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相当好 据了解，糖尿病对脑微血管的影响在很大程度上是未知的。一个 理解这些变化的主要障碍是技术挑战 与活体小血管网络拓扑的完整三维标测相关联。 技术挑战在于在毛细血管尺度上捕捉血管形态的能力，同时 也横跨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，船舶网络在基线方面差异很大 跨动物的结构和对病理扰动的反应，因此血管网络必须 被纵向跟踪以捕捉这些动态。因此，这项建议的目标是 通过开发新的光学显微镜技术来克服这些技术限制 在活体内完成整个皮质微血管网络结构的3D测绘，并使用 这些技术用于量化与分型相关的脑微血管慢性改变 2糖尿病小鼠模型。我们将介绍双光子和三光子荧光的新进展 显微镜将显著提高我们在活体内成像微血管网络的能力 深度可达1.5毫米，覆盖大范围的小鼠皮质。我们将开发一种新的空间 利用长波的最新进展的偏置双色多光子激发方法， 同步超快激光。皮质和皮质下血管系统的大尺度图像将 允许血管网络的全3D矢量化，这将使详细分析 微血管形态随时间的变化。我们将在体内使用这种先进的组合 显微镜、新型超快激光和量化血管网络的计算分析 与2型糖尿病相关的脑微血管结构改变。

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.