Photophysics of single-molecule probes

Experiments at the single molecule level enable the direct measurement of molecular trajectories, allowing investigators to identify rare events and transient intermediates, and to quantify the dynamics of unsynchronized molecular processes. This chapter focuses on the critical role of fluorophore photophysics on both the design of single molecule experiments and the interpretation of the measured signals. The formation of non-emissive (dark) states is an unavoidable property of all fluorescent dyes that results in fluorescence blinking in a variety of timescales. Dark states also contribute to signal saturation, which greatly complicates the analysis of single molecule intensities in terms of accurate FRET efficiencies. The goal of this chapter is to bring attention to the considerations and control experiments that are important to avoid artifacts or misinterpretations of the data, while maximizing the accuracy of the kinetic and structural information obtained from the experiment. The chapter opens with an introductory summary of photophysical concepts and a discussion of how the events that follow photoexcitation ultimately contribute to the measured fluorescence signals. Although emphasis was placed on the interpretation of single-molecule FRET experiments, the concepts and information conveyed in this chapter will be of general interest to researchers working on any area of single molecule fluorescence and super-resolution microscopy. The final section of the chapter provides an overview of the properties of the most common families of fluorophores used in single-molecule research. Researchers are encouraged not to use this information as universal truths, but instead as starting points to plan additional experiments not only to rule out possible artifacts, but also to determine quantities that can be used to maximize the accuracy of the results derived from single-molecule signals.