Comolli Luis R, Downing Kenneth H
Life Science Division, Lawrence Berkeley National Laboratory, CA, USA.
J Struct Biol. 2005 Dec;152(3):149-56. doi: 10.1016/j.jsb.2005.08.004. Epub 2005 Sep 13.
Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality.
电子断层扫描是目前唯一一种能够在未受干扰的细胞环境中直接对大分子进行三维可视化的方法。原则上，断层扫描应能够识别和定位完整细菌内嵌入无定形冰中的主要大分子复合物。为了优化记录数据的条件，使我们接近理论极限，我们在此展示了在液氦和液氮温度下嵌入无定形冰中的新月柄杆菌细胞的剂量耐受性比较。这种革兰氏阴性细菌的内膜和外膜以及S层的周期性结构提供了理想的特征，可用于监测对比度和有序度随剂量的变化。在氦气和氮气温度下，S层的有序度损失在相当的剂量下发生。在两个温度下，细胞和塑料支撑体内都会出现宏观气泡，但在氦气温度下气泡形成得更慢。在氦气温度下，气泡的纹理在初始阶段更细，给人一种标本某些部分对比度反转的印象。气泡在不同的细胞器中以不同的方式演变，这可能是由于它们不同的化学成分和机械性能所致。最后，无定形冰在氦气温度下“流动”，导致标本内标记物的相对位置发生变化并使细胞变形。我们得出结论，对于全细胞的冷冻电子断层扫描，液氮温度可提供更好的整体数据质量。
