Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy

Yongxin Zhao, Octavian Bucur, Humayun Irshad, Fei Chen, Astrid Weins, Andreea L Stancu, Eun-Young Oh, Marcello DiStasio, Vanda Torous, Benjamin Glass, Isaac E Stillman, Stuart J Schnitt, Andrew H Beck, Edward S Boyden

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.