부산대학교 도서관

This chapter reviews the use of fluorescent methods in the study of single molecules, how the foundations of fluorescence are rooted in Einstein’s description of absorption and emission (spontaneous and stimulated), and their quantum-mechanical explanation in terms of transitions between quantized energy levels, as represented in Jablonski diagrams. It describes the non-radiative channels which compete with fluorescent emission, decrease its efficiency, and ultimately destroy the fluorescent molecule. Fluorescence Polarization Spectroscopy, FRET, and FCS are briefly presented. Without reviewing the various available fluorophores, it describes the various illumination methods used to study them, sketching super-resolution methods (STED, STORM, PALM) that have recently allowed fluorophores to be resolved to a few tens of nanometres. Finally, it describes the considerations (bandwidth, signal to noise, signal to background) used in choosing a single-molecule fluorescence detector, and the extraction of the diffusion constant of a fluorophore from the finite time, noisy traces of its positions.