Development and Commercialization of a Sample Preparation System for Time Resolved Cryo-Electron Microscopy

低温电子显微镜(Cryo-EM)是一种对生物分子进行成像的强大技术。 这些样品在使用电子束成像之前被保存在冷冻、水合状态中。 这项技术为X射线结晶学的结构表征提供了一种替代方法 生物样品不能结晶或在其天然状态下不结晶。当冰冷的时候- EM样品制备技术近年来变得更加高效，因为 开发用于进行网格准备的自动样品玻璃化系统，该领域 CRYO-EM受阻于无法在足够快的 时间尺度，以观察许多生物反应的进展，由于固有的滞后 传统的吸墨法。哥伦比亚大学的弗兰克团队开发了一个实验室-- 支持此缺失功能并支持毫秒时间控制的系统原型 网格制备过程中采样生物反应的反应时间。 此快速跟踪STTR提案的目的是开发由 弗兰克组建了一个商业生产单位，并将其作为一种产品推出。初始阶段I 拟议工作中的里程碑将集中于建立一个原型，并增加对反应的控制 确定系统中必要的控制变量并优化样本网格的参数 准备和时间解析。此快速通道提案第二阶段的进一步工作将创建 简化的、用户友好的生产单元，将保持必要的控制 在最初的里程碑中确定，同时优化制造时间并总体减少 生产成本。由此产生的系统将为低温电磁领域增加另一个维度， 为用户提供捕捉反应进程中的瞬时中间体的能力 研究并进一步阐明提出的具有时间分辨率的反应机理。

Cryo-electron microscopy (cryo-EM) is a powerful technique for imaging biological molecules in which samples are preserved in a frozen, hydrated state before imaging using an electron beam. The technique provides an alternative to X-ray crystallography for structural characterization of biological samples that are not crystallizable or do not crystallize in their native state. While cryo- EM sample preparation technique has become more efficient in recent years with the development of automatic sample vitrification systems to perform grid preparation, the field of cryo-EM is hindered by the inability to perform time-resolved experiments on a sufficiently fast time scale to observe the progression of many biological reactions due to the inherent lag of traditional blotting methods. The Frank group at Columbia University has developed a lab- prototype of a system to enable this missing capability and enables millisecond time control over the reaction time of sampling biological reactions in the grid preparation process. The purpose of this Fast-Track STTR proposal is to develop the time-resolved system built by the Frank group into a commercial production unit and launch it as a product. Initial Phase I milestones in the proposed work will focus on building a prototype with added control of reaction parameters to determine necessary control variables in the system and to optimize sample grid preparation and time resolution. Further work in Phase II of this Fast-Track proposal will create a streamlined, user-friendly production unit which will maintain the necessary controls as determined in initial milestones, while optimizing manufacturing time and reducing overall production costs. The resulting system will add another dimension to the field of cryo-EM, providing users with the capability to capture transient intermediates in reaction progression studies and further clarifying proposed reaction mechanisms with time resolution.