Title: Single-molecule techniques in biophysics: a review of the progress in methods and applications Article now published in Reports on Progress in Physics doi: 10.1088/1361-6633/aa8a02 Authors: Helen Miller1, Zhaokun Zhou1, Jack Shepherd, Adam J. M. Wollman and Mark C. Leake2 Affiliations: Biological Physical Sciences Institute (BPSI), University of York, York,YO10 5DD, U.K. 1 These authors contributed equally 2 Correspondence to be spent to [email protected] Abstract 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 several orders of magnitude to 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 kBT, where kB 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 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. 1 1. Introduction Ensemble biophysics methods have produced a wealth of information, but in life, more so than in classical condensed matter, each member of a population is an individual. Single- molecule techniques allow researchers to probe the heterogeneity one molecule at a time to reveal a highly complex bigger picture of biological systems [1,2], general soft condensed matter [3], and biological physics[4], as well as intricate nanoscale biomolecular machines [5] and a range of single molecule behaviours in native living cells [6,7]. We begin with a brief review of the key experiments enabling the observation, perturbation and prediction of the behaviour of single molecules and a discussion of the nanometre length scale single- molecule environment. 1.1. What is ‘single-molecule biophysics’? There are many ensemble average techniques in biophysics that are able to provide valuable information about the mean average state of a biological system. However, by utilizing single-molecule biophysics technique we are able to explore energetically metastable, heterogeneous states one molecule at a time, which is not possible with ensemble average methods. In biology we study the processes and components of ‘life’. ‘Life’ as a philosophical concept has no unique agreed definition, nevertheless, from a physics perspective we can say that the processes involved are typically far from thermodynamic equilibrium. Typically, in studying life we can capture information at a sufficiently meaningful level by investigating properties of single molecules rather than their atomic or sub-atomic constituents: this is typically the smallest length scale at which we can probe to understand biological processes at the level of apparent ‘biological function’ in a relevant organism [8]. The type of ‘molecular heterogeneity’ we encounter will vary depending on the system. In solid systems we might have a static heterogeneity, for example, arising from the defects in a crystal, but often heterogeneity is temporal; in particular when a biomolecule undergoes conformational changes related to its biological role; or spatial if the molecule has interactions over the nanometre length scale with other biomolecules. Single-molecule biophysics techniques allow us to look at biological features of interest, one molecule at a time, and build up a picture of the underlying molecular heterogeneity in the system. Within the relatively small volume of one cell there can be vast differences in environment, for example, in viscosity or in the local concentration of a biomolecule. Single molecule studies aid us in understanding the mechanisms behind the properties we are investigating, as the average may not necessarily correspond to a real, achievable state of the system. To explain this notion we can use an analogy of the average speed of a group of swimmers who join a swimming session at a swimming pool but in separate lanes. We imagine the whole swimming pool to be the biological system we are interested in and the separate lanes as sub-regions of the system which we can sample, with our swimmers as individual biomolecules. If we first average the speed of each swimmer in the whole pool we will find an average swimming speed that might not correspond with the swimming speed of any one person. If we look at the swimmers in each lane one at a time we may also observe spatial 2 heterogeneity; a different average swimming speed in each lane, seen as a variation in swimming speed from one side of the pool to the other. If we were not looking at swimming humans, but individual molecules, we might infer a gradient in viscosity or temperature causing this spatial change in speed, however the underlying cause is instead that of molecular heterogeneity. Our analogy of swimming here emphasizes the key aspect of heterogeneity when taking averages over a population. However, it should also be noted that the role of Brownian fluctuations and thermal forces are important factors in regards to molecular heterogeneity. This is the main source of noise and variability in single-molecule experiments. In many circumstances it is difficult to discriminate molecular heterogeneity from just stochastic noise due to thermal forces. Both temporal and spatial heterogeneity can be present in a system, and single molecule studies allow us to observe these differences. Where ensemble methods provide us with just a mean average value, single molecule methods can, ultimately, generate the average value but also produce a probabilistic distribution of values either side of that mean. A distribution with distinct clusters of measurements separated by gaps might indicate different energetic or conformational states, and the location of the average relative to two distinct clusters might indicate a preference for one state over the other. Probability distributions can highlight deviations from mean average behaviour and are able, with appropriate biological and physical insight, to suggest potential mechanisms for the observed behaviour, far beyond what can be inferred from a simple mean average value obtained by an ensemble, population-level technique. With the potential to glean so much information from single-molecule biophysics there come also extreme technical challenges. For example, as we strive to collect data at higher temporal and spatial resolutions we must aim to reduce the background level for detection to achieve a signal-to-noise ratio greater than one; we must maximise collection efficiency and, perhaps most importantly, perform checks to ensure that what we believe are single molecules, are in fact just that and not multiples. The suite of biophysical techniques that have been developed to study single molecules are not inherently high throughput, but for many of the techniques, recent advances are being made towards multiplexing measurements. In the following pages this review will cover a brief history of the key experiments that have enabled single molecule studies, before summarising the most recent progress in techniques which detect, manipulate, or simulate single molecules. We conclude by looking at the increasing number of correlative techniques which combine at least two single molecule methods and the challenges faced by the field as techniques develop from proof of principle to the study of real, complex biological questions. 3 1.2. Key historical experiments We briefly review some of the key experiments that have carved the current research landscape for the field of single-molecule biophysics, and laid the way for the new developments discussed in detail later. Here we consider single molecules in the condensed phase. Single molecule/atom/ion traps in a vacuum/gaseous state have a longer history that we are do not consider in the context of biophysics. 1.2.1. Detection of single molecules using electron microscopy