The advance of optical super-resolution fluorescence microscopy has revolutionized our vision of the subcellular world. Further improvement in the spatial resolution is of great significance for structural and functional investigations. The recently developed expansion microscopy (ExM), which achieves sub-diffraction imaging via physical expansion of the sample, provides a great opportunity for further resolution enhancement of existing optical super-resolution techniques. However, although such combination seems apparent, several technical obstacles, especially the dramatic loss of fluorescence signal during ExM sample preparation, have hampered this goal. In this work, aiming at this challenge, we have developed new strategies to retain and increase the fluorescence of the expanded sample. With the new labeling methods, we have successfully made the labeling density of expanded samples sufficing the Nyquist sampling criteria for optical super-resolution imaging, such as stimulated emission depletion microscopy (STED) and super-resolution optical fluctuation imaging (SOFI). The newly developed expansion nanoscopic imaging (ExN) approaches, i.e. ExSTED and ExSOFI, demonstrated up to 4-fold resolution enhancement compared to standard STED and SOFI, providing a simple and effective way to realize high resolution imaging both at the cellular and tissue level.

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.

Expansion microscopy (ExM) improves the resolution of fluorescence microscopy by physically expanding the sample embedded in a hydrogel 1-4 . Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples 2-9 . 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 protein labeling using enzymatic tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved 5 nm resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.By physically expanding the sample before image acquisition, ExM has enabled the use of a conventional confocal microscope to achieve ~ 70 nm lateral spatial resolution 2-4, 6, 9, 10 . Recent efforts have further enhanced the resolution of ExM either by increasing the volume expansion ratio 11,12 or by combining ExM with super-resolution microscopy such as Structured Illumination Microscopy (SIM) 5,8,13 and Stimulated Emission Depletion (STED) microscopy 7,14,15 . In all these cases, the homogenization of the specimen through either protease digestion 10 or protein denaturation 2, 3 is essential to achieve isotropic expansion without structural distortion. To retain the spatial information of the target structures, the biomolecules of interest (e.g. protein 2-4 , RNA 1 ) and/or labels (e.g. dye-labeled DNA 10 , dye-labeled antibodies 2, 4 or fluorescent proteins 4 ) are anchored to the hydrogel matrix prior to digestion or denaturation. Nevertheless, digestion and denaturation cause incompletely anchored proteins or protein fragments to be washed out, the polymerization reaction to make the hydrogel produces free radicals that readily destroy fluorophores, and both factors can damage fluorescent proteins. Consequently, more than 50% of the target molecules can lose labeling after expansion 4 , which is a major issue of current ExM methods 16,17 . This label loss is exacerbated when aiming for higher spatial resolution. First, ensuring isotropic expansion at nanometer scale requires more thorough digestion or denaturation, hence more wash-out. Second, superresolution microscopy often requires certain dyes that do not survive hydrogel polymerization reaction. For example, Alexa Fluor (AF) 647, one of the best fluorophores for STORM, suffers > 90% loss of the fluorescent molecules after polymerization and

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