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

长期以来，以亚细胞分辨率和足够的速度捕捉细胞动力学，对生长的生物的完整三维结构进行成像一直是一个无法实现的目标。传统的方法对于几百个细胞来说已经足够了，但是对于探测更大的系统，比如整个胚胎来说，速度太慢并且会造成太多的光损伤。最近，研究人员开发了一种新的方法，“光片显微镜”（LSM），在这种方法中，通过扫描光片激发荧光探针的照明，可以在最小的光损伤下实现快速，高分辨率的3D成像。该技术可以在24小时内以亚细胞分辨率获取整个发育中的斑马鱼胚胎的3D图像，每60-90秒创建一个跨越胚胎的3D图像。这种方法有望彻底改变生物成像。该奖项支持一个有两个主要目标的项目。第一个目标是在俄勒冈大学建造一个薄片显微镜，这是美国第一个这样的显微镜。这个仪器对于研究斑马鱼和其他生物现象模型的研究人员来说是非常宝贵的。它将阐明早期胚胎发生中细胞迁移与骨骼发育之间的联系；允许绘制细菌种群与宿主免疫细胞之间相互作用的全生物图谱，这是正常和患病生理学的关键因素；使特定突变如何在长时间尺度上影响肠神经系统发育的研究成为可能；和更多。第二个目标是扩展薄片显微镜的功能，创造下一代薄片显微镜。该项目将开发(1)一种能够进行三维暗场成像的仪器，这对于可视化整个生物体中的非荧光纳米颗粒分布尤其重要，对于解决纳米技术毒理学问题至关重要；(2)一种能够结合荧光LSM和差干涉对比显微镜的仪器，它将揭示表达特定荧光蛋白的细胞的局部环境。这项提议的更广泛影响有三个方面。首先，快速3D成像将使跨越生物科学领域的前所未有的进步，如上所述，以及物理科学，例如软材料的结构和动力学。其次，该项目将为博士后、研究生和本科生提供宝贵的跨学科培训。第三，由LSM成像产生的3D数据的视觉冲击力非常适合教育和推广，因为它们将胚胎发生和自组装等主题的插图从静态或原理漫画转变为充满活力和“真实”的3D电影。拟议项目的数据将被纳入针对非科学专业大学本科生的生物物理学新课程，以及针对社会经济弱势高中生的为期一周的日间夏令营。该项目的成果——一种具有暗场和微分干涉对比成像能力的功能性薄片显微镜——将在俄勒冈大学（Eugene， OR）上市。

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).