DEVELOPMENT LOW TEMPERATURE AFM/FREEZE FRACTURE AND ETCH

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DESCRIPTION: (Adapted from the applicant's abstract) The atomic force microscope (AFM) uses a sharp probe to obtain the surface topology of the specimen, which has been applied to a broad range of biological materials. Despite its ability of high spatial resolution on hard materials, the resolution obtained on hydrated biological samples has been much lower, except for a few special cases. A major limiting factor in achieving high resolution is the softness of these materials and the relatively large probe force used in an AFM. Even with 0.l nN probe force, the pressure at the probe-sample contact can still be thousands of times the atmospheric pressure at nm resolution. Since the probe force is limited by the thermal noise and other factors, cryonic temperature AFM has been suggested as an alternative to overcome these limitations. In the previous project period, the investigators claim to have successfully constructed a cryo-AFM in liquid nitrogen vapor under ambient pressure. It was demonstrated that this was the preferred approach over a vacuum-based system, because surface contamination, a major problem for vacuum-based cryo-AFMs, is completely eliminated. High resolution AFM images of biological macromolecules were obtained at temperatures below l00 K, demonstrating the potential of cryo-AFM for structural research. Most importantly, direct measurements on individual IgG and DNA indicate that the Young's modulus, a measure of the stiffness of a material, is l,000-10,000 times greater at cryogenic temperatures, providing the most important validation of the cryo-AFM for structural biology. To date, this is the only functional cryo-AFM suitable for biological research. In this renewal period, the investigators plan to continue their effort in the development of cryo-AFM for structural biology. In addition to continued instrumental improvement, the methodology for high-resolution biological cryo-AFM will be the main focus, which includes both the specimen preparation technique, such as freeze fracture, and the fabrication of super-sharp AFM tips. To achieve these objectives, they will construct a self-contained contamination-free specimen preparation station and will modify an existing scanning electron microscope for depositing super-sharp tips on a cantilever. With these instruments, they will characterize the necessary procedures for preparing high-quality biological samples. Their ultimate goal is to achieve a surface resolution of better than l nm on macromolecules and to have the ability to image the fractured surfaces so that the trans-membrane domains of an integral membrane protein can be directly evaluated without other treatments, such as heavy metal shadowing. Such a high resolution will enable to study the conformational changes of proteins and protein-protein, as well as protein-nucleic acids associations. Other applications of the cryo-AFM include the structure of a cell surface and 3-D (sectional) imaging of cells and organelles with controlled etching and removal of exposed materials. These unique capabilities make the cryo-AFM a versatile, yet powerful, structural probe for biology.