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Table 1 Summary of ExM methods based on the target biomolecules

From: Nanoscale fluorescence imaging of biological ultrastructure via molecular anchoring and physical expansion

Target biomolecule Method name Chemical anchor(s) Crosslinker(s) Expansion factor (single round unless otherwise noted) Commercial availability of the anchor(s) References
Proteins proExM Acryloyl-X, SE N,N′-Methylenebisacrylamide ~ 4–4.5× Yes [37]
ExM compatible with conventional fluorescently labeled antibodies and fluorescent proteins Methacrylic acid N-hydroxysuccinimide ester (MA-NHS), glutaldehyde (GA) N,N′-Methylenebisacrylamide ~ 4× Yes [38]
MAP Acrylamide and formaldehyde N,N′-Methylenebisacrylamide ~ 4× Yes [45]
Original ExM/iExM Acrydite-modified oligonucleotides N,N′-Methylenebisacrylamide, N,N′-(1,2-dihydroxyethylene) bisacrylamide (DHEBA), N,N-bis(acryloyl)cystamine (BAC) ~ 4.5–5.5×, ~ 16–22× (two rounds), ~ 53× (three rounds) No [34, 46]
pan-ExM Acrylamide and formaldehyde N,N′-(1,2-dihydroxyethylene) bisacrylamide (DHEBA), N,N′-methylenebisacrylamide ~ 3.8–4×, ~ 13–21× (two rounds) Yes [47]
TREx Acryloyl-X, SE N,N′-Methylenebisacrylamide ~ 10× Yes [48]
X10 ExM Acryloyl-X, SE N,N-Dimethylacrylamide (DMAA) ~ 9.5×–11.5× Yes [49, 50]
NIFS hydrogel MA-NHS N,N-Ethylenebisacrylamide (EBIS) ~ 9× Yes [51]
ExR Acryloyl-X, SE N,N′-methylenebisacrylamide ~ 4×, ~ 20× (two rounds) Yes [52]
Tetra-gel (TG) NHS-azide Step-growth polymerization (no crosslinking); the polymerization proceeds via click reaction between the azide and alkyne groups of the monomers ~ 3.0–3.5×, ~ 10–16× (two rounds), ~ 40× (three rounds) Yes [53, 54]
LR-ExM Trifunctional anchors (HOOC-MA-biotin, HOOC-MA-DIG, BG-MA-biotin, BG-MA-DIG, and BC-MA-DIG) N,N′-Methylenebisacrylamide ~ 4.3–4.7× No [55]
PhASE-ExM Acryloyl-X, SE 8-arm and 10 kDa PEG-SH, N,N′-methylenebisacrylamide ~ 3.25× Yes [56]
U-ExM Acrylamide and formaldehyde N,N′-Methylenebisacrylamide ~ 4× Yes [57, 58]
Nucleic acids ExFISH Label X N,N′-Methylenebisacrylamide ~ 3.3× No (Synthesized from Label-IT and AcX) [59]
EASI-FISH MelphaX N,N′-Methylenebisacrylamide ~ 2–3× No (Synthesized from Melphalan and AcX) [60]
ExSeq Label X N,N-Methylenebisacrylamide ~ 4× No (Synthesized from Label-IT and AcX) [61]
Lipids mExM Membrane probe pGk5b N,N′-Methylenebisacrylamide ~ 3× No [62]
LExM Trifunctional LExM reagent N,N′-Methylenebisacrylamide ~ 4–8× No [63]
Sphingolipid ExM α–NH2–ω–N3–C6–ceramide and GA N,N′-Methylenebisacrylamide  ~ 4 and 10× No [64]
Multiplexed labeling TRITON Trivalent TRITON linker N,N′-Methylenebisacrylamide ~ 3.2× No [40, 65]
Click-ExM Interaction between biotin, streptavidin, and Acryloyl-X, SE (or GA) N,N′-Methylenebisacrylamide ~ 4.5× No [66]
Physical entanglement eMAP Entanglement by the polymer chains (no covalent anchoring) N,N′-Methylenebisacrylamide ~ 4 and 10× Yes [67]
ELAST Entanglement by the polymer chains (no covalent anchoring) N,N′-Methylenebisacrylamide ~ 2–3× Yes [68]