ZERNIKE PHASE-CONTRAST IMAGING

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： 我们正在研究使用Zernike相位板（Danev和Nagayama，Ultramicroscopy，88：243-252，2001）来增强冷冻水合样品的电子层析成像的相位对比度。 相位板放置在物镜透镜的后焦平面上，在我们的情况下（在400 kV下），由一个50 μ m的物镜孔径组成，覆盖有一个34 nm厚的碳膜，其中包含一个1 μ m的中心孔。 未散射的电子和以低角度（代表非常低的空间频率）散射的电子穿过中心孔，而样本散射的大多数电子穿过碳膜并经历½/2的额外相移，大大增加了对比度。 使用相位板，在物镜透镜聚焦的情况下进行成像，并且可以在比使用高物镜透镜欠焦的常规相位对比成像可能的更宽的空间频率范围上获得优异的相位对比。 对于典型的碳膜Zernike相位板，约2和20 nm之间的信息传递几乎恒定在约85%（由于相位板内的散射，传递不是100%）。 对于使用10-20 μ m的典型欠焦值的常规相衬成像，振荡CTF中的零点出现在d = 4和d = 6 nm之间的空间频率处，导致有价值的结构信息的损失。 使用相位板时，整体对比度大约增加一倍，在某些空间频率下增加多达五倍。 这对于低温电子断层扫描是至关重要的，因为它允许以较低的电子剂量记录倾斜图像。 每个图像的较低剂量意味着可以记录更多的倾斜图像，从而导致更高的分辨率，而不会出现与较高电子剂量相关的损坏。

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: We are investigating the use of the Zernike phase plate (Danev and Nagayama, Ultramicroscopy, 88:243-252, 2001) to enhance phase contrast for electron tomography of frozen-hydrated specimens. The phase plate is placed in the back focal plane of the objective lens, and in our case (at 400 kV) consists of a 50 ¿m objective aperture covered with a 34-nm-thick carbon film containing a 1-¿m central hole. Unscattered electrons, and those scattered at low angles (representing very low spatial frequencies) pass through the central hole, while most electrons scattered by the specimen pass through the carbon film and undergo an additional phase shift of ¿/2, greatly increasing the contrast. Using the phase plate, imaging is done with the objective lens in focus, and excellent phase contrast can be obtained over a wider spatial-frequency range than is possible with conventional phase-contrast imaging, which uses high objective lens underfocus. With a typical carbon-film Zernike phase plate, the transfer of information between about 2 and 20 nm is almost constant at about 85% (transfer is not 100% because of scattering within the phase plate). With conventional phase contrast imaging using typical underfocus values of 10-20 ¿m, zeroes in the oscillating CTF occur at spatial frequencies between d = 4 and d = 6 nm, causing loss of valuable structural information. Overall contrast is approximately doubled with a phase plate, with the increase of as much as five times at certain spatial frequencies. This is critically important for cryo-electron tomography because it allows tilt images to be recorded at a lower electron dose. The lower dose per image means that more tilt images can be recorded, resulting in higher resolution without the damage associated with a higher electron dose.