the mean values over all FOVs in the corresponding condition and error bands are the FOV-level standard deviations. (b) Mean SNR plotted as a function of frame rate, for SNR calculation see Methods. (c) mean ERLB plotted as a function of frame rate, ERLB is defined as in ‘Empirical estimate of linking precision’ with error bars representing FOV-level standard deviations (Methods). (d) Evaluation of bleaching rate in KEAP1-HaloTag SMT at variable frame rates. Fraction of detections remaining was plotted across frame rate for a given time series. The fraction of detections remaining was defined as the number of detections in each frame divided by the number of detections in the first frame. Exponential fits (blue text below frame rate) were performed with respect to the model \(f(t)={c}_{0}+(1-{c}_{0}){e}^{-{kt}}\), where t is frame index, k is bleaching rate and c0 is the unbleached fraction, using an iterative least-squares routine. The number of FOV replicates per frame rate was as follows: n = 88 (100 Hz), n = 88 (200 Hz), n = 132 (400 Hz), n = 198 (800 Hz), and n = 264 (1250 Hz) with each FOV containing approximately 40–50 cells at 100 Hz.
