Advanced multidimensional optics to investigate biological complexity at the single-molecule level in living, functional cells

细胞的物理分子基础是什么？生物体中的单分子介观特性如何扩大到影响整个生物体的功能？我们能在理解生物分子的物理特性和生物超级计算机的最终表现之间架起一座桥梁吗？在一个理性的、可预测的环境中，生物超级计算机就是单个细胞。这些都是未来以生物为灵感的物理研究面临的主要挑战，但我建议通过发展激进的新技术来解决这些挑战。本提案描述了一种特别通用的、超灵敏的定制显微镜的构建和进一步发展，该显微镜能够实时监测活细胞中功能分子复合物的动态定位和化学计量，并具有超分辨率光学性能。活细胞中的大多数重要活动都是由蛋白质完成的，蛋白质如此之小，以至于10亿个蛋白质可以装进一个句号。许多这些过程都需要蛋白质的集合来组装成功能性的生物机器。这种被提议的显微镜将使我们能够精确地确定一台机器有多少部件，它们是如何组装和拆卸的，它们是如何与彼此和周围环境机械地相互作用的，它们的分子稳定性是如何动态的，以及不同生物过程的机器是如何在整个细胞水平上合作产生强大的复合效应的。只有通过研究引发和指导细胞事件的关键相互作用，才能全面了解活细胞的机制，尽管到目前为止，生物系统的剪切复杂性已经导致精确的单分子实验要求太高，而不是集中在使用相对粗糙的整体集成的单个系统的研究上-平均测量实际上使用来自数千个分子的求和信号。我现在的建议是使用更强大和精确的单分子技术，在一个活的功能细胞中同时监测几个生物系统，从自下而上的分子物理学水平进行研究。但是，要在实验上实现这一目标，需要一种严谨而专门的物理研究方法。该显微镜将由一个激光器组成，该激光器可以输出多种波长的可控偏振态，可以交替激发附着在同一生物样品中几种不同蛋白质上的各种彩色荧光偶极子标签，荧光发射使用高量子效率热电冷却宽场光子计数技术进行成像。短波长激发将允许控制的光活化改性分子染料允许超越光学远场衍射极限的超分辨率能力。我的光学物理方法提供了一种最小扰动、非侵入性的方法来探测功能性活细胞，这是大多数现有的生物启发物理学的单分子方法所不具备的特点。这将使在真实、完整、原始的生物学背景下研究分子功能成为可能。所开发的技术将为物理学家、生物学家、化学家、工程师、数学家和计算机科学家带来巨大的跨学科利益，在生物传感和制药行业的天然细胞药物验证方面具有强大的商业开发潜力。在相对低复杂性的细菌细胞上进行体内成像优化将为复杂多细胞系统的更具挑战性的研究铺平道路，最终在涉及疾病调查和建立治疗方法的人类细胞系的医学研究中达到顶峰。

What is the physical, molecular basis of the cell? How do single-molecule mesoscopic properties in a living organism scale up to effect whole-organism functionality? Can we bridge our gap in understanding between the physical characteristics of bio-molecules to the ultimate manifestation of a biological super-computer which is the single cell, in a rational, predictive context? These are the major outstanding challenges to the future of biologically-inspired physics research, but ones which I propose to address by development of radical new technologies. This proposal describes the construction and further development of an exceptionally versatile, ultra-sensitive custom-built microscope capable of monitoring dynamic localization and stoichiometry of functional molecular complexes in living cells to a precision of single molecules in real-time with super-resolution optical performance. Most of the vital activities in living cells are carried out by proteins, so small that 1 billion could fit on a full-stop. Many of these processes require collections of proteins to assemble together into functional biological machines. This proposed microscope will allow us to determine precisely how many components a machine has, how they assemble and disassemble, how they mechanistically interact with each other and their surroundings, how dynamic their molecular stability is and how machines of different biological processes co-operate to produce potent, compounded effects at the level of the whole cell. A full insight into the mechanisms of living cells can be achieved only by investigating the key interactions that elicit and direct cellular events, though to date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements which use in effect the summed signals from many thousands of molecules. What I now propose is to monitor several biological systems simultaneously in a living functioning cell using more powerful and precise single-molecule techniques, investigating from a bottom-up molecular physics level. But, to achieve this experimentally requires a rigorous and dedicated physics-research approach. The microscope will consist of a laser that can output multiple wavelengths of controllable polarization states which can alternate excitation for a variety of coloured fluorescent-dipole tags attached to several distinct proteins in the same biological sample, with fluorescence light emissions being imaged using high quantum-efficiency thermoelectrically cooled wide-field photon counting technology. Short wavelength excitation will allow controlled photo-activation of modified molecular dyes permitting exceptional super-resolution capability beyond the optical far-field diffraction limit. My optical-physics approach offers a minimally-perturbative, non-invasive means to probe functional living cells, features not readily available to most existing single-molecule approaches of biologically-inspired physics. This will enable the study of molecular function within the true, intact, original biological context. The techniques developed will be of immense interdisciplinary benefit for physicists, biologists, chemists, engineers, mathematicians and computer scientists, with a strong potential for commercial exploitation in bio-sensing and native-cell drug validation for the pharmaceutical industry. Following in vivo imaging optimization on relatively low complexity bacterial cells will pave the way towards more challenging studies on complex multi-cellular systems, culminating in medical research on human cell lines involved in the investigation of disease and in establishing therapeutics.