IVEM OPERATION AND IMPROVEMENTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。JEOL JEM-4000FX IVEM配备了LaB6阴极、5轴计算机控制的测角仪和多个样品支架，包括单倾斜和倾斜旋转冷冻转移支架。IVEM最近配备了Gatan GIF2002柱后能量滤光器，该滤光器配备了2048 × 2048像素CCD相机，该相机具有特殊的高灵敏度荧光粉闪烁体。采用TVIPS FastScan电视速率CCD摄像机对低温样品进行低剂量测量。使用Emispec ES Vision系统和内部编写的软件，通过外部计算机控制进行自动低剂量能量过滤断层扫描。ES视觉系统还集成了STEM， EELS和EDX，具有完整的微分析能力。TEM的仪器分辨率为0.14 nm（金晶格），STEM为1 nm， EELS为0.9至2 eV。Zernike相位板的实施一个项目已经开始调查使用Zernike相位板（Danev和Nagayama， Ultramicroscopy, 88:243- 252,2001）来增强相位对比。应用将包括冷冻水合标本的电子断层扫描。相位板放置在物镜的后焦平面上，在我们的例子中，相位板由一个50¿m的物镜孔径组成，上面覆盖着一层34nm厚的碳膜。薄膜的内势改变了？/2，将CTF的形式从正弦变为余弦。有一个1-？碳膜上的M个中心孔，最低频率信息通过该孔而不发生相移。使用相位板，成像是在物镜聚焦的情况下完成的，并且与使用高物镜聚焦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出色的相衬。对于典型的碳膜Zernike相板，2 ~ 20 nm之间的信息转移几乎恒定在85%左右（由于相板内部的散射，转移不是100%）。传统的相位对比成像使用典型的欠聚焦值10-20¿m，振荡CTF中的零点出现在d = 4和d = 6 nm之间的空间频率，导致有价值的结构信息丢失。经过三十年的发展，Zernike相板在TEM中的应用再次被引入（Danev和Nagayama， Ultramicroscopy, 88:243- 252,2001）。相板的采用一直被推迟，主要是因为早期tem的真空系统不够清洁，无法避免由于相板污染而导致的过度充电。Nagayama已经证明，一个清洁的真空系统，或加热相板，可以克服充电问题。马可先生参观了位于日本冈崎的长山实验室，以获得使用相位板的经验。这与Nagayama博士组织的“生物电子显微镜前沿”研讨会的特邀演讲同时进行。在特殊的多孔物镜孔径上制作了泽尼克相板。中心孔在奥尔巴尼纳米技术公司使用我们计划用于TRD#1的聚焦离子束仪器进行研磨。完成的相板在安装于显微镜前用等离子体蚀刻清洗。采用零损耗能量滤波和400kV加速电压，记录了各种冷冻水合和干燥样品以及碳膜的图像。获得了如何使用相板的经验。绘制了功率谱和傅立叶环相关图，并对相板的行为进行了表征。在零离焦时可以获得良好的相衬。与典型的冷冻水合样品电子断层扫描条件下的高欠焦图像相比，相位板图像在高空间频率和全面高对比度下具有更好的转移。初始实验中使用的中心孔直径为1¿m，当适当居中时，在d = 9 nm处切断相移的低空间频率。新相位板的中心孔为0.5¿m，可以将相移低频扩展到约d = 20 nm。在一份NSF拨款提案中，我们与LBL的Dieter Typke博士和RPI的J. Jay McMahon共同研究人员打算采用上述碳泽尼克相板，用于低温电子断层扫描倾斜系列的收集。同时，我们提出了一种布尔施静电相板的制作方法。Boersch相板使用一个中心环电极来相移轴上未分散的电子。它优于Zernike型，因为它避免了相板本身内部的散射，从而避免了对比度的轻微下降。此外，由于电极激励是可调的，它可以在一个加速电压范围内使用。到目前为止，还没有报道过布尔施相板的制造，我们的计划是使用RPI的微加工设施来实现这一目的。

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 JEOL JEM-4000FX IVEM is equipped with a LaB6 cathode, a 5-axis computer-controlled goniometer, and several specimen holders, including single-tilt and tilt-rotation cryo-transfer holders. The IVEM was recently equipped a Gatan GIF2002 post-column energy filter equipped with a 2048x2048-pixel CCD camera that has a special high-sensitivity phosphor scintillator. A TVIPS FastScan TV-rate CCD camera is used for low-dose survey of cryo specimens. Automated low-dose energy-filtered tomography is carried out via external computer control using an Emispec ES Vision system and software written in-house. The ES vision system also integrates STEM, EELS, and EDX for full microanalytical capability. The instrumental resolution is 0.14 nm (gold lattice) for TEM, 1 nm for STEM, and 0.9 to 2 eV for EELS. Implementation of the Zernike phase plate A project has started to investigate the use of the Zernike phase plate (Danev and Nagayama, Ultramicroscopy, 88:243-252, 2001) to enhance phase contrast. Applications would include 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 34nm-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. After three decades, use of the Zernike phase plate for TEM is being re-introduced (Danev and Nagayama, Ultramicroscopy, 88:243-252, 2001). Adoption of the phase plate has been delayed primarily because the vacuum systems of early TEMs were not clean enough to avoid excessive charging due to contamination of the phase plate. Nagayama has shown that a clean vacuum system, or heating of the phase plate, can overcome the charging problem. M. Marko visited the Nagayama lab in Okazaki, Japan to get experience with use of a phase plate. This was in conjunction with invited talk at a symposium on ¿Frontiers in Biological Electron Microscopy¿ that was organized by Dr. Nagayama. Zernike phase plates were made on special multi-hole objective apertures. The central holes were milled at Albany Nanotech using the focused ion beam instrument we plan to use for TRD#1. Completed phase plates were cleaned by plasma etching before installation in the microscope. Images were recorded from various frozen-hydrated and dry specimens, as well as carbon films, using zero-loss energy filtering and 400kV accelerating voltage. Experience was gained in how to use the phase plate. Power spectra and Fourier ring correlations were plotted, and the behavior of the phase plate was characterized. Good phase contrast at zero defocus could be obtained. Compared to high-underfocus images taken under conditions typical for electron tomography of frozen-hydrated specimens, the phase plate images had better transfer at high spatial frequencies and all-over higher contrast. The central hole used in the initial experiments, 1 ¿m in diameter, cut off the phase-shifted low spatial frequencies at d = 9 nm when properly centered. New phase plates were made with a 0.5 ¿m central hole, which should extend phase-shifted low frequencies to about d = 20 nm. In an NSF grant proposal, with co-investigators Dr. Dieter Typke of LBL and J. Jay McMahon of RPI, we intended to adapt the carbon Zernike phase plate, described above, for collection of cryo-electron tomographic tilt series. At the same time, we proposed to fabricate a Boersch electrostatic phase plate. The Boersch phase plate uses a central ring electrode to phase-shift the on-axis, unscattered electrons. It is superior to the Zernike type because scattering within the phase plate itself is avoided, thus avoiding a slight decrease in contrast. In addition, since the electrode excitation is adjustable, it can be used over a range of accelerating voltages. Fabrication of a Boersch phase plate has so far not been reported, and our plan was to use the microfabrication facilities at RPI for this purpose.