Nature and Nanotechnology likewise employ nanoscale machines that self-assemble into structures of complex architecture and functionality. Fluorescence microscopy offers a non-invasive tool to probe and ultimately dissect and control these nanoassemblies in real-time. In particular, single molecule fluorescence resonance energy transfer (smFRET) allows us to measure distances at the 2-8 nm scale, whereas complementary super-resolution localization techniques based on Gaussian fitting of imaged point spread functions (PSFs) measure distances in the 10 nm and longer range. Here, I will describe a method for the intracellular single molecule, high-resolution localization and counting (iSHiRLoC) of microRNAs (miRNAs), a large group of gene silencers with profound roles in our body, from stem cell development to cancer. Microinjected, singly-fluorophore labeled, functional miRNAs were tracked as individual diffusing particles inside single human cells. Observed mobility and mRNA dependent assembly changes suggest the existence of two kinetically distinct assembly/disassembly processes, thus bringing into focus a unifying molecular mechanism for the ubiquitous RNA silencing pathway. In addition, I will describe how we have utilized super-resolution fluorescence microscopy to monitor walks of molecular spider nanorobots on tracks defined by programmable DNA scaffolds called origami, one molecule/particle at a time; how we can “PAINT” individual tracks to characterize their idiosyncrasies at super-resolution; and how we can use DNA origami as “rafts” to deliver loads to and probe the basal membrane of single human cells.