IVEM OPERATION AND IMPROVEMENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： IVEM(JEOL JEM-4000FX)主要用于冷冻水化标本的TRD，即电子断层扫描。IVEM配备了LaB6阴极、5轴计算机控制测角仪和多个双倾斜和倾斜旋转试件工作台，以及GaTan单倾斜和倾斜旋转低温转移工作台。2002年，我们完成了Gatan GIF2002柱后能量滤光片的安装，配备了一台2048x2048ccd摄像机，并安装了一台TVIPS FastScan电视速率ccd摄像机，用于在极低的电子剂量下跟踪光束敏感的样品。自动电子断层扫描是通过外部计算机控制提供的。仪器分辨率为0.14 nm(金点阵)。STEM模点对点分辨率为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相板(Danev和Nagayama，超微显微镜，88：243-252,2001)来增强冷冻水化样品的电子断层扫描的位相对比度。位相板被放置在物镜的后焦平面上，在我们的例子中，由一个50？m的物镜孔径组成，上面覆盖着一层34 nm厚的碳膜。该薄膜的内电势在400千伏时使相位移动？/2，使CTF的形式从正弦变为余弦。在碳膜上有一个1？m的中心孔，最低频率的信息通过这个孔没有相移。 利用相位板，在物镜聚焦的情况下进行成像，与使用高物镜下焦距的传统相衬成像相比，可以在更宽的有用空间频率范围内获得良好的相衬。对于典型的碳膜Zernike相板，大约2到20 nm之间的信息传输几乎是恒定的，大约85%(由于相板内的散射，传输不是100%)。对于使用10-20m的典型欠焦值的常规相位对比度成像，振荡CTF中的零点在d=4和d=6 nm之间的空间频率处出现，导致有价值的结构信息丢失。 与理想的相位板相比，整体对比度几乎翻了一番。这对于低温电子断层成像至关重要，因为倾斜图像可以在较低的电子剂量下记录。每幅图像的剂量较低意味着可以记录更多的倾斜图像，从而获得更高的分辨率，而不会受到与较高电子剂量相关的损害。

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.