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

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 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.

Toward Precision Measurement and Manipulation of Single‐Molecule Reactions by a Confined Space

“…AFM, an excellent surface‐sensitive method, has been applied for detection and mapping of force (e.g., elasticity and stiffness) on single molecules . It adopts a thin and compliant microscopic metal cantilever with functionalized tips to probe the surface of a sample.…”

“…Once single‐molecule sensitivity and resolution are enabled by a confined space, the next step is to examine the methods that have been developed to investigate SMRs. These tools can be broadly divided into two categories: either to measure or to manipulate a SMR; the former is further classified as sensing and imaging, while the latter includes tuning and controlling. To clarify, here in this review, sensing mainly refers to the detection of short‐lived intermediates, and can be done with both free and immobilized reactant molecules of interest; while imaging aims to visualize ambiguously the structure and chemical composition of those substances and is mostly done with immobilized molecules by scanning probe techniques.…”

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

“…Every population under investigation, whether it is a cell culture or a protein bulk within a unicellular organism, is a heterogeneous system. Therefore, ensemble averaging masks important effects of subpopulations [1][2][3] , such as drug resistant bacteria or cancer cells [4][5][6] . Single-molecule optical biophysics methods permit real-time visualization of key cellular processes 7 , the action of so-called biological 'nanomachines' 8 , such as signal transduction, gene expression, immune response, mapping cellular genome, etc., providing direct insights into molecular mobility, stoichiometry, copy numbers 9,10 .…”

Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

“…Here we describe a novel approach for achieving dynamic 3D spatial resolution at millisecond time scales and single-molecule detection sensitivity directly in single living eukaryotic cells using astigmatism imaging [10]. We modified a method that generates a narrow field of laser illumination which produces high excitation intensities in the vicinity of single live cells [11]- [14]. This technique is based on introducing astigmatism into the imaging path through insertion of a long focal length cylindrical lens between the microscope emission port and camera detector, which enables extraction of 3D spatial positions of single fluorescent reporter molecules.…”