TD-iSCAT image processing

Even with the enhanced contrast in SBR-iSCAT imaging, it is still difficult to identify intracellular cargos in the cytoplasmic space full of unidentified subcellular objects with similar shapes and contrasts. To capture dynamic cargos from the complex cytoplasmic landscape, we used the time-differential (TD)-iSCAT image processing method (Supplementary Fig. ^{2b, d}) instead, highlighting dynamic cellular features by suppressing quasi-static iSCAT signals from relatively stationary cellular substance. First, we obtained a temporal-median cell image from 20 consecutive frames (Δt = 0.4 s) taken at 50 Hz. By dividing a temporal-median image by another such image taken after the time interval (ΔT) of 0.2 s, a TD-iSCAT image was obtained. In the image, individual cargos which move faster than 200 nm/s appear as a pair of dark and bright spots, indicating the early and late positions in the time interval averaged over for each image (Δt = 0.4 s). In contrast, other static or randomly moving cellular entities fade or disappear due to signal cancellation. Cargos slower than 200 nm/s would be self-canceled because their signals fall in the same pixel (1 pixel ~ 40 nm) within the time interval of 0.2 s. In the cases cargos backtrack, however, they would be counted multiple times.