Scanning Transmission Electron Tomography of Biological Structures

生物结构的扫描透射电子断层扫描

基本信息

批准号：
7967905
负责人：
Richard Leapman
金额：
$ 10.18万
依托单位：
NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
依托单位国家：
美国
项目类别：
财政年份：
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/7967905
关键词：
Address Area Biocompatible Materials Biological Cell Nucleus Cell membrane Cells Cleaved cell Complex Cytoplasm Detection Development Dimensions Electron Microscope Electrons Elements Endoplasmic Reticulum Erythrocyte Membrane Erythrocytes Eukaryotic Cell Event Food Goals Gold Golgi Apparatus Human Image Immunoglobulin Fragments Incidence Label Lipids Malaria Mammalian Cell Membrane Methods Microscope Microtomy Morphogenesis Optics Organelles Parasites Plasmodium falciparum Polymers Procedures Process Proteins Resolution Sampling Scanning Scanning Transmission Electron Microscopy Procedures Series Slice Specimen Staining method Stains Structure Surface Techniques Thick Tomogram Tubular formation Vacuole Work base detector electron tomography interest lens nanoGold nanoparticle pathogen reconstruction rhoptry simulation three-dimensional modeling tomography transmission process

项目摘要

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.

用附着在抗体片段上的重原子簇进行标记是一种有吸引力的技术，可在电子显微镜下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来检测。我们利用大角度环形暗场探测器和小角度轴向明场探测器开发了一种基于定量扫描透射电子显微镜（STEM）的技术。使用暗场技术，我们证明可以在厚度约为 100 nm 的细胞中检测到 11 个金原子簇和包含约 67 个金原子的纳米金簇。 STEM 断层扫描有可能使用带有小重原子簇标记的抗体片段来定位透化细胞中的特定蛋白质。我们的定量分析提供了一个框架，用于确定检测限和定位这些小簇的最佳实验条件。我们还研究了 STEM 断层扫描在真核细胞较厚切片成像中的应用。传统的明场电子断层扫描倾斜系列是通过收集穿过宽光束照射的样本的电子来获得的。使用这种方法，厚度受到严重图像模糊的限制，当经历多次能量损失的电子被显微镜的物镜聚焦时，会发生严重的图像模糊。此外，图像的最大区域受到物镜景深的限制，因此只有部分样品在高倾斜角度下聚焦。使用带有紧密聚焦电子探针的 STEM 进行断层扫描重建可以克服使用传统 TEM 进行断层扫描重建所带来的一些限制。首先，由于入射 STEM 探针可以聚焦在样本中的任何点，因此即使在高倾斜角度下也能对大面积区域进行聚焦成像。其次，由于在 STEM 中，样本后面没有成像透镜，因此厚样本图像中可达到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高角度散射的电子。然而，由于入射电子探针的大会聚角所限定的景深有限，暗场 STEM 技术不太适合对厚生物样本进行成像。通过调整显微镜光学器件将会聚半角减小至约 12 mrad，可以将景深增加十倍或更高。暗场 STEM 应用于厚样品成像的另一个限制特征是，由于光束展宽，截面底部表面的空间分辨率会严重下降。相比之下，我们发现通过仅收集那些散射到低角度的电子，即使用轴向明场探测器，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从 STEM 探针的入射点位移，并且平均而言具有更大的净散射角。因此，这些电子的很大一部分可以从轴向探测器记录的图像中排除，从而提高厚样本底部表面的空间分辨率。我们使用蒙特卡罗电子轨迹模拟量化了分辨率的这种意外提高。受感染红细胞的断层扫描切片显示在分裂过程中存在寄生虫。由于从连续薄片进行传统 3D 重建的过程非常费力，人们对恶性疟原虫细胞器发生和形态发生的动态知之甚少。然而，使用基于 STEM 的断层扫描和轴向探测器可以更快速地重建整个裂殖体，从而可以研究一系列细胞并建立形态事件的序列。来自断层扫描的 3D 模型揭示了几个主要细胞器的空间排列，包括细胞核、菱形、食物泡、高尔基复合体、顶质体和脂质体。还观察到了一堆我们认为迄今为止尚未报道的内质网。除了这些细胞器外，裂殖体周围的三层膜也清晰可辨：寄生虫质膜、寄生虫液泡膜和红细胞膜。其他寄生虫衍生的膜结构（例如，液泡膜的管状延伸、毛雷氏裂和圆形裂）在红细胞胞质内也可见。因此，现在可以使用一种新的超微结构方法来研究人类红细胞内疟原虫发育的复杂动态。因此，我们证明了 STEM 使用轴向检测以 510 nm 左右的空间分辨率对厚切片进行成像的可行性和优势，这与较薄切片（通常为 38 nm）的传统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，并且可以轻松配备明场探测器，预计这将有助于该技术的实施。迄今为止，传统薄片电子断层扫描的大规模应用已经满足了生物样本高分辨率、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将是可能的。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。