When imaging a biological specimen, scientists typically use microscope optics to magnify features they’re studying. A new method called expansion microscopy improves resolution by physically magnifying the specimen itself, neuroscientists at Massachusetts Institute of Technology report (Science 2015, DOI: 10.1126/science.1260088). The method might one day enable researchers to use standard microscopes to image three-dimensional biological networks all the way down to nanoscale resolution.
Edward S. Boyden, Fei Chen, and Paul W. Tillberg physically magnify specimens by synthesizing a polyacrylate hydrogel network inside tissue. The polymer—similar to the one used in disposable diapers—swells when it absorbs water.
The method starts out much like ones used by pathologists who study biopsy samples: The researchers highlight cellular features by staining extracted tissue with antibodies that target particular biomolecules. In the new method, however, these antibodies are attached to oligonucleotides that are in turn attached to a fluorophore and a methacryloyl group.
After staining the tissue, the researchers add hydrogel precursors and cross-linkers to their samples and initiate polymerization. The methacryloyl moiety helps anchor the fluorescent antibodies onto the polymer network. The researchers then use enzymes to digest proteins in the tissue so that rigid cellular structures won’t resist expansion. Finally, they add water, which causes the hydrogel to expand equally in all directions by a factor of approximately 4.5.
The expansion pushes fluorescently labeled structures in the tissue apart, making them easier to resolve with a conventional fluorescence microscope. Because the specimen expands in all directions, the features retain their relative shape and position—they just get bigger. Researchers can determine the original dimensions of tissue features using the expansion factor.
One potential application is in mapping neural circuits. “We showed that we could take a chunk of brain tissue large enough to include many cells and their connections and resolve individual connections,” Boyden says. In images of mouse brain tissue, Boyden and coworkers achieved lateral resolution of about 70 nm.
“The idea to physically magnify biological specimens prior to imaging is simply brilliant. This is a very good and by no means obvious idea,” says Ernst H. K. Stelzer, a microscopy expert at Goethe University Frankfurt, in Germany. Some teams, including Stelzer’s, try imaging 3-D tissue features by chemically clearing away lipids and other opaque molecules. In these cases, Stelzer says, specimens can shrink by about 10%. “Why not let them expand?” he asks.
Boyden’s group is working to achieve larger expansion factors. “We did show that by lowering the cross-linker concentration we could get almost 10-fold expansion in each direction,” Boyden says. “The problem is that the sample becomes extremely fragile.” One way around that problem, he says, could be to use stronger polymers.