Expansion microscopy (ExM) is a novel super-resolution imaging technique that physically expands biological samples to achieve high-resolution imaging. By embedding a swellable polymer network within a specimen and expanding it, ExM enables isotropic magnification, allowing structures smaller than the optical diffraction limit to be resolved. This method uses covalently anchored labels to separate and visualize nanoscale features with high precision. ExM can be performed using conventional diffraction-limited microscopes, achieving lateral resolutions of ~70 nm in cultured cells and brain tissue. The technique involves labeling key biomolecules with tri-functional fluorescent labels that are incorporated into the polymer network, followed by expansion and imaging. ExM provides isotropic expansion of ~4.5-fold, enabling the resolution of structures previously indistinguishable under conventional microscopy. It has been successfully applied to study microtubules, clathrin coated pits, and other cellular structures in both cultured cells and brain tissue. ExM allows for three-color super-resolution imaging of large volumes of brain tissue, with axial resolution of ~100 µm. The method offers several advantages, including the ability to perform super-resolution imaging on diffraction-limited microscopes, enabling scalable imaging with high resolution. ExM also facilitates in situ analysis by providing a well-defined, in vitro-like environment for labeling. The technique is compatible with various imaging modalities and has the potential to enhance the speed and resolution of super-resolution imaging. ExM's physical magnification enables the visualization of nanoscale features across multiple length scales, making it a powerful tool for studying neural circuits and other biological systems.Expansion microscopy (ExM) is a novel super-resolution imaging technique that physically expands biological samples to achieve high-resolution imaging. By embedding a swellable polymer network within a specimen and expanding it, ExM enables isotropic magnification, allowing structures smaller than the optical diffraction limit to be resolved. This method uses covalently anchored labels to separate and visualize nanoscale features with high precision. ExM can be performed using conventional diffraction-limited microscopes, achieving lateral resolutions of ~70 nm in cultured cells and brain tissue. The technique involves labeling key biomolecules with tri-functional fluorescent labels that are incorporated into the polymer network, followed by expansion and imaging. ExM provides isotropic expansion of ~4.5-fold, enabling the resolution of structures previously indistinguishable under conventional microscopy. It has been successfully applied to study microtubules, clathrin coated pits, and other cellular structures in both cultured cells and brain tissue. ExM allows for three-color super-resolution imaging of large volumes of brain tissue, with axial resolution of ~100 µm. The method offers several advantages, including the ability to perform super-resolution imaging on diffraction-limited microscopes, enabling scalable imaging with high resolution. ExM also facilitates in situ analysis by providing a well-defined, in vitro-like environment for labeling. The technique is compatible with various imaging modalities and has the potential to enhance the speed and resolution of super-resolution imaging. ExM's physical magnification enables the visualization of nanoscale features across multiple length scales, making it a powerful tool for studying neural circuits and other biological systems.