An Ultrafast Electron Counting Camera for 100 kV Cryo-EM

项目总结/摘要 单粒子低温电子显微镜（cryo-EM）已成为高分辨率结构分析的重要工具。 研究，包括基础生物学和人类健康研究以及药物发现。目前， 对于这种强大的技术来说，最紧迫的挑战是满足对低温EM的高且不断增长的需求。 考虑到300 kV低温EM极高的资本投资和运行成本，这尤其具有挑战性 设备.随着对冷冻EM的需求远远超过能力，越来越多的人推动“民主化”， 通过开发基于100 kV TEM柱的具有成本效益和高度可及的设备，实现冷冻EM。 尽管最近已经证明了在100 kV下的高分辨率冷冻EM的可行性（Naydenova等人， 2019年），这一想法目前受到缺乏合适的低能量高性能探测器的阻碍。电流 直接检测摄像机针对200和300 kV的操作进行了优化。这些相机的性能在 100 kV非常差，分辨率非常低，并且由于反向散射而具有非常高的噪声。 针对这一问题，我们提出研制一种新的超快电子计数直接探测相机 优化为100 kV。该探测器将基于直接电子的新型超快二进制读出 传感器，能够以高达8，000 fps的内部帧速率进行电子计数（比任何 市场上的其他直接检测器）。我们建议修改100 kV操作的检测器的设计。 我们已经开发了一个100千伏优化直接探测器的基础上直接电子的初步原型， 扫描电子显微镜（SEM）的新传感器，对3 - 30 kV电子敏感。的第一个结果 这个原型传感器证实了我们的新设计提供了高分辨率，最小的反向散射， 在100 kV下的异常电子计数DQE。 在第二阶段的这个项目中，我们将修改我们的新型超快速二进制读出传感器的设计和布局 以优化其100 kV运行。将开发具有这种新传感器的相机系统， 普通的100 kV显微镜，目标是最大限度地减少相机的制造和持续支持 成本新相机将集成在SerialEM中，用于自动数据采集。一个工作流程，高- 将开发100 kV单粒子冷冻EM。最后，将在100 kV下进行单粒子冷冻EM。 使用新相机演示。 该项目的成功将为高分辨率100 kV低温EM创造一个全新的市场。这将 显著增加了冷冻EM的可及性，推动了结构生物学的发展，因为越来越多的研究人员 获取他们需要的工具。此外，扩大冷冻EM的可用性将使研究人员能够更多地 快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 由100千伏电子提供的额外对比度有望推动小样本的低温EM的极限 （<100 kDa）。

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).