Scanning Transmission Electron Tomography of Biological Structures

生物结构的扫描透射电子断层扫描

• 批准号: 10701551
• 负责人: Richard Leapman
• 金额: $ 152.52万
• 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/10701551
• 关键词: 3-Dimensional; A kinase anchoring protein; Area; Biological; Brain; Cell Nucleus; Cell membrane; Cells; Complex; Cryoultramicrotomy; Cysteine; Data; Dendritic Spines; Diffuse; Dose; Drug Metabolic Detoxication; Electron Microscope; Electron Microscopy; Electron energy loss spectroscopy; Electrons; Elements; Endosomes; Exocytosis; Face; Fluorescence; Freeze Drying; Freeze Substitution; Freezing; Golgi Apparatus; Hippocampus (Brain); Image; Ions; Knowledge; Label; Laboratories; Manganese; Measurement; Measures; Methods; Microscope; Microscopy; Microtome - medical device; Modernization; Mutate; Mutation; N-terminal; Neurons; Neurosciences; Optics; Organelles; Photons; Proteins; Rattus; Regulation; Reporting; Resolution; Roentgen Rays; Sampling; Scanning; Scanning Electron Microscopy; Series; Serine; Signaling Protein; Slice; Source; Specimen; Stains; Structure; Subcellular structure; Surface; Synapses; Synaptic plasticity; Techniques; Testing; Thick; Time; Tissues; Transmission Electron Microscopy; Vertebral column; Vesicle; Visualization; Work; X ray spectroscopy; base; brain magnetic resonance imaging; density; detector; electron tomography; fluorescence imaging; frontier; imaging approach; imaging modality; implementation facilitation; interest; knock-down; lens; nanoprobe; nanoscale; novel; palmitoylation; postsynaptic; presynaptic density protein 95; reconstruction; sample fixation; three dimensional structure; tomography; trafficking; transmission process; uptake

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 caused by chromatic aberration of the objective lens, which occurs when electrons undergoing multiple energy losses are focused into different planes by the objective of the microscope. Furthermore, the maximum area of the sample that can be imaged 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 1 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. Stain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including STEM tomography as well as serial blockface electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). We have developed a method to determine the stain density in conventionally prepared plastic sections, based on some straightforward measurements in the TEM. Numbers of stain atoms per unit volume are computed from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. We have used STEM tomography 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. We have also applied bright-field axial STEM tomography to deduce the structural arrangement of A-kinase anchoring protein (AKAP79/150), which helps organizes signaling proteins controlling synaptic plasticity in mammalian brain. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains cysteine residues that are reversibly palmitoylated. Cysteine-to-serine mutations abolishing palmitoylation reduce its endosomal localization and association with the postsynaptic density (PSD). Thick-section STEM tomography revealed more AKAP immunogold-labeled clusters corresponding to endosomes in spines for wild-type AKAP than for cysteine-to-serine mutated AKAP, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Our data suggests that palmitoylation fine-tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have important effects on its regulation of synaptic plasticity (Chen et al., Frontiers in Synaptic Neuroscience, in press). We have demonstrated the feasibility and advantages of axial STEM tomography for imaging thick sections at a spatial resolution of 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 bright-field detectors, which is expected to facilitate implementation of the technique. Our current work shows that it is feasible to reconstruct 1- to 2-micrometer thick volumes of any tissue type that is prepared by fixation and embedding, and larger volumes can be reconstructed by serial thick-section STEM tomography. Correlative microscopy has become an essential technique for determining the relationship between structure and function in cells and tissues on the scale of subcellular organelles and supramolecular assemblies. We have used this approach to correlate images acquired in the transmission electron microscopy with x-ray fluorescence image acquired at the Advanced Photon Source, at Argonne National Laboratory. This workflow has allowed us to localize specific diffusible ions and hence help to elucidate the mechanism for uptake of manganese ions in Mn-enhanced magnetic resonance imaging of brain, a technique that enables the tracing of neuronal connections. Specimens of organotypic rat hippocampal slices were prepared by slam-freezing, and frozen cryosections were cryo-transferred into our TEM operating at a beam energy of 300 keV. Correlative images have provided evidence that divalent manganese ions are concentrated in Golgi vesicles surrounding cell nuclei, consistent with previously reported studies in which exocytosis of these vesicles provides a detoxification mechanism to remove the element from cell bodies. In other images it has been possible to correlate concentrations of manganese in synaptic structures within the hippocampal slices.

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，厚度受到物镜色差引起的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物镜聚焦到不同平面时，就会发生这种情况。此外，样品可成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到大约 1 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。 染色密度是优化从多种 3D 电子显微镜技术获得的超微结构数据质量的重要参数，这些技术包括 STEM 断层扫描以及串行块面电子显微镜 (SBEM) 和聚焦离子束扫描电子显微镜 (FIB-SEM)。我们开发了一种方法，根据 TEM 中的一些简单测量来确定传统制备的塑料切片中的染色密度。每单位体积的染色原子数是根据包含纯嵌入材料和嵌入的感兴趣生物结构的样本区域的亮场强度的测量比率计算的。该确定仅需要了解切片厚度（可以通过切片机设置或低剂量电子断层扫描来估计）以及用于染色样本的重原子的弹性散射截面。 我们使用 STEM 断层扫描来观察培养的大鼠海马切片中的突触棘。首次能够可视化整个突触后密度，并评估当某些重要蛋白质（如 PSD-95）被敲低时出现的超微结构差异。我们还应用明场轴向 STEM 断层扫描来推断 A 激酶锚定蛋白 (AKAP79/150) 的结构排列，该蛋白有助于组织控制哺乳动物大脑中突触可塑性的信号蛋白。 AKAP79/150 通过其 N 端结构域与质膜和内体结合，该结构域包含可逆棕榈酰化的半胱氨酸残基。废除棕榈酰化的半胱氨酸到丝氨酸突变减少了其内体定位以及与突触后密度（PSD）的关联。厚切片 STEM 断层扫描显示，与半胱氨酸至丝氨酸突变的 AKAP 相比，野生型 AKAP 中有更多对应于棘中内体的 AKAP 免疫金标记簇，这与内体运输中 AKAP 棕榈酰化的要求一致。 我们的数据表明，棕榈酰化可微调 AKAP79/150 在树突棘中的纳米级定位、移动性和运输，这可能对其突触可塑性的调节产生重要影响（Chen 等人，突触神经科学前沿，出版中）。 我们已经证明了轴向 STEM 断层扫描以 5 至 10 nm 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 3 至 8 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，这有望促进该技术的实施。我们目前的工作表明，通过固定和包埋制备的任何组织类型可以重建 1 至 2 微米厚的体积，并且可以通过连续厚切片 STEM 断层扫描重建更大的体积。 相关显微镜已成为在亚细胞器和超分子组装体规模上确定细胞和组织结构与功能之间关系的基本技术。我们使用这种方法将透射电子显微镜采集的图像与阿贡国家实验室先进光子源采集的 X 射线荧光图像关联起来。该工作流程使我们能够定位特定的可扩散离子，从而有助于阐明在脑部锰增强磁共振成像中摄取锰离子的机制，这是一种能够追踪神经元连接的技术。通过猛烈冷冻制备器官型大鼠海马切片样本，并将冷冻冷冻切片冷冻转移到我们在 300 keV 束能量下运行的 TEM 中。相关图像提供的证据表明，二价锰离子集中在细胞核周围的高尔基体囊泡中，这与之前报道的研究一致，在这些研究中，这些囊泡的胞吐作用提供了一种解毒机制，可以从细胞体中去除该元素。在其他图像中，可以将海马切片内突触结构中的锰浓度关联起来。