Mass Mapping Of Macromolecular Assemblies

扫描透射电子显微镜（STEM）提供了一种通用的方法来确定分子质量，从而确定大蛋白质组装中亚基的排列。我们在配备场发射源和探针形成电子光学元件的300 kV透射电子显微镜（FEI Tecnai TF30）上实现了STEM质量测绘。在显微镜柱的最后一个投影透镜后的端口安装了一个高效环形暗场探测器。将这种能力添加到先进的电子显微镜中，将使冷冻水化样品的冷冻电子显微镜成为可能，这些样品随后可以在原位脱水并通过STEM成像，以确定相同结构内的质量分布。通过调整显微镜？S投影镜头，可以改变相机的长度，这样可以优化环形探测器选择的散射角范围。我们已经证明，通过结合散射截面的理论模型（最近的NIST数据库），可以量化质量测量。大分子被吸附到一层薄薄的支撑膜上，一个纳米大小的电子探针在样本上扫描，同时收集弹性散射信号。由此产生的数字图像强度是成比例的局部质量密度的标本。图像可以在低电子剂量下记录，而不会对感兴趣的结构造成明显的辐射损伤。通过对已知结构、烟草花叶病毒颗粒和软体动物血青素的分析，在300 kV FEI Tecnai电子显微镜上对该系统进行了测试。

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. We have implemented STEM mass mapping in a 300 kV transmission electron microscope (FEI Tecnai TF30) equipped with a field-emission source and probe forming electron optics. A high-efficiency annular dark-field detector was installed in a port after the final projector lens of the microscope column. Addition of this capability to an advanced electron microscope will enable cryo-electron microscopy of frozen hydrated specimens, which can be subsequently dehydrated in situ and imaged by STEM to determine the mass distribution within the same structures. By adjusting the microscope?s projector lenses, it is possible to vary the camera length so that the range of scattering angles selected by the annular detector can be optimized. We have shown that it is possible to quantify the mass measurements by incorporating a theoretical model for the scattering cross sections (recent NIST Database). Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic-scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images can be recorded at low electron dose without significant radiation damage to the structures of interest. We have successfully tested the mass mapping system on the 300 kV FEI Tecnai electron microscope by analyzing the well known structures, tobacco mosaic virus particles and mollusk hemocyanin. We are now able to perform mass mapping with our 300 kV instrument as well as with our dedicated 100 kV VG Microscopes HB501 STEM. We have applied the technique to a number of projects including characterization of proteins associated with motility of the simple bacterium, Spiroplasma, and to determine the molecular mass of postsynaptic densities.