Title: Single-molecule techniques in biophysics: a review of the progress in methods and applications

Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living biological matter. The key utility of singlemolecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods. Here, we discuss the motivation and technological requirements for single-molecule experiments that have generated a recent range of particularly valuable new techniques, including the mathematics underpinning these approaches. These methods are broadly divided into tools which detect single molecules and those which manipulate them. Into the former category we discuss many of the exciting developments of super-resolution microscopy, which has been transformative for addressing many longstanding questions in the life sciences, and in the latter category we include discussion of recent progress in ‘force spectroscopy’ methods. Force spectroscopy approaches involve single-molecule force transduction techniques that mechanically perturb molecules to probe their viscous and elastic properties, motions and interactions. We also consider exciting recent development in theoretical computational approaches, using in silico methods in the form of single-molecule computational physics techniques in the realms of molecular simulation and data analysis, and examine how simulation and experimentation may be drawn together to give a multi-scale understanding of the behaviour of single molecules. Increasingly, combinatorial single-molecule biophysics techniques are now used, such as correlative atomic force microscopy and super-resolution fluorescence imaging, being developed to probe far more complex biological questions which are closer to their native physiological behaviour. We identify the trade-offs, limitations and applications of these different techniques, and discuss future, exciting new directions.