Towards higher resolution and cellular imaging using cryo-electron tomography

研究大分子复合物的结构对于理解它们如何发挥作用至关重要。此外，在其细胞环境中研究大分子复合物的结构提供了关于其分子功能的最可靠的信息。冷冻电子断层扫描（cryo-ET）是一种功能强大的成像技术，可提供有关细胞结构的三维信息，而无需化学固定和染色伪影。Cryo-ET可达到约10 nm分辨率，最高可达3 nm，亚断层图像可在计算机中进行平均。限制低温ET分辨率的因素是电子剂量限制辐射损伤和厚样品。该分辨率给出了关于蛋白质的整体形状的信息，但不能提供任何分子机制。本论文的研究重点是以微管偶态为测试样品，将低温电子显微镜的分辨率提高到亚纳米级。在这样的分辨率下，二级结构是清晰可识别的，因此，亚基的原子模型可以对接到实验断层图中，以获得对蛋白质-蛋白质相互作用和分子机制的新见解。我的研究还将推动冷冻ET在较厚的样品上工作，即通过使用相位板，直接电子探测器和改进的采集方案来提高信噪比，从而观察部分细胞。这将为我们提供一个独特的能力，打开一个窗口进入细胞，看看细胞过程作为前所未有的分辨率。此外，为了解决细胞过程的动态性质，我的实验室将开发相关的光电子显微镜技术，该技术将cryo-ET的能力与荧光显微镜的特异性识别能力相结合，以精确定位感兴趣的蛋白质在其周围环境中，从而深入了解基本的生物学机制。

Studying the structure of macromolecular complexes is essential for understanding how they function. Furthermore, studying the structure of the macromolecular complexes within their cellular context provides the most reliable information about their molecular function. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that provides three-dimensional information about cellular architecture devoid of chemical fixation and staining artifacts. Cryo-ET can reach ~10 nm resolution and up to 3 nm with subtomogram averaging in silico. The limiting factor in cryo-ET resolution is the electron dose-limiting radiation damage and thick specimen. This resolution gives the information about the overall shape of the proteins but is not able to provide any molecular mechanism. My research focus is to push the resolution of cryo-ET to sub-nanometer resolution using the microtubule doublet as the test sample. At such resolutions, secondary structures are clearly identifiable and consequently, atomic models of subunits can be docked into the experimental tomographic maps to gain novel insights into protein-protein interactions and molecular mechanism. My research will also push cryo-ET to work on thicker sample i.e. looking at part of the cells by improving the signal-to noise ratio using the phase plate, direct electron detector and improved acquisition scheme. This will provide us with a unique ability to open a window into the cell to look at cellular process as unprecedented resolution. Furthermore, to address the dynamic nature of the cellular process, my laboratory will develop correlative light-electron microscopy technique, which combines the power of cryo-ET to resolve ultrastructure with the specific identification power of fluorescence light microscopy to precisely localize proteins of interest in the context of their surroundings to yield insights into fundamental biological mechanisms.