NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 采用组分在网格混合和闪光光解技术的微滴喷涂的一个严重局限性是，反应发生在碳涂层网格的表面，涉及大分子络合物的反应是否会像在试管中一样发生似乎是值得怀疑的。使用孔栅可能会有所帮助，但这并不总是可行的，也不能消除反应物与碳膜或冻结前存在的空气-水界面的相互作用。预混合模式是首选的，但由于它依赖于在网格上沉积反应混合物后用滤纸吸墨，时间分辨率较差(即，在几秒钟的数量级)。理想情况下，动力学实验应在体相体外进行，条件应尽可能接近生理条件，并应以选定的时间间隔采集样品并在EM网格上瞬间冷冻。上一节描述的微型混合器/喷雾器是解决这些问题的一种方法。 在另一种方法中，我们正在努力评估微/纳米制造以及微和纳米流体技术执行快速(毫秒时间尺度)预混合操作模式的可行性，方法是演示一种正在运行的微型制造原型装置，该装置直接在改进的格栅上或在施加到格栅之前混合亚微升体积的反应物，然后近乎瞬时地冻结格栅。成功的实施将使这一方法成为有效发挥作用的大分子组件的时间分辨低温EM的选择方法，并将为更复杂的设备奠定基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: A serious limitation of the microdroplet spraying with on-grid mixing of components and flash photolysis techniques is that the reactions take place on the surface of a carbon-coated grid, and it seems questionable whether reactions involving macromolecular complexes will occur in this environment in the same way as they do in the test tube. Using holey grids could help, but this is not always feasible and does not eliminate interactions of the reactants with the carbon film or at the air-water interface that is present prior to freezing. The pre-mix mode is preferred, but since it relies on blotting with filter paper following deposition of the reaction mixture on the grid, the time resolution is poor (i.e., in the order of seconds). Ideally, kinetics experiments should be conducted in bulk phase in vitro under conditions that are as close to physiological as possible, and at selected time intervals samples should be taken and frozen on EM grids instantaneously. The micro-mixer/sprayer described in the previous section is one approach to solving these problems. In another approach, we are working to assess the feasibility of micro/nanofabrication and micro- and nanofluidics technologies for performing fast (millisecond time-scale) pre-mix modes of operation by demonstration of a functioning microfabricated prototype device that mixes sub-microliter volumes of reactants either directly on a modified grid or before application to the grid, and then freezes the grid near-instantaneously. Successful implementation would establish this as the method of choice for time-resolved cryo-EM of actively functioning macromolecular assemblies, and would lay the groundwork for more sophisticated devices.