Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues

Asano SM*, Gao R*, Wassie AT*, Tillberg PW, Chen F, Boyden ES (2018) Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues, Current Protocols in Cell Biology 80(1):e56. (*, co-first authors)

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Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by _4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. Here we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence in situ hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light-sheet microscopes is provided.

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