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性质如何放大以影响整个生物体的功能？我们能在理解生物分子的物理特征和生物超级计算机的最终表现之间弥合鸿沟吗？生物超级计算机是在理性、可预测的背景下形成的单一细胞。这些都是生物启发物理学研究未来面临的主要突出挑战，但我建议通过开发激进的新技术来解决这些挑战。这项建议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，该显微镜能够监测活细胞中功能分子复合体的动态定位和化学计量比，实时达到单分子的精度，具有超分辨率的光学性能。活细胞中的大多数重要活动都是由蛋白质进行的，蛋白质如此之小，以至于可以在句号上容纳10亿个蛋白质。这些过程中的许多都需要收集蛋白质，才能组装成功能强大的生物机器。这一拟议的显微镜将使我们能够准确地确定一台机器有多少组件，它们如何组装和拆卸，它们如何相互作用和周围环境，它们的分子稳定性有多动态，以及不同生物过程的机器如何合作，在整个细胞水平上产生强大的复合效果。对活细胞机制的全面洞察只能通过调查引发和指导细胞事件的关键相互作用来实现，尽管到目前为止，生物系统的剪切复杂性导致精确的单分子实验过于苛刻，而是专注于使用相对粗略的总体总体平均测量来研究单个系统，这些测量实际上使用了来自数千个分子的总和信号。我现在的建议是，使用更强大、更精确的单分子技术，从自下而上的分子物理学水平进行研究，在一个活的功能细胞中同时监测几个生物系统。但是，要在实验上实现这一点，需要一种严谨而专注的物理研究方法。显微镜将由一台激光组成，它可以输出多个波长的可控偏振态，可以交替激发贴在同一生物样本中几个不同蛋白质上的各种有色荧光偶极标签，并使用高量子效率的热电冷却广域光子计数技术对荧光光发射进行成像。短波长激发将允许对修饰的分子染料进行受控的光激活，从而允许超越光学远场衍射限制的特殊的超分辨率能力。我的光学物理方法提供了一种微扰、非侵入性的方法来探测功能活细胞，这些特征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。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好处，并具有在制药业的生物传感和天然细胞药物验证方面进行商业开发的巨大潜力。在相对较低复杂性的细菌细胞上进行体内成像优化之后，将为对复杂的多细胞系统进行更具挑战性的研究铺平道路，最终将在涉及疾病研究和建立疗法的人类细胞系上进行医学研究。

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.