Fundamentals and applications of single-molecule FRET in biophysical research

Single-molecule FRET (smFRET), a molecular ruler based on dipole-dipole coupling of two fluorescent dyes in proximity of 2-10 nm, has become an established tool to study biomolecular structure and dynamics in vitro and in vivo[1]. I here introduce the principles of the technique with an overview of state-of-the-art applications[1,2], highlighting its role in assessing fundamental biophysical processes such as the temporal coupling of ligand binding and conformational changes in proteins[3].

Finally, I will show how single-molecule fluorescence studies, which are typically conducted in optical laboratories, can become possible at the biochemistry bench. For this we introduce a compact and versatile 3D-printed microscopy platform that allows to assemble many different fluorescence imaging modalities including confocal-, video- and super-resolution microscopy[4].

[1] Lerner et al., Science 359 (2018) eaan1133

[2] Agam et al., Nature Methods 20 (2023) 523-535

[3] Han et al., eLife (2024) in press

[4] Moya et al., Science Advances 10 (2024) ado3427