Nanofabricated Tools for Single Molecule Studies

描述（由申请人提供）：本文提出的技术开发将导致新的光学显微镜方法，该方法具有亚20 nm空间分辨率、单分子检测限、埃级切片能力、同时荧光和力敏感性，以及在生理条件下对活细胞系统进行所有这些测量的能力。目标是开发新的技术和方法，结合联合收割机属性从近场扫描光学显微镜（NSOM），原子力显微镜（AFM），荧光共振能量转移（FRET），以提高成像能力的单分子研究可行的生物组织。在具体目标1和2中开发的NSOM/AFM和NSOM/FRET探针将与许多商业AFM仪器兼容，为目前进行AFM测量的仪器增加重要的新对比机制。一旦在对照样品上进行测试和验证，根据具体目标1和2开发的纳米技术将应用于具体目标3中的核膜研究。这些技术的高分辨率和灵敏度将被用来解开关键的悬而未决的问题，有关的作用和身份的“塞子”观察到核孔复合物，塞子响应内腔钙耗竭，分布和相关性的核孔与钙通道的膜，和塞子运动的动力学在核孔复合物。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无法实现的

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