(a) Schematic illustration of photo-transfer by allyl sulfide exchange expansion microscopy (PhASE-ExM): A cytocompatible hydrogel prepared using allyl-sulfide crosslinking groups (top left) is permeated with the photoexpansion microscopy (PhotoExM) formulation, which contains an 8-arm, 10 kDa PEG-SH as the chain transfer agent, sodium acrylate as the polyelectrolyte monomer, PEG-diacrylamide as the crosslinker, acrylamide, and LAP as the photoinitiator. Upon light irradiation, two processes simultaneously occur. While photopolymerization of PhotoExM formulation yields a swellable polyelectrolyte network (middle right), radical addition fragmentation chain transfer (AFCT) reaction between the allyl-sulfide and thiyl radicals incorporates the chains of the cytocompatible hydrogel to the polymerizing PhotoExM network (middle left), which can be homogeneously expanded with repetitive H2O washes upon termination of light irradiation (bottom), enabling expansion microscopy of organoids cultured in synthetic hydrogels without requiring another degradation step for the hydrogel. (b) Gel evolution of PhASE-ExM hydrogels as measured by in situ photo-rheology: The first 60 s shows the storage modulus (G’) of the allyl-sulfide crosslinked hydrogels permeated with the PhotoExM formulation, and upon light irradiation (Io = 4.5 mW/cm2, λ = 365 nm), PhASE-ExM hydrogels reach > 95% conversion within 70 s. n = 3 independent measurements, dark blue line = mean, blue shaded area = s.d. Yellow shaded area shows the duration of light irradiation. (c) Overall size expansion factor of the PhASE-ExM hydrogels. n = 4 hydrogels, graph presented as mean +/− s.d. (d) Representative images of allyl sulfide crosslinked hydrogels on a glass coverslip (left), which shrinks ~ 30% by size following permeation with the PhotoExM solution (middle) and post-expansion (right). The allyl sulfide crosslinked hydrogels were functionalized with AlexaFluor 488 to determine its retention post-expansion.