image courtesy of the Deisseroth lab

Now You See It

Replacing the lipid-rich infrastructure of the brain with a hydrogel makes it transparent, revealing details of its circuitry.

In neuroscientist Karl Deisseroth’s lab at Stanford University, samples of brain tissue—even entire brains—have a way of disappearing. There’s nothing mysterious about this vanishing act: It’s the result of CLARITY, a technology Deisseroth and his colleagues developed to bring the brain’s complex neural circuits into better view by removing the neurons’ fatty components, which distort images produced by even the most advanced microscopes.

“This is something we’ve wanted to do for a long time,” Deisseroth says. His lab group studies the neural circuits that underlie behavior. Using light-based tools he developed in 2005 that give researchers precise control over specific nerve cells (an approach Deisseroth dubbed optogenetics), his team has identified cells and connections in the brains of mice that are involved in anxiety, drug abuse, social behavior, and depression. Still, these cell-by-cell studies offer too limited a view of brain function for Deisseroth, an HHMI early career scientist and practicing psychiatrist who wants to understand mental illness and improve treatments for it.

“That approach has been useful, but it hasn’t allowed us to come to a deeper, circuit-level understanding of how physiology and behavior arise from the neurons that we target,” he says. “That’s because we don’t know in detail how they’re wired—how they’re connected in tissue, both locally and globally. We can control cells, we can see behaviors—but to turn that into a deep understanding of how the circuitry works has been a challenge.”

The wiring information Deisseroth hopes for has been difficult to come by in part because to examine the brain’s cellular structure under a light microscope, scientists must first slice the tissue into thin, light-accessible sections. Today’s imaging tools can reveal remarkably fine details about the individual sections, but reconstructing a tissue’s original three-dimensional structure is a labor-intensive and error-prone process. Contextual information, which Deisseroth says is essential to understanding the real consequences of a cell’s activity within a functioning brain, is often lost.

The problem, Deisseroth says, is fat. Lipids in cell membranes provide structure and support for information-processing neurons, but because they bend and scatter light, they cloud the view of biological tissues. Furthermore, lipids interfere with a tissue’s permeability to antibodies, which researchers use to label specific molecules and characterize cells. This is true for all biological tissue. But the brain, packed with elongated and elaborately branched membrane-bound cells, is particularly lipid-rich. Removing fat from the brain allows access by light and macromolecules like antibodies. But without it, the tissue loses its structure and stability.

“We knew we needed to build an infrastructure within the tissue,” Deisseroth says. His team, led by postdoctoral researcher Kwanghun Chung, began experimenting with various molecular support structures a few years ago and finally settled on a hydrogel. They found that they could infuse a tissue with the building blocks of the gel (hydrogel monomers) and chemically link them to the proteins, DNA, and RNA inside the tissue. “It leaves the lipids out in the cold,” Deisseroth says. “They don’t crosslink with the polymer.”

When the infused tissue is heated, the gel monomers link together, forming a three-dimensional network that captures proteins and genetic material in their original positions. With the scaffold set in place, lipids can be removed with strong detergents and an electric current.

Tissue treated in this way emerges from the process transparent and accessible to biological labeling molecules. Deisseroth named the procedure CLARITY and his team described it in the May 16, 2013, issue of the journal Nature.

Player for HHMI Bulletin website (based on New HHMI.org Player)
Take a journey through a transparent CLARITY-processed mouse brain. Video courtesy of Karl Deisseroth

CLARITY can transform an intact mouse brain—a pale pink lump of tissue about the size of a pencil eraser—into a transparent form in about five days. Deisseroth acknowledges that the clarified samples are a little harder to keep track of at the lab bench, but under a microscope, they offer a never-before-seen view of the brain’s circuitry—revealing connections between distant parts of the brain while allowing researchers to zoom in on fine details of the cellular structure.

With lipid barriers gone, labeling antibodies can permeate the porous hydrogel, enabling specific proteins and structures to be visualized under the microscope. Further, the hydrogel is stable enough to withstand the harsh treatments needed to remove labels, meaning that researchers can reanalyze the same tissue with a focus on different proteins.

Deisseroth says the ability to image an intact mouse brain will complement the optogenetic tools his lab has developed for manipulating neuronal activity, finally allowing them to directly link what they learn about activity patterns to a deeper understanding of brain anatomy. But that’s not CLARITY’s only application. Deisseroth says the technique should be applicable to any tissue from any organism.

His team has successfully clarified clinical samples of postmortem human brain and glimpsed neuronal connections that weren’t discernible in images reconstructed from a series of thin samples. Other labs are finding their own applications of the technology, such as visualizing cancer cells within the three-dimensional context of a biopsy sample. Deisseroth is encouraging widespread use of the technology, sharing resources online at clarityresourcecenter.org and hosting hands-on training courses in his Stanford laboratory.

Scientist Profile

Investigator
Stanford University
Neuroscience, Physiology

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