Toward fast and deep imaging of living tissue with cellular resolution

摘要 高空间分辨率深部组织成像的一个令人兴奋的最新发展是长波长三- 光子荧光显微镜(下午3点)。自从它首次展示了大脑皮质下结构的成像以来 小鼠大脑，下午3点推动深部组织成像突破双光子深度限制取得快速进展 荧光显微镜(下午2：00)。长波长3 PM可能是最有前途的深度探测新技术 在分散的生物组织内进行成像，并在许多生物医学领域具有潜在的影响 如神经科学、免疫学和癌症生物学。另一方面，存在许多挑战， 必须在下午3点充分发挥其潜力之前克服。因为它是一个高阶的非线性过程，三- 光子激发(3PE)本质上弱于双光子激发(2PE)。下午3点的微弱信号强度 对于动态细胞过程的快速成像尤其有问题。此外，下午3点的激光光源 还没有针对深层组织渗透进行优化，而激发源的复杂性和成本是主要的 3 PM在典型生物医学研究实验室应用的障碍。最后，今天几乎所有下午3点的应用程序 都在大脑里。在下午3点到达解剖边界的其他器官也是同样可能的，但明显 需要展示新位置的活体成像，才能将深度成像能力带给其他 生物系统。这项提案的研究活动将直接解决体内的上述挑战 深部组织下午3点我们将开发新一代3 PM，以改进现有3 PM的性能 可提高两个数量级，并可使用单一激发波长进行多色深层组织成像。我们 将使用低成本、基于光纤的激光系统展示前所未有的成像能力，从而消除 生物实验室部署下午3点的关键障碍。此外，通过将我们的技术应用于各种 对于生物系统，我们将为3pm的应用创造一个有价值的知识库。我们的发展 下一代3PM的发展与2PM的发展平行，在激光方面的共同开发努力， 显微镜和20世纪90年代的生物学应用使2 PM在生物医学研究实验室中无处不在 21世纪初。我们的愿景是让深度、快速的3 PM成为各种生物医学的常规仪器 就像2 PM在生物组织和器官的较浅区域所做的那样。成功者 这一计划的完成将使完整器官在亚细胞水平上的动态过程可视化 和动物模型，这些模型完全超出了任何现有成像技术的范围。

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.