IVEM OPERATION AND IMPROVEMENTS

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来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 摘要： IVEM（JEOL JEM-4000 FX）主要用于TRD 1，冷冻水合标本的电子断层扫描。 IVEM配备了一个LaB 6阴极，一个5轴计算机控制的测角仪，和几个双倾斜和倾斜旋转样品台，以及Gatan单倾斜和倾斜旋转低温转移台。 2002年，我们完成了Gatan GIF 2002柱后能量过滤器的安装，该过滤器配备了2048 x2048 CCD摄像机，并安装了TVIPS FastScan电视速率CCD摄像机，用于在非常低的电子剂量下跟踪束敏感标本。 通过外部计算机控制提供自动电子断层扫描。仪器分辨率为0.14 nm（金晶格）。 STEM模式点到点分辨率为1 nm。 提供完整的分析能力，包括EELS和EDX。 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，在我们的例子中，它由一个50 μ m的物镜孔径组成，上面覆盖着一层34 nm厚的碳膜。 对于400 kV，该薄膜的内部电势使相位偏移<$/2，将CTF的形式从正弦变为余弦。 在碳膜中有一个1-m的中心孔，最低频率的信息通过该孔而没有相移。 使用相位板，成像是在物镜透镜聚焦的情况下完成的，并且与使用高物镜透镜欠焦的常规相位对比成像相比，可以在更宽范围的有用空间频率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。 使用理想的相位板，总对比度大约加倍。 这对于低温电子断层扫描是至关重要的，因为倾斜图像可以在较低的电子剂量下记录。 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: The IVEM (JEOL JEM-4000FX) is primarily used for TRD1, electron tomography of frozen- hydrated specimens. The IVEM is equipped with a LaB6 cathode, a 5-axis computer-controlled goniometer, and several double-tilt and tilt-rotation specimen stages, as well as Gatan single-tilt and tilt-rotation cryo-transfer stages. In 2002, we completed installation of a Gatan GIF2002 post-column energy filter equipped with a 2048x2048 CCD camera, and a TVIPS FastScan TV-rate CCD camera was installed for tracking beam-sensitive specimens at very low electron dose. Automated electron tomography is provided via external computer control. The instrumental resolution is 0.14 nm (gold lattice). The STEM-mode point-to-point resolution is 1 nm. Full analytical capability is provided, including EELS and EDX. 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 consists of a 50 ¿m objective aperture covered with a 34 nm-thick carbon film. The inner potential of this film shifts the phase by ¿/2 for 400 kV, changing the form of the CTF from sine to cosine. There is a 1-¿m central hole in the carbon film, through which the lowest-frequency information passes without phase shift. Using the phase plate, imaging is done with the objective lens in focus, and excellent phase contrast can be obtained over a wider range of useful spatial frequencies 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. The overall contrast is approximately doubled with an ideal phase plate. This is critically important for cryo-electron tomography because tilt images can 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.