Scanning Transmission Electron Tomography of Biological Structures

传统的亮场电子层析成像倾斜序列是通过收集穿过宽束照射的样品的电子来获得的。使用这种方法，厚度受到严重的图像模糊的限制，当经历了多次能量损失的电子被显微镜的物镜聚焦时发生的图像模糊。此外，图像的最大面积受到物镜景深的限制，因此在大倾角时只有一部分样品对焦。 使用带有紧密聚焦电子探针的STEM进行层析重建可以克服使用传统的透射电子显微镜进行层析重建所带来的一些限制。首先，因为入射的STEM探头可以在样本中的任何点聚焦，所以即使在高倾角的情况下，也可以对大片区域进行聚焦成像。其次，因为在STEM中，样品之后没有成像透镜，所以在厚样品的图像中可以获得的分辨率不会因为遭受了多次能量损失的电子而进一步降低。最常用的STEM方法利用环形暗场探测器来收集被散射到高角的电子。然而，由于入射电子探针的大会聚角定义了有限的景深，所以暗视野STEM技术不太适合于厚生物样品的成像。通过调整显微镜光学元件，将会聚半角减小到大约12mrad，可以使景深增加10倍或更高。应用于厚样品成像的暗场STEM的另一个限制特征是，由于光束展宽，向截面底部表面发生的空间分辨率严重下降。相反，我们发现，通过只收集那些被散射到低角度的电子，也就是使用轴向亮场探测器，可以获得更高的空间分辨率。经历多次弹性散射的电子基本上偏离STEM探测器的入射点，平均具有更大的净散射角。因此，这些电子中的很大一部分可以从用轴向探测器记录的图像中排除，从而提高了对厚样品底部表面的空间分辨率。我们用蒙特卡罗电子轨迹模拟量化了这种意想不到的分辨率提高。 我们应用轴向明场STEM断层扫描技术，从小鼠朗格汉斯胰岛重建了1微米厚的未染色的塑料包埋的胰岛β细胞切片。这些重建使我们能够在整个标本体积中可视化β细胞的特征，如胰岛素颗粒、线粒体和高尔基体堆积。这种对细胞体积的更全面的观察提供了细胞器组织的信息，这是传统的断层扫描方法所不能获得的。 我们还使用这项技术对培养的大鼠海马片中的突触脊椎进行了可视化。这是第一次有可能可视化整个突触后的密度，并评估当某些重要蛋白质，如PSD-95被击倒时，超微结构的差异。在其他实验中，描述大鼠视网膜中完整的带状突触并显示这些结构内分泌囊泡的精确组织是可行的。 因此，我们证明了STEM使用轴向探测技术以5~10 nm的空间分辨率成像厚切片的可行性和优势，这与传统的薄层电子断层扫描(通常为3~8 nm)的空间分辨率相当。大多数现代电子显微镜可以在STEM模式下操作，并且可以很容易地配备亮场探测器，预计这将有助于该技术的实施。到目前为止，常规薄层电子断层成像的大规模应用解决了对生物样品的高分辨率、大体积成像的需求。我们目前的工作表明，通过连续的厚切片断层扫描重建完整的细胞器、细胞内病原体甚至整个哺乳动物细胞是可能的。

Conventional bright-field electron tomographic tilt series are obtained by collecting electrons that have traversed a specimen illuminated by a broad beam. Using this approach, the thickness is limited by the severe image blurring that occurs when electrons that have undergone multiple energy losses are focused by the objective lens of the microscope. Furthermore, the maximum area of the image is limited by the depth-of-field of the objective lens, so that only part of the sample is in focus at high tilt angles. Tomographic reconstruction using STEM with a tightly focused electron probe can overcome some of the limitations imposed by tomographic reconstruction using conventional TEM. First, because the incident STEM probe can be focused at any point in a specimen, large areas are imaged in focus even for high tilt angles. Second, because in STEM there are no image-forming lenses after the specimen, the resolution attainable in images of thick specimens is not further degraded by electrons that have suffered multiple energy losses. The most commonly applied STEM approach makes use of an annular dark-field detector to collect electrons that are scattered to high angles. However, the dark-field STEM technique is not well-suited to imaging thick biological specimens because of the limited depth of field defined by the large convergence angle of the incident electron probe. A tenfold or higher increase in depth of field is possible by adjusting the microscope optics to decrease the convergence semi-angle to approximately 12 mrad. Another limiting feature of dark-field STEM as applied to imaging thick specimens is the severe degradation in spatial resolution that occurs toward the bottom surface of a section because of beam broadening. In contrast, we found that much higher spatial resolution can be obtained by collecting only those electrons that are scattered to low angles, that is, by using an axial bright-field detector. Electrons that undergo multiple elastic scattering are substantially displaced from the point of incidence of the STEM probe and have, on average, larger net scattering angles. A large fraction of these electrons can thus be excluded from images recorded with an axial detector, leading to an improvement in spatial resolution toward the bottom surface of thick specimens. We quantified this unexpected improvement in resolution using Monte Carlo electron-trajectory simulations. We have applied axial bright-field STEM tomography to reconstruct one-micrometer thick unstained sections of plastic-embedded beta cells from mouse pancreatic islets of Langerhans. The reconstructions have enabled us to visualize features in the beta cells, such as insulin granules, mitochondria and Golgi stacks, throughout the entire specimen volume. This more comprehensive view of the cell volume provides information about the organization of organelles, which is not attainable using conventional tomography approaches. We have also used the technique to visualize synaptic spines in cultured slices of rat hippocampus. It has been possible for the first time to visualize entire post-synaptic densities and to assess differences in ultrastructure that occur when certain important proteins such as PSD-95 are knocked down. In other experiments, it has been feasible to characterize entire ribbon synapses in rat retina and to visualize the precise organization of secretory vesicles within those structures. Thus we have demonstrated the feasibility and advantages of STEM using axial detection for imaging thick sections at a spatial resolution around 5 to 10 nm, which is comparable to the spatial resolution of conventional electron tomography from thinner sections (typically 3 to 8 nm). Most modern electron microscopes can be operated in STEM mode and can be readily equipped with a bright-field detector, which is anticipated to facilitate implementation of the technique. The demand for high-resolution, large-volume imaging of biological specimens has been addressed so far by the large-scale application of conventional electron tomography of thin sections. Our current work suggests that it will be possible to reconstruct intact organelles, intracellular pathogens and even entire mammalian cells through serial thick-section tomography.