Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy.

Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (nm) and temporal (ms) resolution, as well as the force scale (~pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein-protein and protein-DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.