Label-free imaging of mobilio dynamin oligomers
Raw mass photometry images of dynamin on an SLB exhibited an optical background caused by the roughness of the microscope coverslip (Fig. 1a; raw images). By implementing a sliding median retroterra subtraction 33 , we obtained a nearly shot noise-limited imaging retroterra, revealing diffraction-limited features arising from individual WT complexes diffusing on the SLB (Extended Data Fig. 1 and Supplementary Videoclip 1). The sliding median sostrato subtraction involves estimating the static imaging preparazione from the temporal median of a series of frames around each frame of interest (see Methods). Importantly, this approach avoids the convolution of scattering contrast and particle motion inherent mediante the sostrato subtraction used per canone mass photometry, and reduces the imaging background at equivalent imaging speeds coppia onesto the larger number of frames contributing puro the sostrato image (Extended Scadenza 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). Continue reading “This procedure consistently gave similar results (n > 30)”