An energy discriminating direct detector for multi-color SEM

项目摘要/摘要 了解大脑功能和神经功能障碍的基础是绘制大脑功能和神经功能障碍之间的联系图 神经元，区分不同的细胞和分子群体，并阐明蛋白质-蛋白质 推动神经功能的相互作用。这种研究跨越了广泛的范围，既需要一个大的领域- 图的连接性和高分辨率，以可视化亚细胞和突触内的分子细节。 多色电子显微镜(EM)在纳米级生物超微结构研究中显示出良好的应用前景 同时还能检测到感兴趣的特定分子组分。这项技术类似于多个 彩色荧光显微镜，但放大倍数约为100倍。然而，目前的方法是 多色电磁数据的获取是基于能量滤波的瞬变电磁法(EFTEM)，这在很大程度上限制了其在 神经生物学由于其严重的低吞吐量和有限的视野。 我们建议开发一种新型的用于扫描电子显微镜(SEM)的超高速直接检测相机 以超过100,000帧每秒(Fps)的速度工作并测量探测到的电子的能量。是这样的 摄像头将是一个惊人的飞跃，极大地提高了吞吐量，并实现了复杂的多 彩色EM技术采用连续块面扫描电子显微镜(SBEM)，使突触小泡等小结构， 核小体、核孔和病毒(都是几纳米到10-40纳米)都可以被识别和定量。 我们已经开发了这种新的直接检测扫描电子显微镜相机的第一阶段原型，基于一种低能量- Direct Electron当前一代瞬变电磁直接探测相机的优化版本。初步结果显示 确认对低至2千伏能量的电子的敏感性，显示出远高于 目前最先进的闪烁体耦合扫描电子显微镜摄像机，最重要的是，揭示了我们的新传感器 设计能够辨别被探测电子的能量。这些初步结果被用来最终确定 这里提出了对新的超快像素化直接探测器的要求，其速度要求达到 使这项技术适用于大视场、高分辨率的多色SBEM神经元成像。 在第二阶段，我们将推进这种新型超高速扫描电子显微镜相机的开发和商业化 系统，通过制造和组装新的超高速扫描电子显微镜相机，进一步完善硬件和软件，以 高效处理产生的海量数据并识别多色EM标签，然后 展示了神经元组织的高速多色SBEM。 该项目的成功将创造一种无处不在的荧光显微镜技术的模拟，但在 使用连续块面扫描电子显微镜，分辨率显著提高。这不仅在以下方面有广泛的应用 神经科学研究，但也将延伸到细胞显微镜在广泛的其他生物学领域。 此外，新相机还将启用能量过滤电子背散射衍射(EBSD)，这是 广泛应用于材料科学研究和工业质量控制。因此，作为一种新的使能技术， 我们预计，拟议的探测器将在各种领域产生广泛影响。

Project Summary / Abstract Understanding brain function and neurological disorder is predicated on mapping the connectivity among neurons, distinguishing various cellular and molecular populations, and elucidating the protein-protein interactions that drive neurological function. Such studies span a wide range of scales, requiring both a large field- of-view to map connectivity and high-resolution to visualize subcellular and intrasynaptic molecular details. Multi-color electron microscopy (EM) has shown promise in studying biological ultrastructure at nanometer resolution while also detecting specific molecular components of interest. The technique is analogous to multi- color fluorescence microscopy, but at about ~100× higher magnification. However, the current method for acquiring multi-color EM data is based on energy-filtered TEM (EFTEM), which significantly limits is usefulness in neurobiology due to its severely low throughput and limited field-of-view. We propose to develop a new ultra-fast direct detection camera for scanning electron microscopy (SEM) capable of operating at more than 100,000 frames per second (fps) and measuring the energy of detected electrons. Such a camera will be an astounding leap forward, dramatically improving throughput and enabling sophisticated multi- color EM techniques using serial block-face SEM (SBEM), so that small structures like synaptic vesicles, nucleosomes, nuclear pores, and viruses (all a few nanometers to 10-40 nm) can be identified and quantified. We have already developed a Phase I prototype of this new direct detection SEM camera, based on a low-energy- optimized version of Direct Electron’s current generation TEM direct detection cameras. Initial results have confirmed sensitivity to electrons down to 2 kV energy, showed far superior information content compared to current state-of-the-art scintillator-coupled SEM cameras, and most importantly, revealed that our new sensor design is capable of energy discrimination of detected electrons. These initial results were used to finalize the requirements for the new ultra-fast pixelated direct detector proposed here, the speed of which is required to make the technique useful for large field-of-view, high-resolution multi-color SBEM for imaging neurons. During Phase II we will advance the development and commercialization of this new ultra-fast SEM camera system, by fabricating and assembling the new ultra-fast SEM camera, further refining hardware and software to efficiently handle the enormous volumes of data produced and identify multi-color EM labels, and then demonstrating high-speed multi-color SBEM of neuronal tissue. The success of this project will create an analog of the ubiquitous fluorescence light microscopy technique, but at significantly higher resolution using serial block-face SEM. This will not only have wide ranging applications for neuroscience research but will also extend to cellular microscopy in a wide range of other biological fields. Additionally, the new camera will also enable energy-filtered electron backscattered diffraction (EBSD), which is widely used in materials science research and industrial quality control. Therefore, as a new enabling technology, we anticipate that the proposed detector will have broad impact across a variety of fields.