Three-dimensional Nonlinear Structured Illumination for Live Imaging with 80 nm resolution

Project Abstract This proposal seeks to develop a microscope capable of live imaging with 80 nm lateral resolution through the development of Three-dimensional Nonlinear Structured Illumination Microscopy. Single Molecule Localization Microscopy can offer unprecedented resolution down to 10 nm but requires 10,000 to 100,000 raw images and, therefore, is not amenable to fast live imaging. Stimulated Emission Depletion Microscopy can achieve ~ 50 nm resolution in live cells and rapid imaging over small (~1 µm2) fields of view but because it is a point scanning technique becomes too slow when the field of view increases. STED also requires high intensities reducing its attractiveness for live imaging. RESOLFT microscopy and associated techniques have shown promising results, achieving ~ 80 nm resolution in live samples but are also slow because they require lateral sample scanning. Structured Illumination Microscopy (SIM) is an attractive approach for live super- resolution imaging because it requires relatively few raw images, no lateral scanning of the sample is required, and the excitation intensity can be low. Linear SIM can achieve ~ 120 nm resolution and live imaging has been demonstrated in both two dimensions and three dimensions with the fastest frame rates for 2D SIM over 50 frames per second. Nonlinear SIM can theoretically achieve unlimited resolution and imaging with 40 nm resolution has been demonstrated in two dimensions. Live two-dimensional NSIM has been demonstrated at 2.8 frames per second with 62 nm resolution. Three-dimensional Nonlinear SIM has yet to be demonstrated. The development of live 3D NSIM with 2D frame rates exceeding 4 frames per second will fill a need for high-resolution live imaging over large fields of view up to ~ 100 µm × 100 µm, allowing cellular structures such as filopodia, vesicles, and mitochondria to be resolved in three-dimensions. To accomplish this we will develop a new Structured Illumination Pattern generator optical design and novel Adaptive Optics Techniques to achieve the best optical performance allowing fast aberration-free imaging in cells and thin tissue sections.