Single-Molecule Microscopy in the Life Sciences

This chapter provides some of the essential considerations when building a single-molecule microscope. Next, the key parameters accessible from a single-molecule measurement are discussed: position, orientation, color, and brightness. The chapter begins with an overview of the specific benefits a single-molecule approach provides. There are multiple parameters that may be used to characterize a dye molecule i: for example, the time-dependent position, the orientation of its excitation or emission dipole, its absorption and emission spectra, or its brightness. The recorded single-molecule parameters can be further processed to obtain insights into the biological sample. Based on real-life scenarios, the strengths and potential pitfalls of single-molecule approaches are explained. The transitions between two conformational states of a protein detected via the concomitant change in the Forster resonance energy transfer (FRET) efficiency EFRET, from which the lifetimes of the states can be directly monitored. Finally, the chapter puts these parameters in the context of biological experiments.