I am planning on performing expansion microscopy on relatively thick (300 um) tissue slices and doing a post-expansion stain with some nanoparticles. It seems that the expansion process should make the tissue more permeable, but I am having trouble finding a direct description in the literature which supports or goes against this intuition. Does anyone know whether or not a post-expanded tissue has greater permeability than an untreated fixed tissue sample? Note: my nanoparticles are rod shaped and have dimensions of 27x60 nm, but I could also try spherical 5 nm nanoparticles if needed.
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. C 2018 by John Wiley & Sons, Inc.
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