Tracking and counting single miRNAs using iSHiRLoC. a Psuedocolored, live U2OS cell microinjected with miR-let-7a-1-Cy5 and imaged 4 h post-injection; miRNP particles exhibit very slow (i), corralled (ii), fast (iii), and biased (iv) Brownian diffusion. b Distribution of diffusion coefficient for let-7a-1-Cy5 at various time points. c Pseudocolored, fixed U2OS cell microinjected with miR-let-7a-1-Cy5 and imaged 4 h post-injection (top) and photobleaching steps detected by iSHiRLoC (bottom). d Particle counting time course reveals assembly of multimeric let-7a-1-Cy5 particles (top), but not cxcr4-Cy5 particles (middle). Co-injection of cxcr4-Cy5 with an mRNA target promotes assembly of multimeric cxcr4-Cy5 particles (bottom). e Model established using iSHiRLoC data of miRNA-mediated translational repression and degradation (Pitchiaya et al. 2012)