Three dimensional analysis of dendrites of the CNS neurons.

我们开发了一种从高压电子显微镜立体图像中定量提取三维信息的方法。为了提高结果的精度，我们在日立HVEM H-1250中引入了一个自由旋转变焦光学系统和一个侧面进入旋转倾斜台，倾斜角度有反馈控制机构。该显微镜还配备了一个图像增强器与实时图像处理器相结合，以减少光束对试样的损伤。利用该系统对大鼠齿状回颗粒细胞树突棘的数量、长度和直径进行了测量。采用5 m厚的高尔基体进行测量。在树突近端(P)、中端(m)和远端(d)部分分别获得3.4、2.3和2.0个/ 1mu m的脊柱密度，是以往光镜报道的1.6-2.0倍。由于树突直径从近端到远端逐渐减小，因此在整个树突树中，每单位树突表面积的棘密度大致相等，为0.8/ μ m^2。棘的形状各异，有花萼形、蘑菇形、棒状、细线状等，但光镜下报道的大而短的棘占20- 40%，不足2%。在p、m和d部分，平均三维脊柱长度分别为1.25、1.26和1.24 μ m，约为二维测量的1.4倍。利用形态学参数测量了棘的表面积，发现在每个枝晶部分加入棘后，枝晶的总表面积增加了一倍。很明显，光学显微镜的分辨率不足以精确测量树突棘的形态。

We have developed a method of extracting three dimensional information quantitatively from high voltage electron microscope stereo images. To improve the accuracy of the result, we introduced into Hitachi HVEM H-1250 a rotation free zoom optical system and a side entry rotation tilt stage with a feed back control mechanism for the tilt angle. The microscope is also equipped with an image intensifier in combination with a real time image processor to reduce the beam damage to the specimen.Number, length and diameter of dendritic spines of the granule cell in the rat dentate gyrus were measured with the aid of this system. 5 m thick Golgi preparations were used for the measurement.Spine densities of 3.4, 2.3, and 2.0 per 1mu m at proximal (P), middle (m), and distal (d) portion of the dendrite respectively were obtained, which were 1.6-2.0 fold of the previous light microscopical report. Since the diameter of the dendrite decreases from proximal to distal portion, the spine densities per unit surface area of the dendrite was about equal, 0.8/mu m^2, throughout the dendritic tree.Spines of various shapes , calyx shape,mushroom shape, club shape , thin thread-like etc. were found , however the large stubby spines which were reported to be 20-40 % by light microscopy were rare, less than 2%. Mean three dimensional spine length were 1.25mu m, 1.26mu m, and 1.24 mu m at p, m, and d portions respectively, which were about 1.4 times longer than those measured in two dimensions.The surface area of the spines were measured using morphological parameters , and it was found that the total surface areas of the dendrite was doubled by the addition of the spines at each dendritic portion.It became evident that the resolution of the light microscope was not sufficient for the precise morphometry of the dendritic spine.