Longitudinal Imaging of Cortical Small Vessel Network Structure with Two-Color Multiphoton Fluorescence Microscopy

项目摘要 虽然2型糖尿病在外周器官的小血管后果是相当好的 尽管糖尿病对脑微血管系统的影响在很大程度上是未知的。一 理解这些变化的一个重要障碍是技术挑战 与体内小血管网络拓扑的完整三维映射相关。 技术挑战在于在毛细血管尺度上捕获血管形态的能力， 也跨越整个皮质厚度并延伸到皮质下区域。进一步 使技术挑战复杂化的是，船舶网络的基线变化很大 结构和对动物病理扰动的反应，因此血管网络必须 纵向跟踪以捕捉这些动态。因此，本提案的目标是 通过开发新的光学显微镜技术来克服这些技术限制， 完整的3D映射的整个皮质微血管网络结构在体内，并使用 这些技术可以量化与类型相关的脑微血管系统的慢性改变， 2糖尿病小鼠我们将介绍双光子和三光子荧光的新进展 显微镜，这将显着提高我们的能力，成像微血管网络在体内， 在小鼠皮层的大空间范围内，深度高达1.5 mm。我们将开发新的空间 偏移双色多光子激发方法利用了长波长的最新进展， 同步超快激光器皮质和皮质下血管的大尺度图像将 允许血管网络的完全3D矢量化，这将使详细分析 随着时间的推移，微血管形态。我们将使用这种先进的体内 显微镜、新型超快激光和血管网络的计算分析， 2型糖尿病相关脑微血管结构改变。

Project Summary Although the small vessel consequences of type 2 diabetes in peripheral organs are fairly well understood, the effects of diabetes on cerebral microvasculature are largely unknown. A significant impediment to understanding these changes has been the technical challenges associated with complete three-dimensional mapping of small vessel network topology in vivo. The technical challenge lies in the ability to capture vessel morphology on the capillary scale while also spanning the entire cortical thickness and extending into the subcortical regions. Further complicating the technical challenge is the fact that vessel networks vary significantly in baseline structure and response to pathological perturbations across animals, thus vessel networks must be followed longitudinally to capture these dynamics. Therefore, the goal of this proposal is to overcome these technical limitations by developing new optical microscopy techniques that permit complete 3D mapping of the entire cortical microvascular network structure in vivo, and to use these techniques to quantify chronic alterations in cerebral microvasculature associated with type 2 diabetes in mice. We will introduce new advances to two- and three-photon fluorescence microscopy that will significantly improve our ability to image microvascular networks in vivo at depths up to 1.5 mm over large spatial extents of mouse cortex. We will develop a new spatially offset two-color multiphoton excitation approach that utilizes recent advances in long wavelength, synchronized ultrafast lasers. The large-scale images of cortical and sub-cortical vasculature will allow full 3D vectorization of vascular networks that will enable detailed analysis of the microvascular morphology over time. We will use this combination of advanced in vivo microscopy, novel ultrafast lasers, and computational analysis of vascular networks to quantify the structural changes in cerebral microvasculature associated with type 2 diabetes.