An Ultrafast Electron Counting Camera for 100 kV Cryo-EM

用于 100 kV 冷冻电镜的超快电子计数相机

• 批准号: 10158113
• 负责人: Benjamin Eugene Bammes
• 金额: $ 86.68万
• 依托单位: DIRECT ELECTRON, LP
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158113
• 关键词: 3-Dimensional; Address; Aldehyde-Lyases; Apoferritin; Biological; COVID-19 pandemic; Capital; Collaborations; Computer software; Cryoelectron Microscopy; Data; Detection; Development; Electronics; Electrons; Emergency Situation; Equipment; Evaluation; Fast Electron; Future; Goals; Health; Human; Image; Investments; Measures; Mechanics; Microscope; Mus; Muscle; Noise; Oryctolagus cuniculus; Performance; Peripheral; Phase; Research; Research Institute; Research Personnel; Resolution; Running; Scanning Electron Microscopy; Side; Specimen; Speed; Structure; System; Techniques; Time; Validation; base; cost; cost effective; data acquisition; design; detector; drug discovery; experimental study; innovation; instrument; novel; operation; particle; printed circuit board; prototype; quantum; reconstruction; sensor; software development; structural biology; success; temporal measurement; tool; voltage

Project Summary / Abstract Single-particle electron cryo-microscopy (cryo-EM) has become an essential tool for high-resolution structural studies, both for basic biological and human health research as well as for drug discovery. Currently, one of the most pressing challenges for this powerful technique is satisfying the high and ever-growing demand for cryo-EM. This is especially challenging given the extremely high capital investment and running costs for 300 kV cryo-EM equipment. With demand for cryo-EM far exceeding capacity, there is a growing push for “democratization” of cryo-EM by developing cost-effective and highly-accessible equipment based on 100 kV TEM columns. Although the feasibility of high-resolution cryo-EM at 100 kV has been recently demonstrated (Naydenova, et al., 2019), this idea is currently hindered by the lack of suitable high-performance detectors at this low energy. Current direct detection cameras are optimized for operation for 200 and 300 kV. The performance of these cameras at 100 kV is remarkably poor, with very low resolution and very high noise due to backscattering. To address this problem, we propose to develop a new ultra-fast electron counting direct detection camera optimized for 100 kV. The proposed detector will be based on Direct Electron’s novel ultra-fast binary-readout sensor, which is capable of electron counting at an internal frame rate of up to 8,000 fps (>5× faster than any other direct detector on the market). We propose to modify the design of this detector for 100 kV operation. We have already developed an initial prototype of a 100-kV optimized direct detector based on Direct Electron’s new sensor for scanning electron microscopy (SEM), which is sensitive to 3 – 30 kV electrons. The first results from this prototype sensor confirmed that our new design delivers high resolution, minimal backscattering, and an exceptional electron counting DQE at 100 kV. During Phase II of this project, we will modify the design and layout of our novel ultra-fast binary-readout sensor to optimize it for 100 kV operation. A camera system with this new sensor will be developed for integration on common 100 kV microscopes, with the goal of minimizing both the camera’s manufacturing and on-going support costs. The new camera will be integrated in SerialEM for automated data acquisition. A workflow for high- throughput 100 kV single-particle cryo-EM will be developed. Finally, single-particle cryo-EM at 100 kV will be demonstrated using the new camera. The success of this project will create an entirely new market for high-resolution 100 kV cryo-EM. This will significantly increase the accessibility to cryo-EM, propelling structural biology forward as more researchers have access to the tools they need. Additionally, expanding the availability of cryo-EM will enable researchers to more quickly respond to future human health emergencies, such as the current COVID-19 pandemic. Finally, the additional contrast afforded by 100 kV electrons is expected to push the limits of cryo-EM of small specimens (<100 kDa).

项目摘要 /摘要 单粒子电子冷冻微镜（Cryo-EM）已成为高分辨率结构的必不可少的工具 研究，包括基本生物学和人类健康研究以及药物发现的研究。目前，之一 对于这种强大的技术，最紧迫的挑战是满足对低温EM的高度和不断增长的需求。 鉴于300 kV Cryo-EM的资本投资和运行成本极高，这尤其具有挑战性 设备。随着对低温EM的需求远远超出了能力，越来越多的推动 通过基于100 kV TEM色谱柱开发具有成本效益且高度可访问的设备，通过冷冻-EM开发。 尽管最近已经证明了100 kV处的高分辨率冷冻EM的可行性（Naydenova等人， 2019年），目前由于缺乏适当的高性能探测器而阻碍了这个想法。当前的 直接检测摄像机可用于200和300 kV的操作。这些相机的性能 100 kV非常差，由于反向散射，分辨率非常低，噪音非常高。 为了解决这个问题，我们建议开发新的超快速电子计数直接检测摄像头 针对100 kV进行了优化。拟议的检测器将基于直接电子的新型超快速二进制读取 传感器，能够以高达8,000 fps的内部帧速率进行电子计数（> 5×> 5× 市场上的其他直接检测器）。我们建议修改该检测器的设计以进行100 kV操作。 我们已经开发了基于直接电子的100 kV优化直接检测器的初始原型 扫描电子显微镜（SEM）的新传感器，对3 - 30 kV电子敏感。第一个结果来自 该原型传感器证实了我们的新设计可提供高分辨率，最小的反向散射和 在100 kV处的特殊电子计数DQE。 在该项目的第二阶段期间，我们将修改新型超快速二进制传感器的设计和布局 为100 kV操作进行优化。将开发带有该新传感器的相机系统以集成 常见的100 kV显微镜，以最大程度地减少相机的制造和持续的支持 费用。新相机将集成在Serialem中以进行自动数据采集。高级工作流程 将开发吞吐量100 kV单粒子冷冻EM。最后，在100 kV处的单粒子冷冻EM将是 使用新相机证明。 该项目的成功将为高分辨率100 kV Cryo-EM创造一个全新的市场。这会 由于越来越多的研究人员拥有 访问他们需要的工具。此外，扩大低温EM的可用性将使研究人员能够增加 迅速应对未来的人类健康紧急情况，例如当前的COVID-19大流行。最后， 预计100 kV电子提供的其他对比度将推动小标本的低温EM的限制 （<100 kDa）。