Here, we discuss a collection of cutting-edge techniques and applications in use today by some of the leading experts in the field of correlative approaches in single-molecule biophysics. A key difference in emphasis, compared with traditional single-molecule biophysics approaches detailed previously, is on the emphasis of the development and use of complex methods which explicitly combine multiple approaches to increase biological insights at the single-molecule level. These so-called correlative single-molecule biophysics methods rely on multiple, orthogonal tools and analysis, as opposed to any one single driving technique. Importantly, they span both in vivo and in vitro biological systems as well as the interfaces between theory and experiment in often highly integrated ways, very different to earlier traditional non-integrative approaches. The first applications of correlative single-molecule methods involved adaption of a range of different experimental technologies to the same biological sample whose measurements were synchronised. However, now we find a greater flora of integrated methods emerging that include approaches applied to different samples at different times and yet still permit useful molecular-scale correlations to be performed. The resultant findings often enable far greater precision of length and time scales of measurements, and a more understanding of the interplay between different processes in the same cell. Many new correlative single-molecule biophysics techniques also include more complex, physiologically relevant approaches as well as increasing number that combine advanced computational methods and mathematical analysis with experimental tools. Here we review the motivation behind the development of correlative single-molecule microscopy methods, its history and recent progress in the field.
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