Phase Plate Holder for Transmission Electron Microscopy

描述(申请人提供)：在生物电子显微镜的前沿，有一个强烈的重点是成像细胞，细胞器和大分子的三维结构在他们的“天然”状态。这是通过使用未经化学固定、脱水或染色的冷冻水化样品来促进的。目前，由于电子辐照损伤，分辨率限制在4 nm左右。在透射式电子显微镜中使用聚焦位相衬度成像--通过在物镜的后焦平面中加入位相板来实现--可以提高电子层析成像的分辨率，也可以增加吞吐量。尽管这些改进最近已被初步实验证实，而且相位板的好处几十年来就已为人所知，但技术上的困难一直阻碍着它们的使用。主要的三个困难是：保持相位板在电子光轴的中心，避免污染，以及允许在显微镜下轻松更换相位板。在第一阶段，我们已经开发和测试了一个工作原型，它带有精密定位系统、加热器和电气馈通，用于静电相板。将瞬变电磁法的原始物镜光圈固定器与位相板固定器进行交换，几乎可以使任何低温电子显微镜都配备有位相板。这种支架的成本大大低于任何其他技术，可以在冷冻水合标本的成像方面产生类似的改进。在第二阶段，我们将把这一概念改进为商业产品，并在支架上添加自动居中软件和负载锁，允许在不破坏显微镜柱真空的情况下更换相位板。后一项添加将消除打破真空以插入相板的需要，并使该产品成为一个完整的解决方案，解决了传统上阻止科学家更常规地使用这种表征技术的所有技术问题。 公共卫生相关性：用于透射电子显微镜的位相板支架将在该项目中开发并商业化的位相板支架允许以前所未有的分辨率和对比度水平对生物结构进行常规的透射电子显微镜表征。相位板的常规使用将允许在非晶体材料的3D断层图像中获得细节水平的大幅飞跃。这种透射电子显微镜成像能力被用来获得对细胞结构的全面了解，并允许结构和功能之间的直接关联。

DESCRIPTION (provided by applicant): At the frontier of biological electron microscopy, there is a strong emphasis on imaging the 3-D structure of cells, organelles, and macromolecules in their "native" state. This is facilitated by the use of frozen-hydrated specimens which have not been subjected to chemical fixation, dehydration or stains. The resolution is currently limited to about 4 nm by electron irradiation damage. The use of in-focus phase contrast imaging in the transmission electron microscope (TEM) - accomplished by incorporating a phase plate in the back focal plane of the objective lens - can increase resolution in electron tomography, and also increase throughput. Although these improvements have recently been confirmed by preliminary experiments, and the benefits of phase plates have been known for decades, technical difficulties have always inhibited their use. The three main difficulties are: keeping the phase plate centered on the electron optical axis, avoiding contamination and allowing easy phase plate replacement in the microscope. In Phase I we have developed and tested a working prototype of a phase plate holder with precision positioning system, a heater and an electrical feed-through for use with electrostatic phase plates. Exchanging the original objective aperture holder of the TEM with the phase plate holder allows nearly any cryo-EM to be equipped with a phase plate. The cost of this holder is substantially lower than any other technology that can yield a similar improvement in imaging of frozen- hydrated specimens. In Phase II we will refine this concept into a commercial product and add auto-centering software as well as a load-lock to the holder, allowing replacement of the phase plates without breaking the vacuum in the microscope column. This latter addition will eliminate the need to break the vacuum to insert the phase plate, and makes this product into a complete solution to all technical issues that have traditionally prevented scientists from more routinely using this characterization technique. PUBLIC HEALTH RELEVANCE: Phase plate holder for transmission electron microscopy Relevance The phase plate holder that will be developed and commercialized in this project allows routine transmission electron microscopy characterization of biological structures at unprecedented resolution and contrast levels. The routine use of phase plates will allow the acquisition of levels of details in 3D tomographic images of non-crystalline materials to increase by a large leap. This TEM imaging ability is used to gain a comprehensive understanding of cellular structures and allows direct correlation between structure and function.