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

MRI：开发用于快速、大容量、三维荧光和暗场显微镜的改进扫描光片显微镜

• 批准号: 0922951
• 负责人: Raghuveer Parthasarathy
• 金额: $ 45.85万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0922951&HistoricalAwards=false
• 关键词: MRI; Development; Improved; Scanned; Light

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).

长期以来，以亚细胞分辨率和足够的速度捕捉细胞动力学，对活的、生长的生物体的完整三维结构进行成像一直是一个遥不可及的目标。传统的方法足以检测几百个细胞，但速度太慢，对探测更大的系统，如整个胚胎来说，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，通过激发荧光探针的扫描光片的照明，可以在最小光损伤的情况下进行快速、高分辨率的3D成像。这项技术可以获取整个发育中的斑马鱼胚胎的亚细胞分辨率超过24小时的3D图像，每60-90秒创建一张跨越胚胎的3D图像。这一方法有望给生物成像带来革命性的变化。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台光片显微镜，这是美国第一个此类显微镜。这台仪器对研究斑马鱼和其他生物现象模型的研究人员来说将是无价的。它将阐明早期胚胎发生中细胞迁移和骨骼发育之间的联系；允许在整个生物体范围内绘制细菌种群和宿主免疫细胞之间相互作用的图谱，这是正常和疾病生理学中的一个关键因素；使人们能够研究特定突变如何在大范围和时间尺度上影响肠道神经系统的发育；以及更多。第二个目标是扩展光片显微镜的能力，创造下一代光片显微镜。该项目将开发(1)一种能够进行3D暗视野成像的仪器，这对于可视化非荧光纳米颗粒在整个活体中的分布尤其重要，这对于解决对纳米技术毒理学的担忧至关重要；(2)一种能够结合荧光LSM和差分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。首先，如上所述，快速3D成像将使跨越生物科学和物理科学的领域取得前所未有的进步，例如软材料的结构和动力学。其次，该项目将为博士后研究人员、研究生和本科生提供有价值的跨学科培训。第三，LSM成像产生的3D数据在视觉上令人惊叹，非常适合教育和推广，因为它们将胚胎发生和自我组装等主题的插图从静态或概略的卡通片转变为充满活力的“真实”3D电影。拟议项目的数据将被纳入为非理科本科生开设的生物物理学新课程，以及为社会经济困难的高中生举办的为期一周的日营。该项目的成果--具有暗场和差分干涉对比成像能力的功能性光片显微镜--将在俄勒冈大学(Ougene，OR)面市。