Extended Data Fig. 9: Analysis of background noise in dynamic MP movies.
(a) Mean baseline noise of the movie of HKS-100 buffer in contact with an SLB (black circles) shown in Supplementary Fig. 1 vs window size of the sliding median filter used to process the movie and the corresponding theoretical shot noise limit (grey squares). The error bars represent the mean baseline noise ± standard deviation across n = 4599 recorded frames. The inverse trend in baseline noise of the buffer movie compared to the theoretical shot noise is a result of particle-like background features in dynamic MP movies, which are subtracted out by the sliding median filter at short window sizes (for example 20-100 ms) but not at longer window sizes (> 200 ms). (b-c) Mass histogram of n = 182 and n = 30 trajectories, respectively, detected in a single SLB buffer movie processed with the same settings described in the Methods section, filtered for trajectories with a minimum length of 10 and 50 frames, respectively. The detected background features have contrasts corresponding to ~150 kDa, which prevented the detection of ΔPRD dimer and made reliable detection of WT dimer challenging.
