MRI: Development of an Improved Scanned Light Sheet Microscope for Rapid, High-Volume, Three-Dimensional Fluorescence and Dark-Field Microscopies

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Imaging the complete three-dimensional architecture of living, growing organisms with sub-cellular resolution and sufficient speed to capture cellular dynamics has long been an unattainable goal. Traditional approaches are sufficient for a few hundred cells, but are too slow and cause too much photodamage to probe larger systems, such as whole embryos. Recently, researchers have developed a new methodology, "light sheet microscopy" (LSM), in which illumination by a scanned sheet of light that excites fluorescent probes allows rapid, high resolution 3D imaging with minimal photodamage. This technique can acquire 3D images of entire developing zebrafish embryos with sub-cellular resolution for over 24 hours, creating one embryo-spanning 3D image every 60-90 seconds. This approach promises to revolutionize biological imaging.This award is supporting a project with two main goals. The first goal aims to build a light sheet microscope at the University of Oregon, the first of its kind in the United States. This instrument will be invaluable to researchers studying zebrafish and other models of biological phenomena. It will illuminate the connections between cell migration and skeletal development in early embryogenesis; allow organism-wide mapping of interactions between bacterial populations and host immune cells, a key factor in normal as well as diseased physiology; enable studies of how particular mutations affect the development of the enteric nervous system over large length and time scales; and more. The second goal aims to extend the capabilities of light sheet microscopy, creating the next generation of light sheet microscopes. This project will develop (1) an instrument capable of 3D dark-field imaging, which will be especially important for visualizing non-fluorescent nanoparticle distributions in whole, living organisms, crucial to addressing concerns about nanotechnological toxicology; and (2) an instrument capable of combined fluorescence LSM and differential interference contrast microscopy, which will reveal the local context in which cells expressing particular fluorescent proteins act.The broader impacts of this proposal have three facets. First, rapid 3D imaging will enable unprecedented advances in fields spanning the biological sciences, as noted above, and also the physical sciences, for example the structure and dynamics of soft materials. Second, the project will provide valuable cross-disciplinary training for postdoctoral researchers, graduate students, and undergraduates. Third, the visually striking nature of the 3D data resulting from LSM imaging are ideally suited to education and outreach, as they transform the illustration of topics like embryogenesis and self-assembly from static or schematic cartoons to vibrant and "real" 3D movies. Data from the proposed project will be incorporated into a new course on biophysics for non-science-major college undergraduates and a week-long day camp for socioeconomically disadvantaged high school students. The project outcome-a functional light sheet microscope with dark-field and differential interference contrast imaging capabilities-will be available at the University of Oregon (Eugene, OR).