MIT researchers have developed a method for making extremely high-resolution images of tissue samples at a fraction of the cost of other techniques, yet with similar resolution. The new technique relies on expanding tissue before imaging it with a conventional light microscope. Two years ago, the team showed that it was possible to expand tissue volumes 100-fold, resulting in an image resolution of about 60 nanometers. Now, they have shown that expanding the tissue a second time before imaging can boost the resolution to about 25 nanometers.
This level of resolution allows scientists to see, for example, the proteins that cluster together in complex patterns at brain synapses, helping neurons to communicate with each other. This could help researchers form maps of neural circuits.
To expand tissue samples, the researchers embed them in a dense, evenly generated gel made of polyacrylate, a very absorbent material that's also used in diapers. Before the gel is formed, the researchers label the cell proteins they want to image, using antibodies that bind to specific targets. These antibodies bear “barcodes” made of DNA, which in turn are attached to cross-linking molecules that bind to the polymers that make up the expandable gel. The researchers break down the proteins that normally hold the tissue together, allowing the DNA barcodes to expand away from each other as the gel swells. These enlarged samples can be labeled with fluorescent probes that bind the DNA barcodes. They can then be imaged with commercially available confocal microscopes, whose resolution is usually limited to hundreds of nanometers.
Although in their original expansion microscopy study, the researchers found that they could expand the tissue more than 100-fold in volume by reducing the number of cross-linking molecules that hold the polymer in an orderly pattern, this made the tissue unstable. Instead, in their latest study, the researchers modified their technique so that after the first tissue expansion, they could create a new gel that swells the tissue a second time — an approach they call “iterative expansion.”
Using iterative expansion, the researchers were able to image tissues with a resolution of about 25 nanometers, which is similar to that achieved by high-resolution techniques such as stochastic optical reconstruction microscopy (STORM). The resolution of expansion microscopy does not yet match that of scanning electron microscopy (about 5 nanometers) or transmission electron microscopy (about 1 nanometer). However, these techniques are very expensive and not widely available. Expansion microscopy is much cheaper and simpler to perform because no specialized equipment or chemicals are required, said Ed Boyden, the study's senior author. The method is also much faster and thus compatible with large-scale 3-D imaging.
In their original expansion microscopy study, the researchers were able to image scaffolding proteins, which help to organize the hundreds of other proteins found in synapses. With the new, enhanced resolution, the researchers were also able to see finer-scale structures, such as the location of neurotransmitter receptors located on the surfaces of the “postsynaptic” cells on the receiving side of the synapse.
Boyden believes that combining expansion microscopy with a new tool called temporal multiplexing could help to achieve a map of the organization of the scaffolding and signaling proteins at the synapses between neurons. Currently, only a limited number of colored probes can be used to image different molecules in a tissue sample. With temporal multiplexing, researchers can label one molecule with a fluorescent probe, take an image, and then wash the probe away. This can then be repeated many times, each time using the same colors to label different molecules. “By combining iterative expansion with temporal multiplexing, we could in principle have essentially infinite-color, nanoscale-resolution imaging over large 3-D volumes,” said Boyden.
The researchers also hope to achieve a third round of expansion, which they believe would, in principle, enable resolution of about 5 nanometers. However, right now the resolution is limited by the size of the antibodies used to label molecules in the cell. These antibodies are about 10 to 20 nanometers long, so to get resolution below that, researchers would need to create smaller tags or expand the proteins away from each other first and then deliver the antibodies after expansion