Research Project
9139049
? DESCRIPTION (provided by applicant): Biology is intricately organized at the nanoscale, yet its functional elements, such as the neuronal networks in the brain, often span over distances of centimeters. Ideally, tools for observing these networks would provide high resolution over large volumes. We propose to meet this challenge by developing 3D Tessellation Imaging (3DTI), a new super-resolution microscopy, and combining it with the properties of newly-published Expansion Microscopy (ExM) specimen preparations. The combined technology offers 10×10×10 nm 3D resolution. Potentially, this isotropic high resolution can be achieved in a single exposure per plane- an order of magnitude faster than other super-resolution methods. 3DTI exploits mathematical symmetries to create sparse, crystalline interference patterns. These 3D struc- tured illumination patterns probe ?uorescently-labeled structures within a transparent specimen. Unlike certain other super-resolution techniques, 3DTI is compatible with an essentially arbitrary selection of ?uorescent labels, enabling many-color ?uorescent images. ExM physically augments the imaging potential of tissue samples. It increases the spatial extent of the sample and makes it easier to see structures that were previously too close together or too small to observe. At the same time, ExM converts the sample into a hydrogel which is almost entirely transparent (but with ?uorescently-labeled locations of interest). In this way, EM provides both a scalable increase in resolution and a means to observe deeper into tissue volumes. In this program, we will: 1. Build and validate a 1-color, 1-objective 3DTI super-resolution prototype. 2. Develop an image processing pipeline for raw data from the 3DTI prototype. 3. Demonstrate the powerful synergy of 3DTI with ExM specimens by creating impressive 3D brain tissue imagery unattainable by any other method. The outcome of these efforts will establish a solid foundation for the continued development of a 3DTI instru- ment that will provide a high-resolution brain mapping and tissue mapping tool for biological discovery.
