Variations of Expansion Microscopy. ExM: In the original protocol, the monomer gel solution includes eight components—sodium acrylate; acrylamide; MBAA, NN′-methylenebisacrylamide; PBS, phosphate-buffered saline; APS, ammonium persulfate; TEMED, N,N,N′,N′-tetramethylethylenediamine; 4HT, 4-hydroxy-tempo. Sodium acrylate and acrylamide form the polymer as they are cross-linked by MBAA. 4HT is a polymerization inhibitor that prevents premature gelation before the monomer solution diffuses throughout the sample tissue; TEMED serves as an accelerator; APS serves as the initiator and thus is added last to the solution.7,27 ProExM: Acryloyl-X is added as an anchoring agent that binds proteins to the polymer.15 iExM: An iterative approach to ExM, where the initial polymer is broken down, and a second swellable polymer mesh is formed in the space newly opened by the first expansion. This was implemented using custom-made DNA oligonucleotides conjugated to secondary antibodies and polymer anchoring moieties,18 such that only the specifically labeled structures of interest are attached to the polymer. ExR: AcX was applied in iExM for broad anchoring of proteins and antibodies to the polymer before post-expansion staining.1910× expansion: A higher expansion factor was obtained by replacing acrylamide with N,N-dimethylacrylamide acid (DMAA) as the main monomer together with sodium acrylate.17 20ExM: A single-step twentyfold expansion protocol without iterative steps, using optimized hydrogel integrity and oxygen-controlled gelation.20 TREx: By decreasing the crosslinker and monomer concentrations, the hydrogel maintains structural integrity and increases the expansion factor tenfold in a single gelation step.16 U-ExM: For expanding cultured cells and analyzing proteins at higher resolution. Anchors proteins to the polymer by applying acrylamide and formaldehyde rather than using AcX.28 iU-ExM: Enables high-resolution visualization of cellular structures by combining ultrastructure expansion with iterative expansion, achieving near-SMLM resolution for detailed molecular imaging in tissues and organelles.29 TissUExM: To penetrate thicker tissue, this protocol adds triton to the monomer solution and increases the concentration of acrylamide. In addition, a collagenase VII digestion step is introduced after gelation, followed by a denaturation step and post-expansion staining.30,31 Whole-body ExM: to homogenize and expand an entire zebrafish with multiple tissue types such as bones, cartilage, muscles, and soft tissues, this protocol extended the proteinase-K digestion time, added collagenase I, II, IV digestion, and decalcification with ethylenediaminetetraacetic acid (EDTA).32 ExFISH: Preserves the integrity and spatial positions of RNA molecules in cells and tissue slices. The RNA is stained by fluorescence in situ hybridization (RNA-FISH) amplified with hybridization chain reactions (HCRs) that enable the resolving of single RNA molecules. Multiple rounds of FISH increase the number of target RNA sequences.23 EASI-FISH: Replaces Label-IT in the ExFISH protocol with a smaller molecule linker, melphalan, to retain and anchor nucleic acids to the ExM polymer. This linker is cost-effective, improves signal-to-noise ratio, and optimizes reagent penetration.33 ExLLSM: Addresses microscopy working distance that limits sample thickness with a light sheet microscopy pipeline.34,35 ExCel: Adapted ExM to the nematode C. elegans by adding steps for cuticle permeabilization.36,37 Magnify: Achieves higher expansion factors and enhanced imaging resolution by adding N,N-dimethylacrylamide (DMAA) to the monomer solution alongside acrylamide and sodium acrylate. This protocol combines the high expansion factor of 10× expansion with protein retention strategies from ProExM, allowing for detailed imaging of proteins in large or thick samples.25 ExSeq: Enables spatially resolved RNA sequencing by anchoring RNA molecules to the gel using reagents such as LabelX and maintaining a neutral pH during expansion to preserve RNA integrity. The protocol involves re-embedding the expanded gel in a non-expandable gel to facilitate sequencing procedures.38 ExIGS: Facilitates in-gel sequencing of DNA or RNA molecules with higher expansion factors by using a modified polyacrylate gel incorporating DMAA and Tn5 transposase. By adding rolling circle amplification (RCA) and sequencing-by-synthesis (SBS) buffers with increased gel porosity, it supports the enzymatic diffusion necessary for sequencing. The protocol includes re-embedding the expanded gel in a non-expandable gel for sequencing procedures and is derived from Magnify and ExSeq.39
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