Label-free imaging of mobile dynamin oligomers
Raw mass photometry images of dynamin on an SLB exhibited an optical sostrato caused by the roughness of the microscope coverslip (Fig. 1a; raw images). By implementing verso sliding median sostrato subtraction 33 , we obtained per nearly shot noise-limited imaging preparazione, revealing diffraction-limited features arising from individual WT complexes diffusing on the SLB (Extended Data Fig. 1 and Supplementary Filmato 1). The sliding median preparazione subtraction involves estimating the static imaging retroterra 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 durante the preparazione subtraction used in standard mass photometry, and reduces the imaging sostrato at equivalent imaging speeds due puro the larger number of frames contributing to the background image (Extended Giorno Fig. 1 and Supplementary Fig. 1).
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 . Continue reading “This procedure consistently gave similar results (n > 30)”