Shining the spotlight on functional molecular complexes

Abstract

Single-molecule research is emerging as one of the fastest growing fields within the biosciences. Historically, most of the techniques employed have operated largely in the world of the test tube in which the components of the biological system under investigation have been extracted and purified from cells to reduce them to just the key ingredients under study, and this research has involved novel, pioneering methods of biophysics to obtain single-molecule measurements. What has emerged recently is the technical ability to now perform key single-molecule experiments whilst retaining the native biological context – namely to do single-molecule experiments on functional living cells. This presents essentially a new science of “single-molecule cell biology”, which combines classical cell biology approaches with modern single-molecule biophysics. Here, key recent studies which have pushed back the boundaries of this field are discussed.

Acknowledgements

M.C.L. is supported by a Royal Society University Research Fellowship, a Hertford College Oxford Research Fellowship, and research grants from the BBSRC (BB/F021224/1) and EPSRC (EP/G061009).

Figures and Tables

Figure 1 Fluorescence imaging on single living bacterial cells at the single-molecule level. (A) E. coli cell tethered via a filament in a TIRF field. (B) Sequential brightfield (upper part) and TIRF (lower part) images, cell rotation indicated (yellow cross and arrow), adapted with permission from ref. 4. (C) Slimfield microscopy in which a laser beam underfills the back aperture of an objective lens. (D) Brightfield (grey) and slimfield images (yellow) of a single E. coli cell, replisome complexes indicated (arrows). (E) Photobleach intensity trace, raw (blue) and filtered (red) data indicated (adapted with permission from ref. 7).