  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.
