Scanning Laser Force Microscope with Nanometer Resolution for Dynamic Imaging of Single Biomolecules Under Physiological Conditions

理想的生物显微镜应该具有足够的分辨率来在纳米尺度上对生物大分子进行成像，应该在生理、水条件下进行成像，并且应该具有足够的时间分辨率来跟踪大分子、大分子组装和细胞尺度上的关键动态过程。生物学中一个重要的未解决的问题是无处不在的运动蛋白的微机械功能，它突出了这种仪器的潜在用途。运动蛋白是肌肉收缩、细胞运动、纤毛和鞭毛跳动、有丝分裂、减数分裂、细胞内运输、DNA复制和转录以及蛋白质合成等过程背后的驱动力。可行性研究的目标是开发一种具有分子分辨率的显微镜，该显微镜基于使用单束激光光阱（光镊）扫描蛋白质样品上的纳米硅尖端。捕获激光器将兼作位移/力传感器，记录针尖与样品之间的相互作用力。位移信号将被送入反馈回路，使尖端与样品保持恒定距离，从而对其轮廓进行成像。作为概念验证，细胞骨架蛋白聚合物将被成像以展示空间分辨率。马达蛋白激酶和ncd将在微管上成像，目的是解决马达单个头部的运动及其在微管表面上的跟踪。单头截断电机将被成像进行比较，目的是了解原生kinesin和ncd的两个头部的协同性，并测试单个头部是否可以产生运动。该项目将是一个跨学科的合作项目：1)纳米制造实验室，提供专业知识，开发具有纳米特征和受控表面化学的精确定义硅尖的工艺；2)物理/生物物理实验室，提供仪器制造、显微镜和激光技术方面的专业知识。iii)分子生物学实验室，提供蛋白质生物化学方面的专业知识，并开发使用大肠杆菌表达系统生产具有良好特征的细胞骨架蛋白和运动蛋白以及修饰蛋白的程序。如果成功，该仪器将在微观结构测定方面取得重大进展，与生物研究的要求相匹配。它将使动态成像生物学的基本功能单位，蛋白质，DNA， RNA，“在工作”成为可能。该仪器将有广泛的应用，特别是在生命科学领域，通过“观察和观察事物是如何工作的”，许多基本问题似乎是可以解决的。

The ideal biological microscmpe should have enough resolution to image biological macromolecules on a nanometer scale, should image in physiological, watery conditions, and should have sufficient temporal resolution to follow crucial dynamic processes on the scale of macromolecules, macromolecular assemblies and cells. An important unsolved problem in biology that highlights the potential usefulness of such an instrument is the micromechanical function of the ubiquitous motor proteins. Motor proteins are the driving force behind processes such as muscle contraction, cell locomotion, ciliar and flagellar beating, mitosis, meiosis, intracellular transport, DNA replication and transcription and protein synthesis. The goal of the proposed feasibility study is to develop a microscope with molecular resolution, based on scanning a nanofabricated silicon tip over a protein sample using a single beam laser optical trap (optical tweezers). The trapping laser will double as displacement/force sensor, recording the interaction force between tip and sample. The displacement signal will be fed into a feed-back loop keeping the tip at a constant distance from the sample, thus imaging its contours. As proof-of-concept, cytoskeletal protein polymers will be imaged to demonstrate spatial resolution. The motor proteins kinesin and ncd will be imaged on microtubules, with the goal of resolving the movement of the individual heads of the motors and their tracking on the microtubule surface. Single headed truncated motors will be imaged in comparison, with the goal of understanding the cooperativity of the two heads of native kinesin and ncd, and to test if a single head can produce movement at all. The project will be an interdisciplinary collaboration between i) a nanofabrication laboratory, providing expertise to develop a process to manufacture exactly defined silicon tips with nm features and a controlled surface chemistry, ii) a physics/biophysics laboratory providing expertise in instrument building, microscopy and laser technology, and iii) a molecular biology laboratory providing the expertise in protein biochemistry and developing procedures to produce well characterized cytoskeletal and motor proteins and modified proteins using an E.coli expression system. If successful, the proposed instrument would constitute a major advance in microstructure determination, uniquely matched to the requirements of biological research. It would make it possible to dynamically image the fundamental functional units of biology, proteins, DNA, RNA, "at work". The instrument would have a broad range of applications, especially in the life sciences, and a multitude of fundamental questions appear solvable, by "looking and seeing how things work".