Extended Data Fig. 2: Comparison between WT in solution and on the SLB.
(a) Mean contrast vs mass calibrations obtained from n = 3 standard MP measurements of WT (40 nM, nmonomer = 832, ndimer = 2637, ntetramer = 2404 and nhexamer = 263 particles) and (b) from n = 6 independent 2 min dynamic MP movies (using only trajectories that lasted at least 20 frames) of 20 nM WT on an SLB (ndimer = 227, ntetramer = 1079, nhexamer = 2482 and noctamer = 311 trajectories) acquired on the same day. The error bars in (a) and (b) represent the mean ± s.d. of the contrast of each oligomeric species from the repeat measurements. In some cases the standard deviation was less than 1% causing the contrast error bars to overlap. (c) Oligomeric distribution of WT (100 nM) in HKS-100 buffer measured by standard MP (n = 4 combined measurements with a total of n = 16794 particles). The peaks represent WT monomer (0.5%), dimer (1.0%), tetramer (2.0%) and hexamer (3.0%). (d) Oligomeric distribution of 10 nM WT in HKS-100 buffer diffusing on an SLB obtained from n = 2 combined sets of 3 min dynamic MP movies considering only trajectories that lasted at least 50 frames (n = 1187 trajectories). The contrast measured in dynamic MP movies was consistently ~8% lower than that measured in standard MP. This effect is likely a result of particle motion during image acquisition, which results in motion blurring of the PSF (Supplementary Fig. 3 and Extended Data Fig. 3). This effect increased as we increased frame averaging in dynamic MP movies (Extended Data Fig. 4). The standard MP measurements were acquired at 331 Hz and then processed at a final integration time ~24 ms (effective frame rate ~41 Hz), which enabled the detection of WT monomer. *Peak due to background noise.
