biostudies-literatureShi XAmerican Heart AssociationNIDCR NIH HHSAmerican Heart Association-American Stroke AssociationUniversity of California, San FranciscoNational Institutes of HealthChan ZuckerbergDamon Runyon Cancer Research FoundationDavid and Lucile Packard FoundationNIH HHSNIGMS NIH HHSNational Science Foundatione202105067https://www.ebi.ac.uk/biostudies/studies/S-EPMC8266563biostudies-literatureUnknown220(9)Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.The Journal of cell biologyLabel-retention expansion microscopy.PMC8266563R01GM124334K99 GM126136DP2 OD008479DP2OD00847919PRE3480616R00 GM126136K99GM126136/R00GM126136R01 GM1243341650113DRG2168-13R01 DE029454Chen JTran AAHuang EJLi QLin ZDai ZShi XMcColloch ARKumar DSeiple IBRamirez ADReiter JFWang XFeng SHuang BChow TTfalseLabel-retention expansion microscopy.Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.2021-01-01T00:00:00Z2021 Sep2025-04-19T20:43:18.028Z2025-04-19T20:43:18.028ZS-EPMC82665633422878310.1083/jcb.202105067