Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy
Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy
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DOI：
10.1016/j.mvr.2006.05.003
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发表时间：
2006-07-01
影响因子：
3.1
通讯作者：
Rogers, R. A.
中科院分区：
文献类型：
--
作者：
Dickie, R.;Bachoo, R. M.;Rogers, R. A.
新生微血管网络的三维结构是再生生长、血管病和癌症中血管灌注的关键决定因素。目前用于微血管可视化的方法受限于内皮免疫标记的不充分渗透和不稳定性、用于血管铸造的高粘度聚合物的血管灌注不足以及扫描电子显微镜所需的处理期间组织基质的破坏。本研究的目的是开发整体组织处理方法，用于微血管的3D原位可视化，该方法也与组织微环境中其他感兴趣结构的补充标记兼容。在这里，我们提出的技术，允许成像的微血管共聚焦显微镜，深度达1500 μ m以下的标本表面。我们的方法包括通过静脉注射荧光缀合凝集素标记内皮细胞的管腔表面，并用碳或荧光纳米颗粒/Mercox填充微血管，然后对厚组织切片进行光学清除，以减少光散射，并允许深入样品的微血管形态的3D可视化。值得注意的是，组织间质被保留，允许通过免疫组织化学或核染料同时标记其他结构。结果是各种鼠组织，包括脂肪，肌肉，心脏和大脑在正常健康的条件下，以及在设置的胶质瘤模型生长在皮下空间或原位在脑实质。(c)2006年爱思唯尔公司All rights reserved.
The three-dimensional architecture of the nascent microvascular network is a critical determinant of vascular perfusion in the setting of regenerative growth, vasculopathies and cancer. Current methods for microvessel visualization are limited by insufficient penetration and instability of endothelial immunolabels, inadequate vascular perfusion by the high-viscosity polymers used for vascular casting, and destruction of tissue stroma during the processing required for scanning electron microscopy. The aim of this study was to develop whole-mount tissue processing methods for 3D in situ visualization of the microvasculature that were also compatible with supplementary labeling for other structures of interest in the tissue microenvironment. Here, we present techniques that allow imaging of the microvasculature by confocal microscopy, to depths of up to 1500 mu m below the specimen surface. Our approach includes labeling luminal surfaces of endothelial cells by i.v. injection of fluorescently conjugated lectin and filling the microvasculature with carbon or fluorescent nanoparticles/Mercox, followed by optical clearing of thick tissue sections to reduce light scatter and permit 3D visualization of microvessel morphology deep into the sample. Notably, tissue stroma is preserved, allowing simultaneous labeling of other structures by immunohistochemistry or nuclear dyes. Results are presented for various murine tissues including fat, muscle, heart and brain under conditions of normal health, as well as in the setting of a glioma model growing in the subcutaneous space or orthotopically in the brain parenchyma. (c) 2006 Elsevier Inc. All rights reserved.