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: 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 (at 400 kV) consists of a 50 ?m objective aperture covered with a 34-nm-thick carbon film containing a 1-?m central hole. Unscattered electrons, and those scattered at low angles (representing very low spatial frequencies) pass through the central hole, while most electrons scattered by the specimen pass through the carbon film and undergo an additional phase shift of ?/2, greatly increasing the contrast. Using the phase plate, imaging is done with the objective lens in focus, and excellent phase contrast can be obtained over a wider spatial-frequency range 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. Overall contrast is approximately doubled with a phase plate, with the increase of as much as five times at certain spatial frequencies. This is critically important for cryo-electron tomography because it allows tilt images to 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.
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