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

我们使用蔡司SIGMA-VP扫描电子显微镜和Gatan 3View系统应用连续块面扫描电子显微镜（sgf -SEM）来测量描述朗格汉斯胰岛结构的参数，朗格汉斯胰岛是大小约200至300微米的微观内分泌器官，分泌胰岛素和胰高血糖素来控制血糖。通过分析整个小鼠胰岛，我们发现可以确定(1)α和β细胞的分布，(2)血管和毛细血管周围间隙的组织，以及(3)单个分泌细胞的超微结构。我们的研究结果表明，β细胞的平均体积几乎是α细胞的两倍，线粒体的总体积大约是α细胞的四倍。相反，发现两种细胞类型的核体积大致相等。虽然α和β分泌颗粒的核心直径相似，但β颗粒有明显的光晕，导致其总直径是α颗粒的两倍。血管可视化显示胰岛的每个分泌细胞都与毛细血管间隙接触，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。由于胰岛结构复杂，用透射电镜薄切片很难达到这样的精度。

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.