This procedure consistently gave similar results (n > 30)

Label-free imaging of mobile dynamin oligomers

Raw mass photometry images of dynamin on an SLB exhibited an optical retroterra caused by the roughness of the microscope coverslip (Fig. 1a; raw images). By implementing verso sliding median background subtraction 33 , we obtained verso nearly shot noise-limited imaging retroterra, revealing diffraction-limited features arising from individual WT complexes diffusing on the SLB (Extended Momento Fig. 1 and Supplementary Schermo 1). The sliding median preparazione subtraction involves estimating the static imaging sostrato from the temporal median of per series of frames around each frame of interest (see Methods). Importantly, this approach avoids the convolution of scattering contrast and particle motion inherent in the retroterra subtraction used con canone mass photometry, and reduces the imaging sostrato at equivalent imaging speeds coppia esatto the larger number of frames contributing to the preparazione image (Extended Giorno Fig. 1 and Supplementary Fig. 1).

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a, Schematic diagram of dynamic mass photometry of protein complexes diffusing on an SLB. The images were acquired at 331 Hz and processed with a sliding median filter, which showed individual protein complexes on the bilayer as diffraction-limited spots. b, Histogram of mean trajectory contrasts detected in a dynamic mass photometry movie (n = 1 movie, 4 min) of WT diffusing on an SLB (considering only trajectories of at least 151 ms in length; n = 425 trajectories). c, Contrast–mass calibration curve of the dynamic mass photometry measurement shown in b (n = 1 dynamic mass photometry movie, 4 min) yielding a contrast to mass ratio of 4.40 % MDa ?1 . Error bars represent the mean contrast ± s.e.m. of each oligomeric species (n_dimer = 34, n_tetramer = 85, n_hexamer = 184, n_octamer = 23 trajectories). d, 2D localization error of our PSF-fitting procedure of WT dimers, tetramers, hexamers and octamers plotted as a function of effective exposure time.