This supplement will critically update the instrumentation available for the parent R35 MIRA award, entitled The RNA nanomachines of gene expression dissected at the single molecule level. The central features of the proposed instrumentation, the AbbeLight SAFe M90 plug-in imaging module, are its versatility, turnkey readiness, and ease of use for automated, multicolor, optimized total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO) imaging of live and fixed mammalian cells. These features will not only increase the information content and throughput of our measurements, but also greatly facilitate access to these methods by the diverse group of junior and senior coworkers in the PI's group. Funding will critically complement two recent NIH investments into the parent grant. First, we used a recent administrative supplement to install a Cytosurge FluidFM OMNIUM, a versatile, turnkey instrument for injecting, sampling, and manipulating the content of live mammalian cells. Second, we will use funds from the parent grant to update our 15-year old objective-type TIRF microscope (O-TIRFM) body for our intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) technology. Our MIRA grant aims to dissect the mechanisms of the nanoscale RNA machines of gene expression at the single molecule level, so far focusing on bacterial riboswitches and the yeast spliceosome. Combining the high-performance, ultra-wide-field, multicolor capabilities and automation of the AbbeLight SAFe M90 imaging module with the upgrade of the O-TIRFM body and the FluidFM OMNIUM semi-automated cell injector will transform our ability to incorporate studies of the intracellular miRNA-guided silencing machinery into the parent grant. Building on our group's 25-year expertise in this space, we then aim to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs; and 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms, realizing opportunities for unprecedented discoveries where individual RNA nanomachines interact. In pursuit of these aims, we will address the unifying hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, as exemplified by the fact that transient miRNA:mRNA interactions lead to the regulation of protein translation in mammalian cells. To address this hypothesis, we have developed a suite of intracellular single particle tracking approaches wherein we inject fluorophore labeled RNAs at defined time points and image them at the single molecule level with high spatiotemporal resolution and multiple colors, an approach whose multiplexing and throughput will be drastically expanded by integrating the AbbeLight SAFe...