This article provides a comprehensive guide to super-resolution fluorescence microscopy, a rapidly evolving field that has revolutionized cell biology by overcoming the limitations of conventional light microscopy. The authors, experts from various institutions, discuss the historical context of microscopy, the physical principles behind the diffraction limit, and classical methods to enhance resolution, such as wide-field deconvolution and confocal laser scanning microscopy (CLSM). They then delve into new strategies, including near-field and far-field methods, that have emerged to achieve subdiffraction resolution. Near-field methods, like total internal reflection fluorescence (TIRF) and near-field scanning optical microscopy (NSOM), use evanescent waves to resolve structures within a few hundred nanometers. Far-field methods, such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (PALM/STORM), exploit nonlinear responses or the precise localization of single molecules to achieve higher resolutions. The article also addresses the trade-offs between different techniques, the challenges of live-cell imaging, and future perspectives, emphasizing the ongoing development and potential of these super-resolution techniques in modern cell biology research.This article provides a comprehensive guide to super-resolution fluorescence microscopy, a rapidly evolving field that has revolutionized cell biology by overcoming the limitations of conventional light microscopy. The authors, experts from various institutions, discuss the historical context of microscopy, the physical principles behind the diffraction limit, and classical methods to enhance resolution, such as wide-field deconvolution and confocal laser scanning microscopy (CLSM). They then delve into new strategies, including near-field and far-field methods, that have emerged to achieve subdiffraction resolution. Near-field methods, like total internal reflection fluorescence (TIRF) and near-field scanning optical microscopy (NSOM), use evanescent waves to resolve structures within a few hundred nanometers. Far-field methods, such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (PALM/STORM), exploit nonlinear responses or the precise localization of single molecules to achieve higher resolutions. The article also addresses the trade-offs between different techniques, the challenges of live-cell imaging, and future perspectives, emphasizing the ongoing development and potential of these super-resolution techniques in modern cell biology research.