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

我们已经应用了使用Zeiss SIGMA-VP SEM和Gatan 3View系统的连续块面扫描电子显微镜（SBF-SEM）来测量描述胰岛结构的参数，胰岛是尺寸约200至300微米的微观内分泌器官，其分泌胰岛素和胰高血糖素以控制血糖。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空间的组织，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，而线粒体的总体积大约是α细胞的四倍。相比之下，发现两种细胞类型中的核体积近似相等。虽然α和β分泌颗粒的核心具有相似的直径，但β颗粒具有显著的晕圈，导致总直径是α颗粒的两倍。血管的可视化显示，胰岛中的每个分泌细胞都与毛细血管周围空间接触，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，使用薄切片的TEM不容易达到这样的精度。 通过连续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分析的组合可以大大缩短从包含数十亿体素的大数据集获得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，或者细胞内由细胞器群体包围的总体积，我们已经表明，可以使用体视学方法来估计该细胞器的数量密度或体积分数，以分析通过细胞随机选择的2D切片，并通过在连续切片中描绘质膜来将这种估计与3D细胞体积的精确测量联合收割机相结合。 这种方法的有效性可以很容易地测试，因为整个3D组织体积是在SBF-SEM数据集。 我们应用这种混合3D/2D技术来确定小鼠胰岛α和β细胞中分泌颗粒的数量，并能够估计β细胞的总胰岛素含量。 这些结果与实测值一致。 SBF-SEM垂直于块面的空间分辨率目前被最小切片厚度限制为约25纳米，该最小切片厚度可以使用内置于SEM的试样台中的超薄切片机去除。 我们已经进行了Monte Carlo模拟的块面内的电子轨迹，以确定是否有可能获得子25纳米的z分辨率，通过记录在不同的光束能量的背散射图像，以探测不同的子表面深度内的块。 结果表明，通过结合两个或多个背散射图像的电子与1千电子伏和3.5千电子伏之间的初级能量，实现约10纳米的z分辨率的可行性。 我们已经在定义明确的测试样本上测试了这种能力，现在正在应用该技术以提高z分辨率来确定细胞超微结构。

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.