Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, OX1 3PU, United Kingdom.
Rep Prog Phys. 2018 Feb;81(2):024601. doi: 10.1088/1361-6633/aa8a02.
Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k T, where k is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.
单分子生物物理学改变了我们对生物学的理解，也改变了对生命物理学的理解。与简单的软物质相比，生物物质的环境更加奇特，远离热平衡，涵盖了从单个分子的纳米尺度到细胞、组织和生物体高出几个数量级的多个长度尺度。生物分子通常具有潜在的不稳定性：存在多个亚稳态自由能状态，它们之间的能级仅相差几个热运动尺度 kT 的倍数，其中 k 是玻尔兹曼常数，T 是绝对温度，这意味着在活跃的生物物质相对较热、潮湿的环境中存在复杂的相互转换动力学。单分子生物物理学技术的一个关键优势是它们能够探测分子群体中自由能状态的异质性，这对于传统的整体平均方法来说太具有挑战性。实验和计算技术的平行发展催化了多路相关技术的诞生，以解决以前难以解决的生物学问题。在实验方面，探测器的灵敏度和速度的提高、光源、探针和微流控技术的稳定性和效率的提高推动了进展。我们讨论了这些最近实验的动机和要求，包括基础数学。这些方法大致分为检测分子的工具和操纵分子的工具。对于前者，我们讨论了超分辨率显微镜的进展，它对于解决生命科学中的许多长期存在的问题具有变革性，对于后者，我们包括了在机械扰动分子的“力谱学”技术方面的进展。我们还考虑了单分子计算物理的计算进展，以及如何将模拟和实验结合起来以获得更完整的理解。越来越多的组合技术现在被用于接近天然生理行为的问题，包括原子力显微镜和荧光成像的相关技术。我们确定了这些技术的权衡、限制和应用，并讨论了令人兴奋的新方向。
