Super-resolution fluorescence imaging with single molecules

•Molecular specificity and live-cell compatibility are key fluorescence strengths.•Time-sequential imaging and precise localization of sparse single-molecule emitters yield super-resolution.•Advances in 3D multicolor and fast time-lapse are highlighted.•Dipole-emission-induced position errors and molecular copy number estimation (counting) are recent thrusts.•Compact labeling strategies and bright emitters will be central to further progress. The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20–40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging — in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains — are discussed.