CLARITY allows imaging of an intact mouse brain. Credit: Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University
CLARITY allows imaging of an intact mouse brain. Credit: Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University

Getting Better Views of Brains by Turning Them Invisible

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CLARITY transformation of a mouse brain at left into a transparent but still intact brain at right. Shown superimposed over a quote from the great Spanish neuroanatomist Ramon y Cajal. Credit: Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University

In H. G. Wells’ The Invisible Man, the protagonist Griffin performs a mysterious procedure upon himself to become invisible. In Marvel’s Fantastic Four comics, it’s a dose of “cosmic rays” that gives the Invisible Woman her powers. And at Stanford University, it’s a technique called CLARITY that renders a mouse brain transparent.

Most of the time, we’re probably pretty glad that we can see mice. But when you want to study their brains, their opaque nature becomes a huge pain in the backside. Say you want to find out where a certain chemical is in the brain, like the amyloid-beta protein that underlies Alzheimer’s disease. It’s easy enough to make molecules that will stick to the protein and glow—that’s called immunolabelling. But since the brain is opaque, you have to cut it into fine slices to see where those glowing markers have stuck, and painstakingly assemble those slices in a computer.

This problem would go away if you could invent a way of making a brain transparent, and that’s exactly what Kwanghun Chung has done. Working with a team of scientists led by uber-tool-maker Karl Deisseroth, Chung, he created a new method called CLARITY*. It renders brains transparent, but just as importantly, it also freezes the brain’s most important components into place. Every neuron, protein, DNA strand and signalling chemical is locked in position, and labelling molecules like antibodies or glowing markers can easily be sent in to detect these components.

First, Chung effectively embalms a cold isolated brain with a liquid combination of formaldehyde and acrylamide. The formaldehyde connects most of the brain’s molecules, like proteins and DNA, to the acrylamide, which hardens into a solid, transparent gel when heated to body temperature. Then, Chung runs an electric current through the brain, which carries off anything that’s not linked to the gel. It’s like taking a pencil drawing of a scene and rubbing out all the edges.

What remains is a phantom brain. It comprises the contents of every cell but not their outlines. It’s transparent because the lipids where the things that were scattering and reflecting light. It can be easily infused with marker molecules since, again, the lipids were acting as a barrier. “You can image entire tissues and reconstruct them in 3D,” says Chung. “You don’t need to slice.”

Here, for example, is the brain of an mouse, which was engineered so that one particular type of neurons gives off a yellowish-green glow. You’re looking at a single image of an intact brain, rather than a composite made from individual slices.

Here’s another clarified mouse brain. You’re looking at the hippocampus—an area involved in learning, memory and spatial navigation. Each colour represents a marker stuck to a different brain molecule. “CLARITY enables immunolabeling and imaging in bulk instead of in thin sections,” says Sebastian Seung from MIT. Or alternatively: “The gain in the brain is mainly in the stain.”

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Credit: Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University

Compared to the usual stain-and-slice methods, CLARITY saves time and effort. It produces cleaner results since cutting a brain into several slices inevitably damages the very tissues scientists are trying to study. And it opens up new possibilities. Previously, if you infused a brain with a marker, you had to destroy it to get any results. Now, scientists can study their “clarified” brains and then keep on adding more markers to them. If you get something interesting, you can keep on running experiments on the same intact brain.

“This is the kind of innovation that will slingshot neuroscience far beyond today,” says Henry Markram, leader of the ambitious Human Brain Project. “This new method of whole-brain imaging across all levels of the brain provides a way to acquire much of the key data we will need.”

CLARITY still has some disadvantages, of course. The main one, according to Chung, is that it’s a tricky technique to get right. The step where the lipids are removed by an electric current was especially difficult, and Chung had to build a custom rig to do the job. “I burned and melted hundreds of brains trying to make it work,” he says. Other labs will need to do the same, but Chung has put all the details of his set-up online so that anyone can duplicate it.

It also takes a lot of time to get marker molecules into the brain. Currently, the only way to do it is to let them diffuse in of their own accord—you basically leave the brain soaking in a pool of markers and wait. It can take a couple of months to fully stain a tiny mouse brain. To do the same for a bigger human or monkey one would be impractical, and Chung is now working on speeding up the process.

And brains are just the beginning. CLARITY works for any tissue or organ. It could turns livers, hearts and lungs transparent as easily as it does neurons. Chung, who is now setting up his own lab, is just scratching the surface of its potential.

PS What does CLARITY stand for? Initially, in the way that scientists like to play fast and loose with the rules of acronyms, it stood for: “Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel”. But since Chung is now working with other gel-forming substances beyond acrylamide, the “A” is not always valid. Hence, it’s just CLARITY now.

Reference: Chung, Wallace, Kim, Kalyanasundaram, Andalman, Davidson, Mirzabekov, Zalocusky, Mattis, Denisin, Pak, Bernstein, Ramakrishnan, Grosenick, Gradinaru & Deisseroth. 2013. Structural andmolecular interrogation of intact biological systems. Nature