| Light microscopy, in particular fluorescence microscopy, has become one of the most widely used techniques for studying biological specimens. The non-invasive nature of light combined with highly specific labeling methods for an ever expanding color palette of fluorescent tags has allowed dynamic, multicolor imaging and greatly expanded our understanding of the inner workings of the cell. Despite numerous advantages, conventional light microscopy has limited spatial resolution, typically 200-300 nanometers (nm) in the x-y dimension and 500-700 nm in the z-dimension, due to the diffraction of light. Recently, a variety of super-resolution fluorescence microscopy approaches have been developed that offer an order of magnitude improvement in spatial resolution over conventional methods. We have previously developed one such method, stochastic optical reconstruction microscopy (STORM), based on sub-diffraction localization of single molecules. Here, we describe the extension of three-dimensional (3D) STORM to both multicolor imaging and thicker, whole-cell samples ∼ 3 micrometers in depth. We also describe the development of live-cell STORM, with spatial resolutions of ∼20-30 nm laterally, and time resolutions as fast as 0.5 seconds. Live-cell STORM is further combined with 3D and multicolor imaging to examine the nanoscale arrangement of both intra and extra-cellular machinery. These advances, which combine new approaches to labeling, image acquisition and data analysis, greatly enhance the ability of STORM to address biological questions and capitalize on the strengths of light microscopy.;Finally, we use the same sub-diffraction localization of isolated fluorescent emitters for tracking single particles. Using this technique in conjunction with biochemical assays, we describe a novel intracellular trafficking pathway taken by cell surface proteoglycans and a variety of gene delivery vectors which use them as primary receptors. |
