Chemical design and imaging validation of tetra-gel (TG)-ExM for high-isotropy expansion. a Possible structural defects of hydrogels synthesized via free-radical chain-growth polymerization. b Chemical components of TG. c HSV-1 virions with directly labelled envelope proteins, expanded by TG- (left) and PAAG-based (right) two-round iterative expansion. Scale bar (for both panels), 1 μm (TG, 10.3 μm; PAAG, 15.3 μm). d Single-particle averaged images of HSV-1 virions after TG- (top) and PAAG-based (bottom) two-round iterative expansion (TG, n = 396; PAAG, n = 362 virions). Scale bars, 100 nm. e Standard deviation (σ) of HSV-1 virion radius (R) within individual virions for TG- and PAAG-based iterative expansion (p = 2.4 × 10−24, two-sided Wilcoxon rank sum test; TG, n = 352; PAAG, n = 330 virions).