Light microscopy is a widely used imaging modality in biological research. However, the spatial resolution of light microscopy, classically limited by diffraction to a few hundred nanometers, is substantially larger than typical molecular length scales in cells. Hence many subcellular structures cannot be resolved by conventional light microscopy. We recently developed a new form of super-resolution fluorescence microscopy, stochastic optical reconstruction microscopy (STORM), that breaks the diffraction limit. STORM uses single-molecule imaging and photo-switchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules. This approach allows the localization of fluorescent probes with nanometer precision and the construction of sub-diffraction-limit images. Using this method, we have achieved multicolor and three-dimensional (3D) imaging of live cells and tissues with nanometer-scale resolution. In this talk, I will discuss the general principles, recent technological advances and biological applications of STORM.