Super-resolution imaging provides the ability to measure (i.e., resolve) objects that are smaller than the ~200 nanometer resolution limit of visible light. This can provide more detailed examinations into cellular metabolism and subcellular compartments, potentially leading to new advances in renewable energy production from microbial and plant sources. To date, however, super-resolution has been restricted to fixed (i.e., not living) samples processed in a particular manner, while single-molecule (SM) live imaging is possible only for very limited periods of time due to photodamage incurred by high-intensity illumination. Furthermore, out-of-focus light restricts the depth within a sample at which SM imaging can be performed. This SBIR/STTR Phase II project aims to combine SM super-resolution microscopy with a patented Light Sheet Fluorescence Microscopy (LSFM) technology that enables the use of high-resolution objective lenses previously incompatible with LSFM. LSFM is both a more photon-efficient method of illumination (less photodamage) and an effective means of limiting out-of-focus light by only illuminating the in-focus specimen plane. While the technology that forms the basis of this proposal is the only LSFM technology that is natively compatible with the high-resolution lenses necessary for SM imaging, proper fluorophore behavior in SM imaging requires significantly more laser power than traditional LSFM demands. In order to achieve these higher powers, the Phase I project successfully re-engineered the illumination path to pair it with stronger lasers and generate a narrower light sheet, concentrating power previously wasted outside the field of view. The resulting prototype used a series of custom focusing mirrors and achieved SM imaging in live cells for an order of magnitude longer duration without phototoxicity, as well as super-resolution of fixed cells. The proposed Phase II work involves building an improved second-generation prototype, demonstrating the ability to achieve 3D dSTORM and 3D SM tracking, and building several prototypes to send to alpha users for data collection and user feedback. The illumination method of the resulting system will increase the longevity of samples, broaden compatibility of fluorophores with SM imaging (including low-abundance labels and metabolic probes), and enable super-resolution of structures deeper in cells, thereby opening up new experimental space for SM imaging.