Lateral Interference Tilted Excitation (LITE)

Research Institution

University of North Carolina at Chapel

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 STTR Phase I project aims to combine SM super-resolution microscopy with a patented Light Sheet Fluorescence Microscopy (LSFM) technology that uniquely 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 illumination path needs to be paired with stronger lasers and re-engineered to generate a narrower light sheet, which will concentrate power currently wasted outside the field of view. This will be achieved using a series of custom focusing mirrors. The proposed work will involve the optical design and prototyping work necessary to establish a functional prototype, as well as evaluation of its performance using a variety of biological samples. In future work, this proof of concept will be leveraged for entry into the market as a first-of-its-kind super-resolution light sheet instrument. The illumination method of the 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.