DEVELOPMENT LOW TEMPERATURE AFM/FREEZE FRACTURE AND ETCH

描述：（改编自申请人的摘要）原子力 原子力显微镜（AFM）使用尖锐的探针来获得纳米颗粒的表面拓扑结构。 标本，已被应用于广泛的生物材料。 尽管它在硬材料上具有高空间分辨率的能力， 在水合生物样品上获得的分辨率已经低得多， 除了一些特殊情况。 一个主要的限制因素，在实现高 分辨率是这些材料的柔软度， AFM中使用的力。 即使使用0.1 nN的探针力， 探针与样品的接触仍然是大气的数千倍 分辨率为nm。 由于探针力受到热应力的限制， 噪声和其他因素，低温原子力显微镜已被建议作为一种 另一种方法是克服这些局限性。 在上一个项目期间，调查人员声称已经成功地 构建了一个低温原子力显微镜在液氮蒸气在环境压力下。 它 证明了这是优于基于真空的 系统，因为表面污染，一个主要的问题，真空为基础的 冷冻原子力显微镜被完全淘汰了 高分辨率AFM图像 在低于100 K的温度下获得生物大分子， 展示了cryo-AFM用于结构研究的潜力。 最 重要的是，对个体IgG和DNA的直接测量表明， 杨氏模量是一种材料硬度的量度，它是1000 - 10000 在低温下的温度要高出一倍，提供了最重要的 cryo-AFM的结构生物学验证。 到目前为止，这是 只有功能cryo-AFM适用于生物研究。 在此更新期间，调查人员计划继续努力， cryo-AFM在结构生物学中的应用 除了 不断改进工具， 生物冷冻原子力显微镜将是主要的焦点，其中包括标本 制备技术，如冷冻断裂，以及制造 超锋利的AFM针尖 为了实现这些目标，他们将建立一个 独立的无污染样品制备站， 改造现有的扫描电子显微镜以沉积超锐 悬臂上的尖端 有了这些仪器，他们将描述 制备高质量生物样品的必要程序。 他们的 最终的目标是在100 μ m上实现优于1 nm的表面分辨率。 大分子，并具有成像断裂表面的能力， 一个完整的膜蛋白的跨膜结构域可以 直接评估，无需其他处理，如重金属阴影。 如此高的分辨率将使得能够研究 蛋白质和蛋白质-蛋白质，以及蛋白质-核酸关联。 低温原子力显微镜的其他应用包括细胞表面的结构 和细胞和细胞器的3D（截面）成像， 以及去除暴露的材料。 这些独特的功能使 cryo-AFM是一种多功能，但功能强大的生物学结构探针。

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.