High-stability solid-state cooled Cryo-TEM transfer holder

高稳定性固态冷却冷冻 TEM 传输支架

• 批准号: 9908270
• 负责人: Daan Hein Alsem
• 金额: $ 15万
• 依托单位: HUMMINGBIRD PRECISION MACHINE COMPANY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-05 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908270
• 关键词: 3-Dimensional; Biological; Biological Process; Biological Testing; Cell physiology; Cells; Cellular Structures; Charge; Colorado; Cool-X-A; Coupling; Data; Data Collection; Detection; Development; Electrons; Gases; Generations; Hour; Hydration status; Image; Knowledge Discovery; Life; Liquid substance; Macromolecular Complexes; Methods; Microscope; Microscopy; Modeling; Names; Nitrogen; Organelles; Performance; Preparation; Principal Investigator; Proteins; Research; Resolution; Role; Rotation; Sampling; Schools; Scientist; Side; Source; Specimen; Speed; Structure; Surface; Techniques; Technology; Temperature; Testing; Three-Dimensional Imaging; Tomogram; Transmission Electron Microscopy; Work; cryogenics; cryostat; density; design; drug discovery; electron optics; evaporation; innovation; novel strategies; prototype; solid state; vibration

Project Summary Project Title: High-stability solid-state cooled Cryo-TEM transfer holder Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Daan Hein Alsem Summary: Cryo-TEM or electron cryotomography (ECT) produces high-resolution 3D views of biological samples in hydrated and functional state. This microscopy technique has a leading role in structural characterization efforts of biological samples, including: intact cells, organelles, macromolecular complexes, and proteins, facilitating basic knowledge and drug discovery. Over the last decade, the resolution of cryo-TEM has seen an unprecedented improvement down to below 3 Angstrom because of the advent of direct-electron detection cameras, which are orders of magnitude more sensitive than previous TEM camera technology and new high-throughput loading mechanisms in the latest generation of dedicated, but expensive, Cryo-TEM microscopes. However, the resolution achieved with these new dedicated TEMs is not better than the resolution capability of standard TEMs installed twenty years ago. There are still hundreds of these previous models of TEMs used for imaging of biological specimens. These labs can (and in many cases have) purchase the latest generation sensitive cameras for these microscopes, which makes that these older microscopes are at that point not limited by their electron-optics, or their camera, but by the cryo-TEM sample holder. The cryo- TEM holder technology used today relies on evaporation of LN2 in a geometrically asymmetric Dewar attached to the sample to cool the sample, which causes image instability and vibration, specifically when changing the tilt angle when acquiring tomograms. Because of shifting mass in the LN2 Dewar and the LN2 encountering less cooled surfaces when tilting resulting in excess boiling, the image stability in these TEM holders is far from perfect. This project will provide scientists with a TEM cryo-transfer sample holder that eliminates these image instability and vibration limits and allows the previously installed cryo-TEMs to achieve the performance similar to or matching the latest generation of dedicated and very expensive cryo-TEMs. We are proposing to achieve this by using a cryo-TEM sample transfer holder that has a rotationally symmetric solid-state cooling block to cool the sample. Because of the symmetry there are no changes in forces on the holder when tilting and the solid-state cooling method assures there is no excess boiling when LN2 is moving around during tilting of the specimen. This eliminates sample instability and vibration that arise during tilting of the holder during acquisition of cryo-TEM tomograms. As an added benefit, this innovation also provides a much longer cold life of the sample (>12 hours) than current generation Cryo-TEM holders. We will achieve this by using an ultra-cooled solid-state cold mass that has no boiling or liquid mass but is instead cold- charged to temperatures below liquid nitrogen temperatures (~50K) to provide long cold-life of the TEM. This adds up to a TEM specimen holder that can provide higher image stability and operate longer without having to be cold-recharged. This product is aimed at scientists in the hundreds of cryo-TEM labs where purchasing an expensive dedicated cryo-TEM is not a realistic proposal. These labs will still propel their research with this new side-entry TEM cryo transfer holder as it will give them a better quality and higher resolution cryo-TEM image that is similar to what they would achieve if they would purchase a dedicated TEM.

项目摘要 项目名称：高稳定性固态冷却Cryo-TEM转移保持器 公司名称：蜂鸟精密机械有限公司dba蜂鸟科技 主要研究者：Daan Hein Alsem 总结： Cryo-TEM或电子冷冻断层扫描（ECT）可生成生物组织的高分辨率3D视图， 处于水合和功能状态的样品。这种显微镜技术在以下方面具有主导作用： 生物样品的结构表征工作，包括：完整细胞，细胞器， 大分子复合物和蛋白质，促进基础知识和药物发现。超过 在过去的十年里，低温TEM的分辨率得到了前所未有的提高， 低于3埃，因为直接电子检测相机的出现，这是订单 比以前的TEM相机技术和新的高通量更敏感的数量级 在最新一代专用但昂贵的Cryo-TEM显微镜中使用了装载机制。 然而，使用这些新的专用TEM实现的分辨率并不比 20年前安装的标准TEM的能力。目前仍有数以百计的 用于生物样本成像的TEM模型。这些实验室可以（在许多情况下 我已经）为这些显微镜购买了最新一代的敏感相机，这使得 这些老式的显微镜在这一点上并不受电子光学或相机的限制， 通过低温TEM样品保持器。今天使用的低温TEM保持器技术依赖于 在连接到样品的几何不对称杜瓦瓶中蒸发液氮，以冷却样品。 样品，这会导致图像不稳定和振动，特别是在改变倾斜角度时 当获取断层图像时。由于液氮杜瓦瓶中的质量转移和液氮遇到 倾斜时冷却较少的表面导致过度沸腾，这些TEM中的图像稳定性 持有人远非完美。该项目将为科学家提供TEM冷冻转移样品 保持器，消除了这些图像不稳定性和振动限制，并允许以前的 安装cryo-TEM，以实现与最新一代 专用且昂贵的低温TEM。 我们建议通过使用低温TEM样品转移保持器来实现这一点， 旋转对称的固态冷却块来冷却样品。因为那里的对称性 当倾斜时，保持器上的力没有变化， 当液氮在样品倾斜过程中四处移动时，没有过度沸腾。这 消除采集过程中倾斜保持器时产生的样本不稳定性和振动 冷冻TEM断层图像作为一个额外的好处，这项创新还提供了一个更长的冷 样品寿命（>12小时）比当前一代的Cryo-TEM支架更长。我们将通过以下方式实现这一目标： 使用不具有沸腾或液体质量而是冷的超冷固态冷质量， 充电温度低于液氮温度（~ 50 K），以提供长的冷寿命， 透射电镜这使得TEM样品保持器可以提供更高的图像稳定性， 更长的时间，而无需冷充电。这个产品的目标是数百名科学家， 低温TEM实验室，其中购买昂贵的专用低温TEM是不现实的建议。 这些实验室仍将推动他们的研究与这种新的侧面进入TEM低温转移保持器，因为它 将为他们提供更好的质量和更高分辨率的低温TEM图像， 如果他们购买一个专用的TEM。