Single-Molecule Microscopy in the Life Sciences

Single-molecule microscopy is currently revolutionizing the life sciences. It appears to perfectly meet the demands of living matter: by singling out the individual entities, complexity becomes accessible to quantitative approaches. This chapter gives an overview of state-of-the-art single-molecule microscopy. We start by describing the potential of single-molecule methods in general: to follow unsynchronized processes, analyze heterogeneous samples, unravel structure-function relations, image nanostructures, and detect extremely rare events. Next, we provide the newcomer with a detailed discussion on technological requirements. Finally, we ask the questions: which parameters can we measure and what can they tell us in a biological experiment? We discuss the determination of single-molecule positions in three dimensions, orientation, and color. These parameters can be used to obtain insights into the association state of a biomolecule, its conformational state, or its mobility.