Scanning Transmission Electron Tomography of Biological Structures

结合抗体片段的重原子簇标记技术是利用电子显微镜中的电子断层扫描技术来确定细胞中特定蛋白质的三维分布的一种有吸引力的技术。然而，标签的小尺寸使得传统的亮场电子断层扫描很难检测到它们。我们利用大角度环形暗场探测器和小角度轴向亮场探测器，发展了一种基于定量扫描电子显微镜(STEM)的技术。使用暗场技术，我们已经证明了在厚度约为100 nm的细胞中可以检测到包含大约67个金原子的11-金原子团和纳金原子团。 干细胞断层扫描有可能通过用小的重原子团标记的抗体片段来定位渗透性细胞中的特定蛋白质。我们的定量分析为确定探测极限和定位这些小星团的最佳实验条件提供了一个框架。 我们还研究了STEM断层扫描技术在真核细胞厚切片成像中的应用。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，因此Drak场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。 感染红细胞的断层图像切片显示，在精神分裂症的过程中有寄生虫。恶性疟原虫器官发生和形态发生的动力学还不是很清楚，因为传统的从连续薄层切片进行三维重建的繁琐过程。然而，使用基于STEM的断层扫描和轴向探测器，可以更快地重建整个裂殖体，这使得可以研究一系列细胞并建立形态事件的序列。由断层图像得到的3D模型揭示了几个主要细胞器的空间排列，包括核、棒状体、食物液泡、高尔基复合体、质外体和脂体。我们还观察到了迄今为止未见报道的内质网堆叠。除了这些细胞器外，裂殖体周围还有三层膜：寄生虫的质膜、寄生虫的液泡膜和红细胞膜。其他寄生虫来源的膜结构(例如，空泡膜的管状延伸、毛瑞斯裂隙和圆形裂隙)也在红细胞胞浆内可见。因此，一种新的超微结构方法现在可以用来研究疟疾寄生虫在人红细胞内发展的复杂动力学。 因此，我们已经证明了STEM使用轴向探测以510 nm左右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38 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。基于轴向STEM的层析成像也可以用于多相聚合物、生物材料和其他软材料的三维表征。

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.