This book provides a comprehensive overview of the field of single-molecule fluorescence microscopy and spectroscopy of molecular machines. It is addressed to both physical scientists (who are familiar with the fluorescence methodology but unfamiliar with the biological molecules, mechanisms and questions) and life scientists (who are familiar with the biology but unfamiliar with the large variety of available fluorescence methods). This is possible due to the background of the two authors, Achillefs Kapanidis (ANK) (biochemist) and Mike Heilemann (MH) (biophysicist). The book does not try to address the huge number of applications in non-biological areas; instead it focuses on many celebrated applications of single-molecule fluorescence on molecular machines and functional studies, with special emphasis on real-time studies of kinetics and mechanisms.