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 [unreadable]m objective aperture covered with a 34 nm-thick carbon film. The inner potential of this film shifts the phase by [unreadable]/2 for 400 kV, changing the form of the CTF from sine to cosine. There is a 1-[unreadable]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 [unreadable]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.