NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要： 微滴喷雾与组件的网格上混合和闪光光解技术的一个严重限制是，反应发生在碳涂层网格的表面上，并且似乎值得怀疑的是，涉及大分子复合物的反应是否会在这种环境中以与试管中相同的方式发生。 使用多孔网格可能有所帮助，但这并不总是可行的，并且不能消除反应物与碳膜或在冷冻前存在的空气-水界面处的相互作用。 的 预混 模式是优选的，但是由于它依赖于在反应混合物沉积在网格上之后用滤纸进行印迹，时间分辨率差（即，以秒的量级）。 理想情况下，动力学实验应在体外本体相中在尽可能接近生理的条件下进行，并且应在选定的时间间隔内采集样品并立即在EM网格上冷冻。前一节中描述的微混合器/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/纳米纤维和微-和纳米流体技术的可行性，用于执行快速（毫秒时间尺度）的预混合操作模式，通过演示一个功能微制造的原型设备，该原型设备直接在修改后的网格上或在应用到网格之前混合亚微升体积的反应物，然后几乎瞬间冻结网格。成功的实施将建立这作为时间分辨的低温EM的方法的选择积极发挥作用的大分子组件，并将奠定基础，更复杂的设备。

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.