During childhood and adolescence, our brain goes through a lot of changes. But studying those changes in juvenile mice is challenging because scientists don't have a way to repeatedly image the same animal's neural pathways as they grow.
Now, by simply rubbing a solution into a juvenile mouse's scalp, researchers at Stanford can make the skin transparent to all visible light, allowing them to image the developing connections in a living mouse's brain. And because the technique is reversible and non-invasive, the researchers can return to the same animal over days and weeks. The work, published Aug. 26 in PNAS, creates new opportunities for research on the developing brain that could improve our understanding of neurodevelopmental disorders and lead to new interventions.
"This opens a literal window to peek into the brain's development," said Guosong Hong, an assistant professor of materials science and engineering and senior author on the paper. "Not only can we image the structures of these neurons, but we can also image the neural activity over time in an animal model. In the future, this approach could enable us to look at how these circuits form during the development of an animal."
Harnessing fundamental laws for new discoveries
Normally, light scatters when it hits skin. Light scattering occurs whenever light waves encounter interfaces between materials with different optical properties. So, under the skin, it also scatters as it encounters lipids, proteins, and molecules inside tissue. Like trying to see through sunlit fog, light scattering causes similar challenges when they attempt to peek inside or through tissues.
"From a physics perspective, we're basically a bag of water with biomaterials," said Mark Brongersma, the Stephen Harris Professor and professor of materials science and engineering and co-author on the paper. "And the mismatch in their optical properties is why we can't see through the skin or scalp."
The key to making skin transparent is by making the water and biomaterials more similar in their optical properties. This can be accomplished by raising the refractive index of the water - how much it bends light - to match the refractive index of the rest of the biomaterials in the body. The researchers found that by mixing a compound called ampyrone into water and rubbing it on the skin of a mouse, they could raise the refractive index of the water in the mouse's skin, turning it transparent. And because ampyrone almost exclusively absorbs ultraviolet light, the inside of the mouse can be seen with the whole visible spectrum.
"The fact that such fundamental optics laws can be applied and work in a biological system is just amazing to me," Brongersma said. "It wasn't clear whether the physics and the chemistry and the biology would all line up to make this happen."
The work builds on the team's groundbreaking discovery of a compound that turns skin transparent to red light, allowing them to view a mouse's internal organs without making an incision. Now, because ampyrone permits the whole visible spectrum of light, the team can see the colors of green and yellow fluorescent proteins that are commonly used to mark neural activity. Young mice have very thin skulls, so this fluorescence can be seen until the mouse is about four weeks old (the equivalent of a human teenager or early adult). The researchers were able to repeatedly image the neurons of sedated juvenile mice and see how neural activity changed in awake mice responding to a puff of air on their whiskers.
"It has been extremely challenging to do this day-by-day imaging in mice that are younger than two weeks because at this age, a mouse's brain and skull are rapidly growing," said Jun Ding, an associate professor of neurosurgery and co-author on the paper. "This is when the synapses are formed and pruned during early development, during learning and memory, during sleep cycles, and during hormone cycles, and we've had no way to look at how these exciting things change at synapse resolution day by day over longitudinal time."
A new field of imaging
Unless the ampyrone solution is reapplied, the effect wears off after about 20 minutes, and the researchers found no evidence that the temporary transparency was causing any harm - in fact, they found more irritation from a saline solution used as a control, possibly because ampyrone has some soothing anti-inflammatory properties.
Still, they are working to identify other molecules that could be even more effective. The solution they are using at the moment is about 70% water and 30% ampyrone, but the researchers would like to achieve the same effect (or better) with a significantly lower concentration.
"We may be able to design and synthesize much more efficient molecules based on ampyrone's structure, such that we can reduce the concentration needed by another tenfold," Hong said.
More efficient molecules could help pave the way for use in people, where this approach may someday reduce the need for some X-rays and CT scans or more invasive testing.
"In some sense, the race is on to combine physics, chemistry, and neuroscience to start designing better molecules that can enable entirely new imaging methodology," Brongersma said.
