Toward fast and deep imaging of living tissue with cellular resolution

以细胞分辨率对活体组织进行快速、深度成像

• 批准号: 10651713
• 负责人: Nozomi Nishimura
• 金额: $ 62.33万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651713
• 关键词: Address; Adoption; Anatomy; Animal Model; Animals; Biological; Biological Process; Biology; Biomedical Research; Blood capillaries; Brain; Brain region; Cancer Biology; Cardiac; Cell Differentiation process; Cell physiology; Cells; Chronic; Color; Data; Databases; Development; Dyes; Face; Fiber; Fluorescence Microscopy; Frequencies; Gases; Generations; Heart; Hippocampus; Image; Imaging Techniques; Immunology; Injury; Intestines; Kidney; Label; Lasers; Location; Mammary gland; Marrow; Measurement; Measures; Microscope; Microscopy; Mus; Muscle; Neurosciences; Operative Surgical Procedures; Organ; Organ Model; Osteocytes; Penetration; Performance; Perfusion; Photons; Physiologic pulse; Preparation; Process; Proteins; Protocols documentation; Research Activity; Resolution; Signal Transduction; Skeletal Muscle; Skin; Source; Structure; System; Techniques; Testing; Time; Tissue imaging; Tissues; Variant; Vision; Visualization; adaptive optics; biological systems; body system; bone; cell motility; cost; daughter cell; design; experimental study; flexibility; fluorophore; frontier; imaging capabilities; improved; in vivo; instrument; intravital imaging; knowledge base; lymph nodes; mechanical load; neural; new technology; next generation; non-invasive optical imaging; novel; programs; scale up; stem cell fate; timeline; tumor; two photon microscopy; two-photon; waveguide

Abstract An exciting recent development for high spatial resolution deep tissue imaging is long wavelength three- photon fluorescence microscopy (3PM). Since its first demonstration of imaging subcortical structures in the mouse brain, 3PM has driven rapid progress in deep tissue imaging beyond the depth limit of two-photon fluorescence microscopy (2PM). Long-wavelength 3PM is perhaps the most promising new technology for deep imaging within scattering biological tissues, and has potential impacts in a large number of biomedical fields such as neuroscience, immunology, and cancer biology. On the other hand, there are a number of challenges that must be overcome before 3PM can reach its full potential. Because it is a higher-order nonlinear process, three- photon excitation (3PE) is inherently weaker than two-photon excitation (2PE). The weak signal strength of 3PM is particularly problematic for fast imaging of dynamic cellular process. Furthermore, the laser sources for 3PM are not yet optimized for deep tissue penetration, and the complexity and cost of the excitation source is a major barrier for the applications of 3PM in a typical biomedical research lab. Finally, nearly all 3PM applications today are in the brains. Reaching anatomical frontiers is equally possible in other organs with 3PM, but explicit demonstrations of intravital imaging in novel locations are needed to bring deep imaging capability to other biological systems. The research activity of this proposal will directly address the above challenges for in vivo deep tissue 3PM. We will develop a new generation of 3PM that will improve the performance of existing 3PM by two orders of magnitude and enable multi-color deep tissue imaging with a single excitation wavelength. We will demonstrate the unprecedented imaging capabilities with a low-cost, fiber-based laser system, removing a key barrier for the deployment of 3PM in biology labs. Furthermore, by applying our techniques to a wide variety of biological systems, we will create a valuable knowledge base for the applications of 3PM. Our development of the next generation 3PM parallels the development of 2PM, where the concerted development effort in lasers, microscopes, and biological applications in the 1990s made 2PM ubiquitous in biomedical research labs by the early 2000s. Our vision is to make deep, fast 3PM a routine instrument for a wide variety of biomedical applications just as 2PM does in the shallower regions of biological tissues and organs. The successful completion of this program will enable visualization of dynamic process at the sub-cellular level in intact organs and animal models that are completely beyond the reach of any existing imaging techniques.

抽象的 高空间分辨率深层组织成像的一项令人兴奋的最新进展是长波长三 光子荧光显微镜（3PM）。自从首次演示皮层下结构成像以来 小鼠大脑，3PM推动深层组织成像突破双光子深度极限 荧光显微镜（2PM）。长波 3PM 或许是深度学习中最有前途的新技术 散射生物组织内成像，并且在许多生物医学领域具有潜在影响，例如 如神经科学、免疫学和癌症生物学。另一方面，也面临着诸多挑战 必须克服 3PM 才能充分发挥其潜力。因为它是一个高阶非线性过程，所以三 光子激发 (3PE) 本质上弱于双光子激发 (2PE)。下午3点信号强度较弱 对于动态细胞过程的快速成像来说尤其成问题。此外，3PM 的激光源 尚未针对深层组织穿透进行优化，并且激励源的复杂性和成本是主要的 3PM 在典型生物医学研究实验室中应用的障碍。最后，今天几乎所有 3PM 申请 都在大脑里。在其他器官中，通过 3PM 达到解剖学前沿同样可能，但明确 需要在新位置进行活体成像演示，以将深度成像能力带到其他领域 生物系统。该提案的研究活动将直接解决体内的上述挑战 深层组织下午 3 点。我们将开发新一代 3PM，以提高现有 3PM 的性能 两个数量级，并能够使用单一激发波长进行多色深层组织成像。我们 将通过低成本光纤激光系统展示前所未有的成像能力，消除了 在生物实验室中部署 3PM 的主要障碍。此外，通过将我们的技术应用于各种 我们将为 3PM 的应用创建一个有价值的知识库。我们的发展 下一代 3PM 的发展与 2PM 的发展并行，其中激光器的协同开发工作， 2PM 在 20 世纪 90 年代的显微镜和生物应用中，在生物医学研究实验室中无处不在 2000年代初。我们的愿景是让深度、快速的 3PM 成为各种生物医学的常规仪器 就像 2PM 在生物组织和器官的较浅区域中的应用一样。成功者 该计划的完成将使完整器官亚细胞水平的动态过程可视化 以及完全超出任何现有成像技术范围的动物模型。