Abstract：
Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.
摘要：
止血,血管损伤的正常生理控制过程，是人类生活的基础。我们都不时遭受轻微的割伤和刺伤。在止血方面，自限性血小板聚集导致形成结构化血栓，其中出血停止来自于从外部盖住孔。这种结构的详细表征可能导致止血和血栓形成之间的区别，一例血小板过度聚集导致闭塞性凝血的病例。这里提出了一种基于成像的穿刺伤口血栓结构的方法，该方法利用了薄切片电子显微镜可视化止血血栓内部的能力。任何基于成像的实验方案中最基本的步骤是良好的样品制备。该方案提供了在小鼠中准备穿刺伤口和富含血小板的血栓以进行后续电子显微镜检查的详细程序。给出了详细的程序，用于原位固定形成的穿刺伤口血栓及其后续处理，以进行电子显微镜染色和包埋。电子显微镜由于其能力而被认为是最终成像技术，当与顺序切片相结合时，以高分辨率可视化血栓内部的细节。作为一种成像方法，电子显微镜给出了无偏采样和实验输出，尺度从纳米到毫米在2或3维。引用了适当的免费软件电子显微镜软件，该软件将支持广域电子显微镜，其中可以混合数百个框架，以对整个穿刺伤口血栓横截面进行纳米级成像。因此，图像文件的任何子区域都可以轻松地放置到完整横截面的上下文中。
