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: 10262661
• 负责人: Richard Leapman
• 金额: $ 151.24万
• 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/10262661
• 关键词: 3-Dimensional; Address; Area; Astrocytes; Biological; Blood Platelets; Brain; Brain imaging; Cell Nucleus; Cells; Centrioles; Cilia; Circadian Rhythms; Cryoultramicrotomy; Data; Defect; Diffuse; Dose; Drug Metabolic Detoxication; Electron Microscope; Electron Microscopy; Electron energy loss spectroscopy; Electrons; Elements; Exocytosis; Face; Fluorescence; Freeze Drying; Freeze Substitution; Freezing; Generations; Goals; Golgi Apparatus; Hippocampus (Brain); Human; Humpback Dolphins; Hypothalamic structure; Image; Ions; Knowledge; Laboratories; Liver; Magnetic Resonance Imaging; Mammalian Cell; Manganese; Measurement; Measures; Membrane; Methods; Microscope; Microscopy; Microtome - medical device; Microtomy; Modernization; Morphology; Mothers; Mus; N-Methyl-D-Aspartate Receptors; Neurons; Optics; Organelles; Phase; Photons; Play; Process; Proteins; Rattus; Reporting; Resolution; Retina; Roentgen Rays; Role; Sampling; Scanning; Scanning Electron Microscopy; Secretory Vesicles; Series; Signal Transduction; Slice; Source; Specimen; Stains; Structure; Subcellular structure; Surface; Synapses; Techniques; Testing; Thick; Three-Dimensional Imaging; Time; Tissues; Transmission Electron Microscopy; Vertebral column; Vesicle; Visualization; Work; X ray spectroscopy; base; biological systems; brain tissue; ciliopathy; density; detector; electron tomography; experimental study; fluorescence imaging; hippocampal pyramidal neuron; imaging approach; imaging modality; interest; knock-down; lens; nanoprobe; nanoscale; novel; pathogen; reconstruction; ribbon synapse; suprachiasmatic nucleus; three dimensional structure; tomography; 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 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 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. The method has been tested on specimens of embedded blood platelets, brain tissue and liver tissue (1). We have applied STEM tomography to perform 3-D imaging of 3-D micrometer thick sections of CA1 regions of mouse hippocampus to determine changes in astrocyte morphology that are associated with circadian rhythm set by the suprachiasmatic nucleus suprachiasmatic nucleus (SCN) of the hypothalamus. It was found that pyramidal neurons change the surface expression of NMDA receptors, and astrocytes change their proximity to synapses. Specifically, fewer astrocytic processes were detected in the D-phase than in the L-phase with a 2-fold increase in the nearest neighbor distance between each post-synaptic density and the closest astrocyte process (J.P. McCauley et al., bioRxiv, 2020) In another application, we 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. 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. We have used STEM tomography to investigate the association of membranes with the mother centriole in the early stages of generation of primary cilia, as these play essential roles in signal transduction. Defects in cilium formation or function cause ciliopathies. The advantage of STEM tomography in this study is its ability to provide 3D reconstructions of the entire centriole assembly. Taken together our work has 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 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. 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. McCauley JP, Petroccione MA, DBrant LY, Todd GC, Affinnih N, Wisnoski JJ, Zahid S, Shree S, Sousa AA, De Guzman RM, Migliore R, Brazhe A, Leapman RD, Khmaladze A, Semyanov A, Zuloaga DG, Migliore M, Scimemi A, 2020, bioRxiv, doi: https://doi.org/10.1101/666073.

传统的明亮场电子断层图倾斜系列是通过收集横穿宽光束照明标本的电子获得的。使用这种方法，厚度受到严重的图像的限制，而严重的图像模糊了，当经历了多个能量损耗的电子被显微镜的客观镜头聚焦。此外，图像的最大面积受到物镜晶状体的深度限制，因此只有一部分样品以高倾斜角为焦点。 使用茎与紧密浓缩的电子探测器使用茎进行整体术重建可以克服使用常规TEM通过层析成像重建所施加的一些局限性。首先，由于事件茎探针可以集中在标本中的任何时刻，因此即使是高倾斜角度，大面积也会成像。其次，因为在茎上没有样品后的图像形成透镜，因此在厚标本的图像中可实现的分辨率不会被遭受多种能量损失的电子进一步降低。最常用的茎方法利用环形深色探测器收集分散到高角度的电子。但是，由于入射电子探针的大收敛角所定义的景深有限，因此深色场茎技术不适合对厚的生物标本进行成像。通过调整显微镜光学元件以将收敛的半角度降低至大约1 mrad，可以通过调整显微镜光学元件来增加十倍或更高的景深。深色场茎的另一个限制特征应用于成像厚标本，这是空间分辨率的严重降解，由于横梁宽阔而朝向截面的底部表面。相比之下，我们发现只能通过使用散布到低角度的那些电子来获得更高的空间分辨率，即通过使用轴向明亮场检测器。 染色密度是优化从几种类型的3D电子显微镜技术获得的超微结构数据质量的重要参数，包括STEM断层扫描以及串行区块侧面电子显微镜（SBEM）以及聚焦离子束扫描电子显微镜（FIB-SEM）。我们已经开发了一种基于TEM中的一些直接测量值来确定常规制备塑料部分中的染色密度。每单位体积的污渍原子数量是根据来自含有纯嵌入材料和感兴趣的嵌入式生物结构的标本区域的测量比率计算出的。该确定仅需要了解截面厚度的知识，该截面厚度可以从微型组设置或低剂量电子断层扫描以及用于染色样品染色的重原子的弹性散射截面。该方法已在嵌入式血小板，脑组织和肝组织的标本上进行了测试（1）。 我们已经应用了茎断层扫描，对小鼠海马的Ca1区域的3-D微米厚的切片进行3-D成像，以确定与suparachiasmamatic suparachiasmaticalicmatic nucleus（SCN）相关的星形胶质细胞形态的变化，这些变化与下丘脑的昼夜节律相关。发现锥体神经元改变了NMDA受体的表面表达，星形胶质细胞改变了其与突触的近端。具体而言，在D期中检测到的星形细胞过程比在L期中较少，而在每个突触后密度和最近的星形胶质细胞过程之间的最近邻居距离增加了2倍（J.P. McCauley等人，Biorxiv，Biorxiv，2020）） 在另一个应用中，我们使用STEM断层扫描可视化大鼠海马培养的切片中的突触刺。 首次有可能可视化整个突触后密度，并评估当某些重要蛋白（例如PSD-95）被击倒时发生的超微结构差异。 在其他实验中，表征大鼠视网膜中的整个色带突触并可视化这些结构内分泌囊泡的精确组织是可行的。 我们已经使用STEM断层扫描来研究原发性纤毛的初期膜与母中心的关联，因为这些在信号转导中起着重要作用。纤毛形成或功能的缺陷引起纤毛病。在这项研究中，STEM层析成像的优点在于它可以提供整个中心装配的3D重建的能力。 综上所述，我们的工作证明了轴向茎断层扫描对于以5至10 nm的空间分辨率对厚部分进行成像的可行性和优势，这与从较薄的截面（通常3至8 nm）的常规电子断层扫描的空间分辨率相媲美。 大多数现代电子显微镜可以在STEM模式下运行，并且可以容易配备明亮的检测器，这预计可以促进该技术的实现。到目前为止，通过大规模应用薄片的传统电子断层扫描，已经解决了对高分辨率，大量生物标本的大型成像的需求。 我们目前的工作表明，可以通过串行厚截面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。 相关显微镜已成为确定细胞和组织尺度上细胞和组织功能之间的关系的重要技术，并具有亚细胞细胞器和超分子组件的规模。我们已经使用了这种方法将在透射电子显微镜中获得的图像与在Argonne National Laboratory的高级光子源中获得的X射线荧光图像相关联。 该工作流程使我们能够定位特定的扩散离子，因此有助于阐明在MN增强型脑部脑中摄取锰离子的机制，该技术可以追踪神经元连接。通过大满贯冻结制备了器官大鼠海马切片的样本，冷冻冷冻切片被冷冻转移到我们的TEM中，以300 keV的梁能量运行。相关图像提供了证据表明，二价锰离子集中在细胞核周围的高尔基囊泡中，这与先前报道的研究一致，在该研究中，这些囊泡的胞吐作用提供了从细胞体中去除元素的解毒机制。 在其他图像中，可以将海马切片中突触结构中的锰的浓度相关联。 McCauley JP, Petroccione MA, DBrant LY, Todd GC, Affinnih N, Wisnoski JJ, Zahid S, Shree S, Sousa AA, De Guzman RM, Migliore R, Brazhe A, Leapman RD, Khmaladze A, Semyanov A, Zuloaga DG, Migliore M, Scimemi A, 2020，Biorxiv，doi：https：//doi.org/10.1101/666073。