The fluorescence microscope is the principal tool for investigating the spatial organization of bio-molecular processes. Instrumentation and techniques have evolved considerably since the first realizations of fluorescence microscopy. The technology is constantly being advanced to image faster, deeper, less invasively, and more quantitatively, driving discovery of both biological and clinical mechanisms. In this book, we discuss the various approaches, which can be successfully used in live cells, the tradeoffs in resolution, speed, and ease of implementation, which one must make for each approach, and the quality of results that one might expect from each technique. We introduce the recent development of optical microscopy that has achieved spatial resolution beyond the diffraction limit. Super-resolution imaging techniques outperforming the classical diffraction limits are photoactivated localization microscopy (PALM), stochastic optical resolution microscopy (STORM), stimulated-emission depletion microscopy (STED), three-dimensional structured illumation microscopy (3D-SIM) and total internal reflection fluorescence microscopy (TIRF). These super-resolution microscopy techniques have permitted detailed studies of organelles, vesicles, microtubules, DNAs, proteins, and other biochemical particles on the nanometer scale. Super-resolution microscopy has opened the door to true nanoscale mapping of biological components.