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

通过串行块面 SEM 进行三维细胞和组织重建

• 批准号: 9361491
• 负责人: Richard Leapman
• 金额: $ 53.17万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9361491
• 关键词: Agreement; Alpha Cell; Alpha Granule; Architecture; Area; Beta Cell; Big Data; Blood Glucose; Blood Vessels; Caliber; Cell Volumes; Cell membrane; Cell surface; Cells; Cytoplasmic Granules; Data; Data Set; Diamond; Dimensions; Electrons; Endocrine Glands; Face; Glucagon; Hybrids; Image; Individual; Insulin; Islets of Langerhans; Measurement; Measures; Microscopic; Microtomy; Mitochondria; Monte Carlo Method; Mus; Nuclear; Organelles; Population; Resolution; Scanning; Scanning Electron Microscopy; Secretory Cell; Secretory Vesicles; Slice; Specimen; Staging; Staining method; Stains; Surface; System; Techniques; Testing; Thick; Time; Tissues; Transmission Electron Microscopy; blood vessel visualization; cell fixing; cell type; density; electron tomography; improved; islet; nanometer; nanoscale; reconstruction; tissue reconstruction; transmission process

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.

我们使用蔡司 SIGMA-VP SEM 和 Gatan 3View 系统应用串行块面扫描电子显微镜 (SBF-SEM) 来测量描述胰岛结构的参数，胰岛是尺寸约为 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 α 细胞的两倍，线粒体总体积约为 α 细胞的四倍。相比之下，两种细胞类型的核体积大致相等。尽管α和β分泌颗粒的核心直径相似，但β颗粒具有明显的光环，导致总直径是α颗粒的两倍。血管可视化显示，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，通过分析少量胰岛可以获得一致的结果。由于胰岛结构复杂，使用薄片 TEM 无法轻松实现如此精确的精度。 通过串行块面扫描电子显微镜（SBF-SEM）对组织体积超微结构进行 2D 和 3D 分析相结合，可以大大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，或细胞内细胞器群体所包围的总体积，我们已经证明，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分析随机选择的细胞 2D 切片，并通过在连续切片中描绘质膜，将此类估计与 3D 细胞体积的精确测量结合起来。 由于 SBF-SEM 数据集中提供了整个 3D 组织体积，因此可以轻松测试这种方法的有效性。 我们应用这种混合 3D/2D 技术来确定小鼠朗格汉斯胰岛的 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。 这些结果与测量值一致。 目前，SBF-SEM 垂直于块面的空间分辨率仅限于约 25 纳米，其最小切片厚度可使用 SEM 样品台内置的超薄切片机去除。 我们对块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光束能量的反向散射图像来探测块内不同的次表面深度来获得亚 25 纳米 z 分辨率。 结果表明，通过组合一次能量在 1 keV 至 3.5 keV 之间的电子的两个或多个反向散射图像，可以实现约 10 纳米的 z 分辨率。 我们已经在明确的测试样本上测试了这种能力，现在正在应用该技术来确定具有改进的 z 分辨率的细胞超微结构。