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: 8933881
• 负责人: Richard Leapman
• 金额: $ 21.86万
• 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/8933881
• 关键词: Active Biological Transport; Address; Area; Biological; Blood Platelets; Cells; Complex; Crowding; Detection; Diffusion; Electron Microscope; Electrons; Freeze Substitution; Freezing; Goals; Hippocampus (Brain); Image; Imagery; Incidence; Mammalian Cell; Measures; Membrane; Microscope; Microtomy; Modeling; Neuraxis; Neurons; Optics; Organelles; Platelet Activation; Process; Proteins; Publishing; Rattus; Resolution; Retina; Retinal; Sampling; Scanning; Scanning Transmission Electron Microscopy Procedures; Secretory Vesicles; Sensory; Series; Signal Transduction; Site; Slice; Specimen; Structure; Surface; Synapses; Synaptic Vesicles; System; Techniques; Testing; Thick; Time; Tissues; Tomogram; Vertebral column; Vesicle; Visual Pathways; Work; analog; auditory pathway; base; density; detector; electron tomography; knock-down; lens; pathogen; presynaptic; protein structure; reconstruction; research study; retinal rods; ribbon synapse; simulation; three dimensional structure; tomography; transmission process; vestibular pathway

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. 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. We have applied axial (bright-field) STEM tomography to investigate the structure of synaptic ribbons, which are presynaptic protein structures found at many synapses that convey graded, analog sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbons surface to reach the presynaptic release sites at its base. To address these questions, we have tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. STEM tomography gave us 3D structures of rat rod bipolar cell terminals in 1-micrometer thick sections of retinal tissue at an isotropic spatial resolution of approximately 3 nm. The resulting structures were then incorporated, along with previously published estimates of vesicle diffusion dynamics, into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. We have also used the technique 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. In another application, we are applying STEM tomography to determine the three-dimensional arrangement of the complex membrane systems that are present in blood platelets, including the open canalicular system, dense tubules, and secretory granules. Collecting STEM tomograms from whole platelets provides a new way to visualize the structural changes that occur on activation of platelets that have been prepared by rapid freezing and freeze-substitution. Thus we have demonstrated the feasibility and advantages of STEM using axial detection for imaging thick sections at a spatial resolution around 5-10 nm, which is comparrable 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.

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。 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。 我们应用轴向（明场）STEM 断层扫描来研究突触带的结构，突触带是在许多突触中发现的突触前蛋白质结构，在视觉、听觉和前庭通路中传递分级的模拟感觉信号。带通常固定在突触前膜上并被束缚的突触小泡包围，被认为可以调节或促进小泡递送至突触前膜。然而，没有直接证据表明囊泡如何与带相互作用，或者一旦附着，就沿着带表面移动到达其基部的突触前释放位点。为了解决这些问题，我们测试了视网膜杆双极细胞带状突触的被动囊泡扩散模型。 STEM 断层扫描为我们提供了 1 微米厚的视网膜组织切片中大鼠视杆双极细胞末端的 3D 结构，各向同性空间分辨率约为 3 nm。然后将所得结构与先前发表的囊泡扩散动力学估计一起纳入数值模拟中，准确再现电生理学测量的囊泡释放/补充速率和囊泡池大小。模拟表明，在生理真实条件下，拥挤在带表面的囊泡扩散会产生流场，增强囊泡向突触前膜的递送，而不需要主动运输机制。 我们还使用该技术来可视化大鼠海马培养切片中的突触棘。 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。 在其他实验中，描述大鼠视网膜中整个带状突触的特征并可视化这些结构内分泌囊泡的精确组织是可行的。 在另一个应用中，我们应用 STEM 断层扫描来确定血小板中存在的复杂膜系统的三维排列，包括开放的小管系统、致密小管和分泌颗粒。 从整个血小板中收集 STEM 断层图提供了一种新的方法来可视化通过快速冷冻和冷冻替代制备的血小板活化时发生的结构变化。 因此，我们证明了 STEM 使用轴向检测以 5-10 nm 左右的空间分辨率对厚切片进行成像的可行性和优势，这与传统电子断层扫描的较薄切片（通常为 3 至 8 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。