ABSTRACT
This chapter focuses on fluorescence and mechanical methods that have been used to probe the kinetics of reactions at the single-molecule level. Single-molecule measurements are usually performed on reactants that are at equilibrium or in a steady-state. Fluorophores selected for single-molecule detection usually emit in the yellow to infrared region. A problem with fluorescence detection that became apparent as a result of single-molecule investigations is that of blinking fluorophores. Single-molecule measurements are conducted using small volumes and usually involve some form of optical microscopy. The transitions for a typical chemical reaction follow a Markov process in that each molecule undergoes a transition to another state at a time that is independent of its history. When protein structural changes occur on the fast time scale compared with the catalytic activity, the heterogeneity is averaged out so that each molecule displays a single exponential distribution of waiting times with a well-defined mean value.
