Nanofabricated Tools for Single Molecule Studies

描述(由申请人提供)：这里提出的技术开发将导致新的光学显微镜方法，具有低于20 nm的空间分辨率、单分子检测极限、Angstrom水平切片能力、同时荧光和力敏感性，并能够在生理条件下对活的细胞系统进行所有这些测量。其目标是开发结合近场扫描光学显微镜(NSOM)、原子力显微镜(AFM)和荧光共振能量转移(FRET)属性的新技术和方法，以增强单分子研究活生物组织的成像能力。将在特定目标1和2中开发的NSOM/AFM和NSOM/FRET探头将与许多商用AFM仪器兼容，为目前正在进行AFM测量的仪器增加了重要的新对比度机制。一旦在对照样品上进行测试和验证，在特定目标1和2下开发的纳米技术将被应用于特定目标3的核膜研究。这些技术的高分辨率和高灵敏度将被用来揭开关键的悬而未决的问题，这些问题涉及在核孔复合体中观察到的“塞子”的作用和身份，对腔钙耗竭的塞子响应，核孔与膜中钙通道的分布和相互关系，以及核孔复合体中的塞子运动的动力学。这些实验将在生理条件下的活性核膜上进行，这是迄今为止近场技术所不可能做到的。

DESCRIPTION (provided by applicant): The technique development proposed here will result in new optical microscopy methods that have sub-20 nm spatial resolution, single molecule detection limits, Angstrom level sectioning capabilities, simultaneous fluorescence and force sensitivity, and the ability to carry out all of these measurements on living cellular systems under physiological conditions. The goal is to develop new techniques and methods that combine attributes from near-field scanning optical microscopy (NSOM), atomic force microscopy (AFM), and fluorescence resonance energy transfer (FRET) to enhance imaging capabilities for single molecule studies viable biological tissue. The NSOM/AFM and NSOM/FRET probes to be developed in specific aims 1 and 2 will be compatible with many commercial AFM instruments, adding important new contrast mechanisms for those currently conducting AFM measurements. Once tested and validated on control samples, the nanotechnology developed under specific aims 1 and 2 will be applied to studies on the nuclear membrane in specific aim 3. The high resolution and sensitivity of these techniques will be utilized to unlock key outstanding issues concerning the role and identity of the "plug" observed in nuclear pore complexes, the plug response to lumenal calcium depletion, the distribution and correlation of nuclear pores with calcium channels in the membrane, and the dynamics of plug motion in the nuclear pore complex. These experiments will be done on viable nuclear membranes under physiological conditions, something heretofore not possible with the near-field technique