Iterative expansion microscopy

Jae-Byum Chang, Fei Chen, Young-Gyu Yoon, Erica E Jung, Hazen Babcock, Jeong Seuk Kang, Shoh Asano, Ho-Jun Suk, Nikita Pak, Paul W Tillberg, Asmamaw T Wassie, Dawen Cai, Edward S Boyden

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5××Iterative expansion microscopyJae-Byum Chang1,2, Fei Chen3, Young-Gyu Yoon1,4, Erica E Jung1, Hazen Babcock5, Jeong Seuk Kang6, Shoh Asano1, Ho-Jun Suk7, Nikita Pak8, Paul W Tillberg4, Asmamaw T Wassie3, Dawen Cai9 &Edward S Boyden1,3,10,11We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ~4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.