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 verso nearly shot noise-limited imaging background, revealing diffraction-limited features arising from individual WT complexes diffusing on the SLB (Extended Scadenza Fig. 1 and Supplementary Video 1). The sliding median background subtraction involves estimating the static imaging background 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 sopra the background subtraction used sopra canone mass photometry, and reduces the imaging preparazione at equivalent imaging speeds paio preciso the larger number of frames contributing preciso the retroterra image (Extended Tempo Fig. 1 and Supplementary Fig. 1).
a, Schematic diagram of dynamic mass photometry of protein complexes diffusing https://datingranking.net/it/meetmindful-review/ 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 (ndimer = 34, ntetramer = 85, nhexamer = 184, noctamer = 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. Data are given as the mean localization errors in 2D ± the combined s.d. of the mean errors in x and y of particle trajectories detected during the dynamic mass photometry movie in b (n = 1 movie, 4 min), processed with different amounts of frame averaging (ndimer = 34, 51, 60, 52, 73; ntetramer = 82, 102, 98, 97, 94; nhexamer = 177, 229, 224, 208, 173; noctamer = 22, 29, 37, 38, 33 trajectories for total exposure times of 3.0, 6.0, 9.1, 12.1 and 15.1 ms, respectively). e, Mass trace and histogram of a WT es). f, Corresponding particle trajectory. g, Corresponding cumulative probability of particle displacements during 1 frame (t = 3 ms) and the fits to a two-component model (equation 4). Scale bars, 500 nm.
For the chosen system, the detected particles exhibited clearly differing signal intensities (Fig. 1a, filtered images, and Supplementary Schermo 1). Filtering for trajectories that remained bound preciso the SLB for at least 50 frames, corresponding sicuro verso residence time of 151 ms (Supplementary Fig. 2), and plotting the mean contrast of the remaining 425 trajectories revealed verso contrast distribution with equally spaced peaks, as expected for different oligomeric states (Fig. 1b). The contrast values of these particles increased linearly with mass (Fig. 1c) and matched well with the expected contrasts of WT dimers, tetramers, hexamers and octamers based on norma mass photometry measurements (Extended Momento Fig. 2a,b and Supplementary Table 1), demonstrating that dynamic mass photometry can simultaneously image, track and measure the mass of diffusing biomolecular complexes on SLBs. Additionally, the oligomeric distribution of WT on the SLB displayed a shift to higher oligomeric states compared with the solution distribution measured using canone mass photometry (Extended Data Fig. 2c,d).