DEVELOPMENT LOW TEMPERATURE AFM/FREEZE FRACTURE AND ETCH

描述：(改编自申请者的摘要)原子力 显微镜(AFM)使用锋利的探头来获得表面拓扑结构 标本，已广泛应用于各种生物材料。 尽管它能够在硬材料上实现高空间分辨率，但 在水合生物样品上获得的分辨率要低得多， 除了几个特殊情况。实现高水平的主要限制因素 分辨率是指这些材料的柔软程度和相对较大的探头 原子力显微镜中使用的力。即使有0.l nN的探测力， 探测器与样品的接触仍可能是大气的数千倍 在纳米分辨率下的压力。由于探测力受温度的限制 噪声和其他因素，低温原子力显微镜已被建议作为一种 克服这些限制的替代方案。 在之前的项目期间，调查人员声称成功地 在常压下构建了液氮蒸气中的低温原子力显微镜。它 证明了这是一种比基于真空的 系统，因为表面污染是真空系统的一个主要问题 低温原子力显微镜被完全消灭了。高分辨率AFM图像 在低于100K的温度下获得生物大分子， 展示了低温原子力显微镜在结构研究中的潜力。多数 重要的是，对个体免疫球蛋白和DNA的直接测量表明 杨氏模数是衡量一种材料硬度的指标，L为000-10,000 在低温下更大，提供了最重要的 低温原子力显微镜在结构生物学中的验证。到目前为止，这是 只有适用于生物研究的功能性低温原子力显微镜。 在这一更新期间，调查人员计划继续努力， 用于结构生物学的低温原子力显微镜的发展。除了……之外 持续的工具改进，高分辨率的方法 生物低温原子力显微镜将是主要的焦点，其中包括两个标本 制备技术，如冷冻断裂，以及制造 超锋利的AFM提示。为了实现这些目标，他们将建立一个 自给自足的无污染样品制备站和意志 对现有扫描电子显微镜进行改装以沉积超锐 悬臂上的小费。有了这些工具，他们将描绘出 准备高质量生物样品的必要程序。他们的 最终目标是达到比L纳米更好的表面分辨率 并有能力对破裂的表面进行成像 一个完整的膜蛋白的跨膜结构域可以是 直接评估，没有其他处理，如重金属阴影。 如此高的分辨率将使研究构象变化成为可能。 蛋白质和蛋白质-蛋白质以及蛋白质-核酸相互作用。 低温原子力显微镜的其他应用包括细胞表面的结构 细胞和细胞器的3-D(切片)成像，可控蚀刻 以及去除暴露的材料。这些独特的功能使 低温原子力显微镜是一种多功能的，但功能强大的生物学结构探测器。

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.