Intracellular single molecule microscopy reveals time and mRNA dependent microRNA assembly

Supplementary Figure 1 Precision and signal intensity of live cell imaging, and cxcr4 control experiments Supplementary Figure 2 Tracking the diffusion of individual PBs in live HeLa cells Supplementary Figure 3 Live cell images after incubation for varying amounts of time after microinjection Supplementary Figure 4 Relative deviation (RD) analysis of diffusion coefficients over time Supplementary Figure 5 Stepwise photobleaching in fixed cells Supplementary Figure 6 Representative images and resulting stepwise photobleaching distributions from fixed cells incubated for various amounts of time after microinjection Supplementary Figure 7 miRNAs assembly is mRNA dependent Supplementary Video legend 1 Videos of both Cy3 and Cy5 labeled miRNAs diffusing in living HeLa cells Supplementary Video legend 2 Different types of motion exhibited by miRNAs in living HeLa cells Supplementary Video legend 3 Live cell imaging control experiments Supplementary Video legend 4 Pseudo-colored video showing photobleaching of let-7-a1-Cy5 miRNAs in fixed cells, imaged 2 hrs after injection Supplementary Table 1 Number of cells and particles used in both live and fixed cell analysis Supplementary Figure S1 │ Precision and signal intensity of live cell imaging, and cxcr4 control experiments. (A) Precision of single particle localization. Histogram depicting the displacement of formaldehyde-fixed let-7-a1-Cy5 particles about their origin (n = 100 randomly selected particles). The location of each particle was tracked over time until it photobleached. The histogram was fit with a Gaussian function, which resulted in a mean of ~30 nm and a standard deviation of ~50 nm. Large jumps (>100 nm) were observed only when there were large fluctuations of intensity. Localization precision of immobilized beads (inset) was ~4 nm, largely owing to their higher signal-to-noise ratio (n = 100 randomly selected particles). (B) Signal intensities of particles undergoing diffusion in living cells. Intensity time trajectories of let-7-a1-Cy3 (green) and let-7-a1-Cy5 (red) particles are shown. Trajectories of both mobile and static particles are shown, as indicated in each trajectory. A significant proportion of particles had a relatively constant intensity during diffusion before disappearing. We infrequently observed large fluctuations in intensity as a particle was diffusing, possibly due to diffusion in the axial dimension (z-axis). We also occasionally observed stepwise changes in intensity of static particles, possibly due to photobleaching (see, for example, second trajectory from the top on the left side). (C-E) Intracellular diffusion of cxcr4 miRNA, with (C) DIC image and (D) the corresponding pseudocolored image of a cell microinjected with cxcr4-Cy3 and imaged …