IDBR: Type A Adaptive Optical Structured Illumination Microscope (AOSIM)

University Of California-Santa Cruz, Santa Cruz CA

Investigators

Abstract

An award is made to the University of California Santa Cruz to develop an Adaptive Optical Structured Illumination Microscope (AO-SIM) for in-vivo super-resolution imaging. While super-resolution imaging has revolutionized optical microscopy by overcoming the diffraction limit in far-field imaging, it has yet to transform dynamic live in vivo imaging through thick tissue, which is of broad interest to the biological research community. The development of an AO-SIM in this project is a novel combination of three different imaging techniques that will enable this; structured-illumination to double the resolution from 220 nm to 110 nm, tomography to increase the corrected volume, and a woofer-tweeter adaptive optical system sufficient for an order of magnitude increase in the depth of correction. This capability will be transformative since live super-resolution imaging has only been demonstrated at superficial depths (i.e. 1-10 microns), and there is a clear and broad need for it at deeper depths within the biological research community. Active areas of research using super-resolution microscopy include cellular architecture, membrane heterogeneity and dynamic protein assembly. Specific examples of research that will benefit from the instrument developed in this project include live, deep-tissue in vivo studies of chromosome structure and function during meiosis in C. elegans and the regulation of chromatin structure and gene expression in Drosophila. Both of these research topics can be addressed with the 1 Hz imaging speed targeted for the instrument. The instrument development team is well qualified to conduct the project as they have previously developed the first adaptive optical microscope that used direct wavefront sensing to achieve live, dynamic (0.6 sec AO correction time) diffraction limited (220 nm) imaging in thick (>90 ìm) biological tissues. The approach used for fast direct wavefront measurement in adaptive optical image correction is also applicable to other forms of super-resolution and structured illumination imaging including Stimulated Emission Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), and Selective Plane Imaging Microscopy (SPIM). The instrument development team has access to the required resources including the W.M. Keck Center for Adaptive Optical Microscopy (CfAOM), the UC Center for Adaptive Optics (CfAO), and the UCO/Lick Laboratory for Adaptive Optics (LAO), all located on the UC Santa Cruz campus. This project enables live super-resolution images of samples in difficult to access locations providing new information for a host of biosystems. It also provides training in cutting edge optical science for students at UC Santa Cruz, a Hispanic-Serving Institution (HSI). The research team members are active participants in programs that help sustain our broader impact, providing research experiences to aspiring scientists, with emphasis on serving underrepresented minority students. These include the NSF-funded California State Summer School for Mathematics and Science (COSMOS) program, the Institute for Scientist & Engineer Educators (ISEE), and the NIH-funded Minority Access to Research Careers and Initiative for Maximizing Student Development. COSMOS, a 4-week summer residential program for high school scholars, provides an intensive experience that encourages promising young scholars to continue their interest in science and mathematics. COSMOS students work side-by- side with outstanding researchers and university faculty, covering topics that extend beyond the typical high school curriculum. Our AO microscopy research results are presented regularly at these summer sessions and COSMOS students have been visiting the AO microscopy lab for the last 5 summers. ISEE also supports a new generation of science, technology, engineering and mathematics (STEM) professionals. The program is structured around three major themes: inquiry, diversity and equity, and assessment. ISEE mobilizes current and future STEM professionals to teach and mentor hundreds undergraduates and postgraduates each year. The resulting next-generation instrument will be made available for users at the W.M. Keck Center for Adaptive Optical Microscopy (CfAOM) (http://cfaom.soe.ucsc.edu/), a research center focused on the application of adaptive optics in biological imaging. The results of the project will be presented at conferences and workshops attended by both biologists and instrument development communities. The research results will be published in leading journals, patent applications and in an image gallery (iGallery) on the CfAOM website. Web links will be established to the iGallery from the leading biological microscopy courses and super-resolution vendor technology web pages in order to advertise the new imaging capability to the biological research community. The original vendor of structured illumination microscopes, Applied Precision, a GE Healthcare Company, has expressed an interest in a commercialization partnership to broadly disseminate the AO-SIM once it is successfully developed. This award is being made jointly by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Chemical Measurements and Imaging, in the Division of Chemistry (Math and Physical Sciences Directorate).