Blowing up the brain to reveal its finer details

A new method allows researchers to visualise brain tissue at the nanoscale using ordinary light microscopes

Scalable 3D super-resolution microscopy of a portion of the mouse hippocampus showing neurons (green) and synapses (blue) and red). Scale bar = 100 microns. Fei Chen/ Paul Tillberg/ Ed Boyden/ MIT.

Children of all ages enjoy growing and hatching dinosaur eggs. These fun little toys are made of a material that is absorbant and expandable. When placed in a glass of water, the rubber dino begins to expand, and the kids’ excitement begins to mount as it grows and breaks through its shell over the next few days…

Researchers at the Massachusetts Institute of Technology (MIT) have developed a new method that expands brain tissue in exactly the same way, physically magnifying it so that nanoscale structures can be seen with an ordinary light microscope.

Ever since its invention in the 17th century, the microscope has enabled scientists to see things that were otherwise invisible to the naked eye, such as the cells that make up our bodies and the tiny creatures found in a drop of pondwater. Ordinary light microscopes cannot, however, resolve objects smaller than about 200 nanometres (nm, or millionths of a millimetre), or approximately half the wavelength of visible red light, so anything of this size or smaller appears blurred.

The development of super-resolution microscopy in the 20th century broke this so-called diffraction limit. These newer methods brought the resolution of light microscopes down to around 20nm, using fluorescent ‘marker’ molecules that bind to cellular proteins of interest and reveal their location, but these machines are expensive and operating them requires a lot of technical expertise.

The new method, described in an advance online publication in the journal Science, allows for super-resolution imaging with conventional light microscopes.

“We were working very hard using classical super-resolution methods like electron microscopy to look at brain circuits,” says Ed Boyden, who developed the technique with his colleagues Fei Chen and Paul Tillberg. “They were very hard to use and can only visualize objects in two dimensions, so we started thinking it would be useful to expand everything.”

They began thinking of ways to physically expand the tissue samples themselves, bringing them to the work of the late Toyoichi Tanaka, a pioneering MIT physicist who discovered “smart gels” in the 1970s. Smart gels contain water or some other fluid within a matrix of chain-like polymer molecules, and respond to changes in temperature, light, and various other forces and stimuli with dramatic changes in size or shape.

Flythrough of a volume rendering of a 10,000,000 cubic micron portion of the mouse hippocampus showing neurons expressing fluorescent protein and synapses marked with antibodies against the pre-synaptic protein Bassoon (blue), and the post-synaptic protein Homer1 (red). From Chen, et al. (2015). Fei Chen/ Paul Tillberg/ Ed Boyden/ MIT.

One of the most promising of Tanaka’s studies involved a salty molecule called sodium acrylate. Which polymerises to form a dense mesh, and swells up when exposed to water. Because of these properties, it is used to make super-absorbent materials for nappies (diapers).

The new method, called expansion microscopy, involves first treating brain tissue with fluorescently-labelled antibodies that bind to specific cellular proteins, then infusing it with a solution containing sodium acrylate and several other chemicals that help the salt molecules cross-link with each other. This expands the tissue to nearly 5 times its normal size, while leaving the relative positions of proteins and other cellular structures largely intact.

Boyden and his colleagues used the method to perform super-resolution imaging of a 10 million cubic micrometer chunk of tissue from the mouse hippocampus, stained with antibodies against three different neuronal proteins, using a standard confocal fluorescence microscope. They also used it to estimate the distance between two protein molecules on opposite sides of a synapse, and their measurements corresponded closely with those obtained earlier with a traditional super-resolution method.

“We get roughly 4-5-fold expansion right now, [which] gives a 60-70 nm resolution,” says Boyden. “That’s great, but protein molecules and other biological structures we’re interested in are much smaller than that. The more the tissue can be expanded, the finer the detail that can be resolved with conventional microscope methods. As it is, the method can expand tissues more than 4-5 fold, but beyond that point the mesh becomes fragile and unstable.

“One thing we want to do is figure out how to expand the polymers even more,” says Boyden. “Another priority for us is to build stronger polymers, or find a way of reinforcing them in the expanded state, and we’re now screening lots of chemicals to find ones that retain their strength after expansion.”

Expansion microscopy is the latest in a series of new methods developed to probe brain structure and function in increasing detail. It could be used in combination with existing super-resolution microscope techniques, to add molecular-level information to the data sets produced by them, which would be a great help in current efforts to characterise the cell types in the brain and map neural circuits.

As well as improving upon the new method, Boyden and his colleagues are exploring ways in which it can be combined with other staining methods, and with RNA and DNA sequencing technologies. They are also applying the method to human tissue obtained from a brain bank. “We want to see how circuits change in diseases,” says Boyden, “because this might help us to understand brain disorders at the circuit level, and bridge the gap between molecular psychiatry and neural circuit dysfunction.”

Reference: Chen, F., et al. (2015). Expansion Microscopy. Science, doi: 10.1126/science.1260088.