IVEM OPERATION AND IMPROVEMENTS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。摘要：JEOL JEM-4000FX IVEM配备了一个LaB6阴极，一个5轴计算机控制的测角仪，以及几个样品夹具，包括单倾斜和倾斜旋转的低温转移夹具。IVEM最近配备了Gatan GIF2002柱后能量过滤器，配备了2048x2048像素的CCD摄像头，具有特殊的高灵敏度荧光粉闪烁体。TVIPS FastScan TV-rate CCD相机用于低温样品的低剂量测量。自动低剂量能量过滤断层扫描是通过使用Emispec es Vision系统和内部编写的软件通过外部计算机控制进行的。ES VISION系统还集成了STEM、EELS和EDX，以实现完全的微量分析能力。仪器分辨率分别为0.14 nm(金点阵)、1 nm(STEM)和0.9-2 eV(电致发光强度)。Zernike相位板的实施A项目已经开始调查Zernike相位板的使用(Danev和Nagayama，超微显微镜，88：243-252,2001)以增强位相对比度。应用将包括冷冻水合标本的电子断层扫描。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400千伏时使位相移动？/2，使CTF的形式由正弦变为余弦。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。三十年后，用于透射电子显微镜的Zernike相板正在被重新引入(Danev和Nagayama，超微显微镜，88：243-252,2001)。相位板的采用被推迟，主要是因为早期TEM的真空系统不够干净，无法避免由于相位板的污染而过度充电。长山已经证明，清洁的真空系统，或相位板的加热，可以克服充电问题。M.Marko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。这是与长山博士组织的生物电子显微镜前沿研讨会的特邀演讲相结合的。Zernike位相板是在特殊的多孔物镜上制作的。在Albany Nanotech使用我们计划用于TRD#1的聚焦离子束仪器对中心孔进行了研磨。在安装到显微镜之前，完成的相位板通过等离子蚀刻进行了清洗。使用零损失能量过滤和400KV加速电压，记录了各种冷冻水化和干燥样品以及碳膜的图像。总结了如何使用相位板的经验。绘制了功率谱和傅里叶环关联式，并对相板的行为进行了表征。在零散焦时可以获得良好的位相对比度。与冷冻水化样品的电子断层扫描的典型条件下拍摄的高欠焦图像相比，位相板图像在高空间频率和全面高对比度下具有更好的传递效果。最初实验中使用的直径为1？m的中心孔，当适当居中时，切断了d=9 nm处的相移低空间频率。新的相位板中心有一个0.5°m的孔，可以将相移的低频扩展到d=20 nm左右。在NSF的一份拨款提案中，我们与LBL的Dieter Typke博士和RPI的J.Jay McMahon博士一起，打算改装如上所述的碳泽尼克相板，用于收集冷冻电子断层扫描倾斜系列。同时，我们还提出了制作Boersch静电位相板的设想。Boersch相位板使用中心环电极对轴上未散射的电子进行相移。它优于Zernike类型，因为避免了位相板本身的散射，从而避免了对比度的轻微降低。此外，由于电极激励是可调的，因此可以在一定范围的加速电压范围内使用。到目前为止，还没有关于Boersch相板的制造的报道，我们的计划是使用RPI的微制造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.