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.
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