By providing spatial localization on the nanometer scale, eliminating the need for ensemble averaging, and permitting non-invasive intracellular investigations, single-molecule imaging has brought much insight to biophysics. A particularly enticing application for single-molecule imaging is the capability to investigate live cells and to examine structure and dynamics in the natural environment. To obtain true superresolution, control of the emission of the single molecules provides a way to maintain a sparse concentration of emitters for any frame so that sequential imaging leads to a final reconstruction with information beyond the optical diffraction limit. In this paper, we discuss several single-molecule- based fluorescence methods that are possible, and indeed often enabled, by having live cell specimens.© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.