NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。摘要：微滴喷射和闪光光解技术的一个严重限制是，这些反应发生在碳涂层网格表面，涉及大分子复合物的反应是否会像在试管中一样在这种环境下发生，这似乎是值得怀疑的。使用多孔网格可能会有所帮助，但这并不总是可行的，并且不能消除冷冻前存在的反应物与碳膜或空气-水界面的相互作用。“预混合”模式是优选的，但由于它依赖于反应混合物沉积在网格上后用滤纸吸干，时间分辨率很差（即，以秒为单位）。理想情况下，动力学实验应该在体外的体相中进行，在尽可能接近生理的条件下，在选定的时间间隔内，样品应该立即被采集并冷冻在EM网格上。我们将努力展示微/纳米制造和微流体技术执行快速（毫秒时间尺度）预混合操作模式的可行性，通过演示一个功能微制造原型设备，该设备直接在修改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。成功的实施将使其成为时间分辨低温电子显微镜的首选方法，并为更复杂的设备奠定基础。这项工作是与RPI由Omkaram Nalamasu领导的纳米技术小组合作完成的，Omkaram Nalamasu也是NYSTAR材料科学与工程特聘教授，也是纳米制造技术的先驱。此外，还有Toh-Ming Lu教授（Ray Palmer Baker杰出物理学教授兼Rensselaer先进互连系统技术中心主任）和Pulickel Ajayan教授（材料科学与工程教授，碳纳米管领域的先驱）。负责两个机构之间主要互动的RPI工程师是J. Jay McMahon。

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 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. We will work to demonstrate feasibility of micro/nanofabrication and microfluidics 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. This work is being done in collaboration with the nanotechnology group at RPI led by Omkaram Nalamasu, who is also the NYSTAR distinguished professor of Materials Science & Engineering and a pioneer in nanofabrication technology. Additionally, Professors Toh-Ming Lu (Ray Palmer Baker Distinguished Professor of Physics and Director of Rensselaer¿s Center for Advanced Interconnect Systems Technologies) and Professor Pulickel Ajayan, Professor of Materials Science and Engineering and a pioneer in the area of carbon nanotubes. The RPI engineer responsible for the major interaction between the two institutions is J. Jay McMahon.