Three dimensional aberration corrected scanning transmission electron microscopy

描述（由申请人提供）：像差校正三维（3D）扫描透射电子显微镜（STEM）能够在没有倾斜载物台的情况下对样本进行高分辨率3D成像。以类似于共焦光学显微镜的方式，通过改变物镜透镜焦点逐层扫描样品，从而记录焦点系列。优化的3D STEM预计将表现出显着的优势，超过传统的薄切片的倾斜系列透射电子显微镜（TEM），如1）更好的分辨率，2）没有机械倾斜，3）成像大面积薄切片的能力，和4）更快的3D数据收集。最近，我们已经获得了包含3 T3细胞的常规薄切片的3D图像，其显示出0.6 nm的横向分辨率和60 nm的轴向分辨率。我们的计算预测，传统的薄切片的轴向分辨率可以提高到7 nm。我们的目标是充分了解图像形成机制，评估3D STEM在辐射损伤方面的可行性，并优化3D STEM在生物医学相关薄切片成像方面的性能。具体目的是：1）确定测试样本的3D STEM分辨率。决定3D STEM分辨率的因素尚未得到很好的理解。需要研究的一个重要方面是嵌入介质对分辨率的电子散射的影响。我们将在没有辐射损伤和极端样品异质性的情况下，检查实际尺寸的测试样品的横向和轴向分辨率。3D STEM的蒙特卡罗模型将通过这些实验进行校准，并用于进一步详细检查3D STEM图像形成和光束-样品相互作用。2)在常规薄切片上确定3D STEM的分辨率。我们将优化样品参数和显微镜设置，以获得1 nm的横向分辨率和7 nm的轴向分辨率的常规薄切片含有3 T3细胞。除了目标1中研究的因素外，在生物样本上获得的分辨率还受到染色剂颗粒大小、染色剂密度局部变化和辐射损伤的影响。我们将开发一个理论模型的电子剂量有限的分辨率来预测最佳的显微镜设置为每个样品厚度。然后，我们将应用3D STEM对几个生物医学相关的样本进行成像。3)通过反卷积提高分辨率。使用3D STEM记录的图像将包含来自焦平面的焦点内信息以及焦点外贡献。这是一种在光学显微镜的宽视场焦点系列中常见的效应。我们将开发一个迭代反卷积策略，包括约束的基础上的知识，在样品中的电子散射的三维STEM。我们的目标是将目标2下获得的数据集的分辨率提高2-3倍。3D STEM可用于研究大分子和隔室的纳米尺寸组装体的复杂组织（例如，核糖体、蛋白酶体、高尔基体和线粒体）。了解这些结构是如何组织的，从而在拥挤的3D细胞体积中发挥作用，可以应用于例如帮助开发新的治疗方法或改善现有的治疗方法。

DESCRIPTION (provided by applicant): Aberration corrected 3-dimensional (3D) scanning transmission electron microscopy (STEM) is capable of high-resolution 3D imaging of specimens without a tilt stage. In a manner similar to confocal light microscopy, the sample is scanned layer by layer by changing the objective lens focus so that a focal series is recorded. Optimized 3D STEM is expected to exhibit significant advantages over tilt-series transmission electron microscopy (TEM) for conventional thin sections, such as 1) better resolution, 2) absence of mechanical tilt, 3) the capability of imaging large area thin sections, and 4) faster 3D data collection. Recently, we have obtained 3D images of conventional thin sections containing 3T3 cells showing a lateral resolution of 0.6 nm and an axial resolution of 60 nm. Our calculations predict that the axial resolution on conventional thin sections could be improved to 7 nm. Our goals are to fully understand the image formation mechanisms, to evaluate the feasibility of 3D STEM with respect to radiation damage, and to optimize the performance of 3D STEM for the imaging of thin sections of biomedical relevance. The specific aims are to: 1) Determine the resolution of 3D STEM of test specimens. The factors determining the resolution of 3D STEM are not yet well understood. An important aspect that needs to be investigated is the influence of electron scattering by the embedding medium on the resolution. We will examine the lateral and axial resolution for test specimens of realistic dimensions in the absence of radiation damage and extreme sample heterogeneity. A Monte Carlo model of the 3D STEM will be calibrated with these experiments and used to examine 3D STEM image formation and beam-sample interactions in further detail. 2) Determine resolution of 3D STEM on conventional thin sections. We will optimize the sample parameters and the microscope settings to obtain a lateral resolution of 1 nm and an axial resolution of 7 nm for a conventional thin section containing 3T3 cells. In addition to the factors investigated in Aim 1 the resolution obtained on biological specimens is influenced by the size of the grains of the stain, by local variations of the stain density, and by radiation damage. We will develop a theoretical model of the electron dose limited resolution to predict the optimal microscope settings for each sample thickness. We will then apply 3D STEM to image several samples of biomedical relevance. 3) Improve resolution by deconvolution. The images recorded with 3D STEM will contain both the in-focus information from the focal plane as well as out-of-focus contributions. This is an effect commonly found in wide-field focal series from optical microscopy. We will develop an iterative deconvolution strategy for 3D STEM including constrains based on knowledge of electron scattering in the sample. We aim to improve the resolution of the datasets obtained under Aim 2 by a factor of 2-3. 3D STEM can be used to study the complex organization of the nanometer-sized assemblies of macromolecules and compartments (e.g., ribosomes, proteasomes, Golgi apparatus, and mitochondria) within Eukaryotic cells. Understanding how these structures are organized, and thereby function, within the crowded 3D volume of the cell can be applied, for example, to aid the development of new therapeutics, or improve existing ones.