Abstract: Dissecting the inner workings of a cell requires imaging methods with chemical specificity, single-molecule sensitivity, molecular-scale resolution, and dynamic imaging capability such that molecular interactions inside the cell can be directly visualized. Fluorescence microscopy is a powerful imaging modality for investigating cells largely owning to its molecular specificity and dynamic imaging capability. 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 and dynamics cannot be resolved by conventional fluorescence microscopy. We recently developed a super-resolution fluorescence microscopy method, stochastic optical reconstruction microscopy (STORM), which 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 has allowed multicolor and three-dimensional imaging of living cells with nanometer-scale resolution and enabled discoveries of novel sub-cellular structures. In this talk, I will discuss the general concept, recent technological advances and biological applications of STORM.