Optical histology: a method to visualize microvasculature in thick tissue sections of mouse brain.
Optical histology: a method to visualize microvasculature in thick tissue sections of mouse brain.
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DOI：
10.1371/journal.pone.0053753
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Choi B
中科院分区：
文献类型：
--
作者：
Moy AJ;Wiersma MP;Choi B
微脉管系统是参与递送组织存活所必需的营养物和气体的血管网络。微血管的研究通常涉及免疫组织学方法。虽然免疫组织学可用于可视化特定感兴趣区域的微米尺度的微血管，但它并不适合可视化器官中的整体微血管结构。因此，免疫组织学的使用排除了器官的整个微脉管系统的可视化，并且因此阻碍了对与由于各种疾病状态引起的组织变化一致发生的微脉管系统的整体变化的研究。因此，迫切需要一种简单、相对快速的技术，以便于整个组织的微血管网络的可视化。使用称为“血管涂染”的方法，用荧光亲脂性染料DiI对小鼠的全身血管系统进行染色。收获脑或其他感兴趣的器官并固定在4%多聚甲醛中。然后将器官切成1 mm的切片，并使用专有的光学透明剂FocusClear进行光学透明或使其透明。在光学透明化之后，使用共焦荧光显微镜对DiI标记的组织微脉管系统进行成像，并且将相邻的图像堆叠平铺在一起以产生组织切片中的微脉管系统的深度编码图。我们证明了使用光学透明增强了组织成像深度和血管密度的估计。使用我们的“光学组织学”技术，我们可视化了小鼠大脑中850 µm深度的微血管。这里展示的是小鼠大脑1 mm厚切片中的微血管图。使用组合的光学透明和光学成像技术，我们设计了一种方法来增强厚组织中微血管的可视化。我们相信这项技术可能被用于生成整个器官中微血管的三维地图。
The microvasculature is the network of blood vessels involved in delivering nutrients and gases necessary for tissue survival. Study of the microvasculature often involves immunohistological methods. While useful for visualizing microvasculature at the µm scale in specific regions of interest, immunohistology is not well suited to visualize the global microvascular architecture in an organ. Hence, use of immunohistology precludes visualization of the entire microvasculature of an organ, and thus impedes study of global changes in the microvasculature that occur in concert with changes in tissue due to various disease states. Therefore, there is a critical need for a simple, relatively rapid technique that will facilitate visualization of the microvascular network of an entire tissue. The systemic vasculature of a mouse is stained with the fluorescent lipophilic dye DiI using a method called “vessel painting”. The brain, or other organ of interest, is harvested and fixed in 4% paraformaldehyde. The organ is then sliced into 1 mm sections and optically cleared, or made transparent, using FocusClear, a proprietary optical clearing agent. After optical clearing, the DiI-labeled tissue microvasculature is imaged using confocal fluorescence microscopy and adjacent image stacks tiled together to produce a depth-encoded map of the microvasculature in the tissue slice. We demonstrated that the use of optical clearing enhances both the tissue imaging depth and the estimate of the vascular density. Using our “optical histology” technique, we visualized microvasculature in the mouse brain to a depth of 850 µm. Presented here are maps of the microvasculature in 1 mm thick slices of mouse brain. Using combined optical clearing and optical imaging techniques, we devised a methodology to enhance the visualization of the microvasculature in thick tissues. We believe this technique could potentially be used to generate a three-dimensional map of the microvasculature in an entire organ.
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