Three dimensional aberration corrected scanning transmission electron microscopy

描述(申请人提供)：像差校正的三维(3D)扫描透射电子显微镜(STEM)能够在没有倾斜阶段的情况下对样品进行高分辨率的3D成像。以类似于共聚焦光学显微镜的方式，通过改变物镜焦点来逐层扫描样品，从而记录焦点序列。经过优化的3D STEM有望显示出与常规倾斜系列透射电子显微镜(TEM)相比的显著优势，例如1)更高的分辨率，2)没有机械倾斜，3)大面积薄层成像的能力，4)更快的3D数据采集。最近，我们已经获得了包含3T3细胞的常规薄片的3D图像，其横向分辨率为0.6 nm，轴向分辨率为60 nm。我们的计算预测，常规薄片的轴向分辨率可以提高到7 nm。我们的目标是充分了解成像机制，评估3D STEM在辐射损伤方面的可行性，并优化3D STEM用于生物医学相关薄层切片的成像性能。具体目的是：1)确定试件的三维STEM分辨率。决定3D STEM分辨率的因素还不是很清楚。需要研究的一个重要方面是包埋介质的电子散射对分辨率的影响。我们将在没有辐射损伤和极端样品非均质性的情况下，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，并用于更详细地检查3D STEM成像和束-样品相互作用。2)确定常规薄层三维扫描电子显微镜的分辨率。我们将优化样品参数和显微镜设置，以获得包含3T3细胞的传统薄片的横向分辨率为1 nm，轴向分辨率为7 nm。除了目标1中研究的因素外，在生物样品上获得的分辨率还受到着色剂颗粒大小、着色剂密度的局部变化以及辐射损伤的影响。我们将开发一个电子剂量限制分辨率的理论模型来预测每个样品厚度的最佳显微镜设置。然后，我们将应用3D STEM对几个与生物医学相关的样本进行成像。3)通过反褶积提高分辨率。用3D STEM记录的图像将包含来自焦平面的聚焦信息以及离焦贡献。这是在光学显微镜的广视场焦距系列中常见的效应。我们将开发一种迭代反卷积策略，用于3D STEM，包括基于样品中电子散射知识的约束。我们的目标是将在目标2下获得的数据集的分辨率提高2-3倍。3D STEM可用于研究真核细胞内大分子和隔室(如核糖体、蛋白酶体、高尔基体和线粒体)的纳米级组装的复杂组织。了解这些结构是如何组织的，从而在拥挤的3D细胞体积内发挥作用，可以用于例如帮助开发新的治疗方法，或改进现有的治疗方法。

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.