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Here, we discuss a collection of cutting-edge techniques and applications inuse today by some of the leading experts in the field of correlative approachesin single-molecule biophysics. A key difference in emphasis, compared withtraditional single-molecule biophysics approaches detailed previously, is onthe emphasis of the development and use of complex methods which explicitlycombine multiple approaches to increase biological insights at thesingle-molecule level. These so-called correlative single-molecule biophysicsmethods rely on multiple, orthogonal tools and analysis, as opposed to any onesingle driving technique. Importantly, they span both in vivo and in vitrobiological systems as well as the interfaces between theory and experiment inoften highly integrated ways, very different to earlier traditionalnon-integrative approaches. The first applications of correlativesingle-molecule methods involved adaption of a range of different experimentaltechnologies to the same biological sample whose measurements weresynchronised. However, now we find a greater flora of integrated methodsemerging that include approaches applied to different samples at differenttimes and yet still permit useful molecular-scale correlations to be performed.The resultant findings often enable far greater precision of length and timescales of measurements, and a more understanding of the interplay betweendifferent processes in the same cell. Many new correlative single-moleculebiophysics techniques also include more complex, physiologically relevantapproaches as well as increasing number that combine advanced computationalmethods and mathematical analysis with experimental tools. Here we review themotivation behind the development of correlative single-molecule microscopymethods, its history and recent progress in the field.

Correlative approaches in single-molecule biophysics: a review of the progress in methods and applications