Three-Dimensional Cell and Tissue Reconstruction by Serial Block Face SEM

我们应用蔡司Sigma-VP扫描电子显微镜和Gatan 3View系统的连续块面扫描电子显微镜(SBF-SEM)测量了描述朗格汉斯胰岛结构的参数，这些内分泌器官大小约200至300微米，分泌胰岛素和胰升糖素以控制血糖。通过对整个小鼠胰岛的分析，我们表明可以确定(1)α和β细胞的分布，(2)血管和毛细血管周围空间的组织，以及(3)单个分泌细胞的超微结构。我们的结果表明，β细胞的平均体积几乎是阿尔法细胞的两倍，线粒体的总体积大约是阿尔法细胞的四倍。相反，发现两种细胞的核体积大致相等。虽然阿尔法和贝塔分泌颗粒的核心直径相似，但贝塔分泌颗粒有明显的光晕，导致其总直径是阿尔法颗粒的两倍。血管显影显示胰岛内各分泌细胞均与毛细血管周围间隙接触，平均接触面积为细胞表面积的9.5%。我们的数据表明，通过分析少量的胰岛可以得到一致的结果。由于胰岛的结构复杂，使用薄层切片的透射电子显微镜很难达到这样的精度。 通过序列块面扫描电子显微镜(SBF-SEM)获得的组织体积超微结构的2D和3D分析相结合，可以极大地缩短从包含数十亿体素的大数据集中获取定量信息所需的时间。因此，为了分析特定类型的细胞器的数量，或者被细胞内的细胞器群体包围的总体积，我们已经表明，使用体视学方法来分析穿过细胞的随机选择的2D切片，并通过描绘连续切片中的质膜来将这种估计与3D细胞体积的精确测量结合起来，是可能的。这种方法的有效性可以很容易地测试，因为整个3D组织体积可以在SBF-SEM数据集中获得。我们应用这种3D/2D混合技术测定了小鼠朗格汉斯胰岛α细胞和β细胞中分泌颗粒的数量，并已能够估计β细胞的总胰岛素含量。这些结果与实测值相吻合。 目前，垂直于块面的SBF-SEM的空间分辨率被限制在大约25纳米，这是可以使用内置在扫描电子显微镜样品台中的超微切割器去除的最小切片厚度。我们对块体表面内的电子轨迹进行了蒙特卡罗模拟，以确定是否有可能通过记录不同束流能量下的背向散射图像来探测块体内不同的亚表面深度，从而获得低于25纳米的z分辨率。结果表明，通过组合两个或更多初级能量在1keV到3.5keV之间的电子的背向散射图像，获得大约10纳米的z分辨率是可行的。我们已经在定义明确的测试样本上测试了这种能力，现在正在应用这项技术来确定细胞的超微结构，并提高了z分辨率。

We have applied serial block-face scanning electron microscopy (SBF-SEM) using a Zeiss SIGMA-VP SEM and a Gatan 3View system to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs about 200 to 300 micrometers in size, which secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9.5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections. A combination of 2D and 3D analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy (SBF-SEM) can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyze the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, we have shown that it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyze randomly selected 2D slices through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive slices. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the SBF-SEM data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in alpha and beta cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of beta cells. These results are in agreement with measured values. The spatial resolution of SBF-SEM normal to the block face is currently limited to approximately 25 nanometers by the minimum slice thickness that can be removed using the ultramicrotome that is built into the SEM's specimen stage. We have carried out Monte Carlo simulations of electron trajectories within the block face to determine whether it is possible to obtain sub-25 nanometer z-resolution by recording backscattered images at different beam energies to probe different sub-surface depths within the block. Results show the feasibility of achieving a z-resolution of around 10 nanometers by combining two or more backscattered images for electrons with primary energy between 1 keV and 3.5 keV. We have tested this capability on well-defined test specimens, and are now applying the technique to determine cellular ultrastructure with improved z-resolution.