Analysis of the three dimensional organization of glia cell processes by X-ray- and high voltage electron -microscopy

利用X射线和高压电子显微镜的优点，我观察了大鼠中枢神经系统不同部分胶质细胞突起的三维组织细节。用X射线显微镜可以观察到厚度超过100的非常厚的样品，甚至在水中，利用高压电子显微镜，可以高分辨率地观察到厚度高达5微米的相当厚的样品。此外，由于X射线显微镜和电子显微镜的焦深很大，可以进行立体观察，从而可以分析从样品深度获得的信息。借助高压电子显微镜，获得了一些重要的新发现。1)灰质中的原浆星形胶质细胞形成致密的三维网络，轴突和树突的终支及其突触嵌入其中。网状结构由薄叶或r…组成。更多厚度小于50 nm的带状结构。这些细枝直接从大枝和细胞体中长出。它们贴靠在毛细血管的外表面，除了由相当厚的星形细胞突起形成的胶质片外，还融合成一层薄的胶质片，通常称为毛细血管末端足。2)光镜下可见星形细胞突起覆盖神经元表面，星形细胞突起覆盖胞体和大树突。网状物的大小与神经末梢的大小大致一致。高压电子显微镜显示覆盖胞体和大树突的一层由多角形隔室或细胞组成的层。细胞壁由一个非常薄的星形细胞突起组成。这些细胞中的每一个都被认为包裹在神经末梢周围。有趣的是，除了神经细胞胞体上的突起外，突起的形状类似于小脑的Bergmann胶质细胞和视网膜的Muller细胞，它们被认为是起源于放射状胶质细胞。3)尽管灰质和白质中纤维状星形胶质细胞的整体形态不同，与原浆星形胶质细胞不同，但其末梢分支的形状原则上相似，呈细小的小叶或带状，与邻近的神经元或血管结构形状相适应。结果发现，高压电子显微镜立体显微镜厚高尔基染色材料是目前观察中枢神经系统胶质细胞突起三维组织细节的最有效和可靠的方法。高尔基染色也与X射线显微镜下的胶质细胞突起形成了足够的对比。X射线显微镜的一大优点是能在一个良好的聚焦图像中观察到100微米厚以上的标本，但其二维分辨率与光学显微镜大致相同，不足以分辨星形胶质细胞精细的末端突起的细节。对X射线显微镜的实际分辨率提出了更高的要求。湿法制备的X射线显微技术将是未来胶质细胞形态研究中的一项有用技术。较少

Taking advantage of the merits of X-ray- and high voltage electron-microscopy, I examined details of the three dimensional organization of glia cell processes in various parts of the rat central nervous system.With the X-ray microscope one can observe very thick specimens of over 100 in thickness and even in water, and with the high voltage electron microscope, a high resolution observation of rather thick specimens of up to 5mum in thickness is possible. In addition , stereo observation can be done because the depth of focus of the X-ray microscope and electron microscope is very large, and in this way the information obtained from the depth of the specimens can be analyzed.Several important new findings were obtained with the aid of high voltage electron microscopy. 1) The protoplasmic astrocyte in the gray matter formed a dense three dimensional network in which terminal branches of axons and dendrites, and their synapses were embedded. The network was composed of thin leaflets or r … More ibbon-like structures of less than 50 nm in thickness. These fine branches arose directly from large branches and cell soma. They abutted on the outer surface of the capillary and fused to form a thin glial sheet in addition to that formed by rather thick astrocytic processes generally called capillary end feet. 2) The surfaces of the neurons studded with many nerve terminals are covered by astrocytic processes which formed networks covering the somata and large dendrites as seen with the light microscope. The sizes of the mesh roughly coincide with those of nerve terminals. High voltage electron microscopy revealed a layer consisting of polygonal compartments or cells which covered the soma and large dendrites. The walls of cells were consisted of a very thin astrocytic process. Each of these cells was supposed to wrap around a nerve terminal. It is interesting to note that the shapes of the processes other than those on the nerve cell somata resemble to those of the Bergmann glia cell in the cerebellum and Muller cell in the retina which are thought to originate from the radial glia cells. 3) Although global shapes of the fibrous astrocytes in the gray matter and white matter differed to each other and differed from those of the protoplasmic astrocytes, the shapes of their terminal branches were similar in principle, thin and fine leaflets or ribbonlike, and they seemed to adapt themselves to the shapes of neighboring neuronal or vascular structures.It was found that high voltage electron microscope stereoscopy of thick Golgi stained materials was the most effective and reliable method of examining the detail of the three dimensional organization of glia cell processes in the central nervous system at present.Golgi staining also gave enough contrast to glia cell processes for X-ray microscopy. Capability of observing over 100 mum thick specimen in one good focused image was a large merit of the X-ray microscope, however it's two dimensional resolution was about the same to that of the light microscope and was not high enough to resolve the details of fine terminal processes of the astrocytes. Improvement in practical resolution of the X-ray microscope is highly requested. X-ray microscopy of wet preparation will be an useful technique in glia cell morphology in the future. Less