ABSTRACT
Over the past decades, the development of highly sensitive detectors and the synthesis of fluorophores with large cross sections and quantum yields have enabled the detection of single fluorescent molecules. This has allowed life scientists to probe the dynamic properties of single molecules (SMs) in solution, immobilized on solid surfaces, and even inside cells. Measurements of biomolecular properties have thus been obtained with higher precision than ever before. In addition, the observation of SMs has evidenced behaviors otherwise obscured by ensemble averaging. The tradeoffs in performing singlemolecule fluorescence spectroscopy (SMFS) are that observation times are limited by photobleaching of the fluorophores and large datasets are required to ensure statistical significance. This chapter explores the fundamentals, experimental implementation, and observables obtained through SMFS, and their potential biomedical applications. An introduction containing the fundamentals of fluorescence techniques is presented, followed by a description of typical experimental setups, and an overview of the observables that can be accessed through different SMFS techniques. The design and implementation of micro-and nanostructured devices for SMFS is also reviewed. The chapter ends with a discussion of the areas of opportunity for SM biomedical studies.
