Scanning Transmission Electron Tomography of Biological Structures

用附着在抗体片段上的重原子簇标记是一种有吸引力的技术，用于在电子显微镜中使用电子断层扫描来确定细胞中特定蛋白质的3D分布。然而，标签的小尺寸使得它们很难通过传统的明场电子断层扫描检测到。 我们已经开发了一种基于定量扫描透射电子显微镜（STEM）的技术，通过使用一个大角度的环形暗场探测器和一个小角度的轴向明场探测器。 使用暗场技术，我们已经证明，有可能检测到11个金原子团簇和纳米金团簇，其中包含约67个金原子的细胞被切成约100 nm的厚度。 STEM断层扫描具有使用用小的重原子簇标记的抗体片段在透化细胞中定位特定蛋白质的潜力。我们的定量分析提供了一个框架，用于确定检测限和最佳实验条件定位这些小集群。 我们还研究了应用STEM断层扫描成像较厚的真核细胞部分。 传统的明场电子层析倾斜系列通过收集已经穿过由宽束照射的样品的电子来获得。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透镜聚焦时，会发生这种模糊。此外，图像的最大区域受到物镜透镜的景深的限制，使得在高倾斜角下仅部分样品聚焦。 使用具有紧密聚焦的电子探针的STEM的层析成像重建可以克服使用常规TEM的层析成像重建所施加的一些限制。首先，因为入射STEM探头可以聚焦在样本中的任何点，所以即使对于高倾斜角，也可以在焦点处对大区域进行成像。其次，因为在STEM中，在样品之后没有成像透镜，所以厚样品图像中可获得的分辨率不会因遭受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散射到高角度的电子。然而，由于入射电子探针的大会聚角所限定的有限景深，所以Drak-field STEM技术不太适合于对厚生物样本进行成像。通过调整显微镜光学器件以将会聚半角减小到约12 mrad，可以使景深增加十倍或更高。另一个限制功能的暗场干成像厚标本是严重的退化空间分辨率发生在一个部分的底部表面，因为光束展宽。相比之下，我们发现，更高的空间分辨率可以通过收集只有那些被散射到低角度的电子，也就是说，通过使用轴向亮场探测器。 经历多次弹性散射的电子基本上从STEM探针的入射点移位，并且平均而言具有更大的净散射角。因此，这些电子的一大部分可以被排除在轴向探测器记录的图像，从而提高空间分辨率朝向厚标本的底表面。我们使用Monte Carlo电子轨迹模拟量化了这种意想不到的分辨率提高。 感染红细胞的断层切片显示寄生虫在寄生过程中。恶性疟原虫的细胞器发生和形态发生的动力学知之甚少，因为传统的三维重建从连续薄切片的费力的过程。然而，使用具有轴向探测器的基于STEM的断层扫描能够更快速地重建整个细胞，这允许研究一系列细胞并建立形态学事件的序列。由断层图像得到的三维模型揭示了几种主要细胞器的空间排列，包括细胞核、棒状体、食物泡、高尔基复合体、顶质体和脂体。 我们认为是迄今未报道的内质网堆栈也被观察到。 除了这些细胞器，三层膜包围的寄生虫是明确可识别的：寄生虫质膜，寄生虫空泡膜，和红细胞膜。其他寄生虫衍生的膜结构（例如，空泡膜的管状延伸，Maurers裂隙和圆形裂隙）也可见于红细胞胞质内。 因此，一个新的超微结构的方法，现在可用于研究疟疾寄生虫发展的复杂动力学内的人红细胞。 因此，我们已经证明了STEM使用轴向检测以约510 nm的空间分辨率对厚切片进行成像的可行性和优势，这与来自较薄切片（通常为38 nm）的常规电子断层扫描的空间分辨率相当。 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明场检测器，这有望促进该技术的实施。到目前为止，对生物样本的高分辨率、大体积成像的需求已经通过对薄切片的常规电子层析成像的大规模应用而得到解决。 我们目前的工作表明，它将有可能重建完整的细胞器，胞内病原体，甚至整个哺乳动物细胞，通过连续厚切片断层扫描。基于轴向STEM的断层扫描也可用于多相聚合物、生物材料和其他软材料的3D表征。

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography in the electron microscope. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. We have developed a technique based on quantitative scanning transmission electron microscopy (STEM) by making use of a large-angle annular dark-field detector and a small-angle axial bright-field detector. Using the dark-field technique, we have demonstrated that it is possible to detect 11-gold atom clusters and Nanogold clusters containing approximately 67 gold atoms in cells that are sectioned to a thickness of around 100 nm. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters. We have also investigated the application of STEM tomography to image thicker sections of eukaryotic cells. 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 drak-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. Tomogram slices of infected erythrocytes revealed parasites during the process of schizogany. The dynamics of organellogenesis and morphogenesis in Plasmodium falciparum are poorly understood because of the laborious procedure of conventional 3D reconstructions from serial thin sections. Using STEM-based tomography with an axial detector, however, enables more rapid reconstruction of entire schizonts, which allows a series of cells to be studied and the sequence of morphological events to be established. The 3D model derived from the tomograms revealed the spatial arrangements of several major organelles, including nuclei, rhoptries, food vacuole, Golgi complex, apicoplast and lipid body. Stacks of what we believe to be hitherto unreported endoplasmic reticulum were also observed. In addition to these organelles, three layers of membranes surrounding the schizont were clearly identifiable: parasite plasma membrane, the parasitophorus vacuole membrane, and the erythrocyte membrane. Other parasite-derived membrane structures (for example, tubular extensions of the vacuolar membrane, Maurers clefts and circular clefts) were also visible inside erythrocyte cytoplasm. Thus, a new ultrastructural method is now available to study the complex dynamics of malaria parasite development inside human erythrocytes. Thus we have demonstrated the feasibility and advantages of STEM using axial detection for imaging thick sections at a spatial resolution around 510 nm, which is comparable to the spatial resolution of conventional electron tomography from thinner sections (typically 38 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. Axial STEM-based tomography could also be useful for the 3D characterization of multiphase polymers, biomaterials and other soft materials.