New Imaging Technology Provides Fly-Through Views of the Inner Brain Processes

Scientists have improved on an original technique for looking into the intact brain, making it more effective and safer. This new application could help scientists better determine the inner connections of how thoughts, memories, or diseases occur.

In 2013, Karl Deisseroth, a Stanford University (Stanford, CA, USA), a professor of bioengineering and of psychiatry and behavioral sciences, reported on a new way of peering into a brain—removed from the body—that provided remarkable fly-through views of its inner connections. Since then, laboratories worldwide have begun using the technique, called CLARITY (Clear, Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining-compatible, Tissue hYdrogel), with some success, to better understand the brain’s wiring.

However, Prof. Deisseroth reported that with two technologic corrections, CLARITY could be even more widely accepted. The first problem was that laboratories were not set up to effectively carry out the CLARITY process. Second, the most typically available microscopy technology not designed to image the whole transparent brain. “There have been a number of remarkable results described using CLARITY,” Dr. Deisseroth said, “but we needed to address these two distinct challenges to make the technology easier to use.”

In a Nature Protocols journal article published June 19, 2014, Dr. Deisseroth presented solutions to both of those bottlenecks. “These transform CLARITY, making the overall process much easier and the data collection much faster,” he said. He and his colleagues anticipate that even more scientists will now be able to take advantage of the technique to better understand the brain at a fundamental level, and also to investigate the origins of brain diseases.

This study may be the first to be published with support of the US White House BRAIN Initiative, announced in 2013 with the goal of mapping the brain’s trillions of nerve connections and determining how signals zip through those interconnected cells to control our thoughts, memories, movement and everything else that makes humans the way they are. “This work shares the spirit of the BRAIN Initiative goal of building new technologies to understand the brain—including the human brain,” said Prof. Deisseroth, who is also a Stanford Bio-X affiliated faculty member.

When one looks at the brain, what one sees is the fatty outer covering of the nerve cells within, which blocks microscopes from taking images of the subtle connections between deep brain cells. The plan behind CLARITY was to eliminate that fatty covering while keeping the brain intact, complete with all its intricate inner wiring. The way, the investigators eliminated the fat was to build a gel within the intact brain that held all the structures and proteins in place. They then used an electric field to pull out the fat layer that had been dissolved in an electrically charged detergent, leaving behind all the brain’s structures embedded in the firm water-based gel, or hydrogel. This is called electrophoretic CLARITY.

The electric field feature was troublesome for some labs. “About half the people who tried it got it working right away,” Prof. Deisseroth said, “but others had problems with the voltage damaging tissue.” He reported that this kind of challenge is normal when introducing new technologies. When he first introduced optogenetics, which allows scientists to control individual nerves using light, a similar proportion of labs were not initially set up to easily implement the new technology, and ran into challenges.

To help expand the use of CLARITY, the investigators devised an alternate way of pulling out the fat from the hydrogel-embedded brain—a technique they call passive CLARITY. It takes a little longer, but still removes all the fat, is much easier and does not pose a risk to the tissue. “Electrophoretic CLARITY is important for cases where speed is critical, and for some tissues,” said Prof. Deisseroth, who is also a professor. “But passive CLARITY is a crucial advance for the community, especially for neuroscience.” Passive CLARITY requires nothing more than some chemicals, a warm bath and time.

Many groups have begun to apply CLARITY to probe brains donated from people who had diseases like epilepsy or autism, which might have left clues in the brain to help scientists understand and ultimately treat the disease. However, scientists, including Prof. Deisseroth, had been wary of trying electrophoretic CLARTY on these valuable clinical samples with even a very low risk of damage. “It’s a rare and precious donated sample, you don’t want to have a chance of damage or error,” Prof. Deisseroth said. “Now the risk issue is addressed, and on top of that you can get the data very rapidly.”

The second advance had to do this rapidity of data collection. In studying any cells, scientists often make use of probes that will go into the cell or tissue, latch onto a particular molecule, then glow green, blue, yellow, or other colors in response to particular wavelengths of light. This is what generates the colorful cellular images that are so common in biology research. Using CLARITY, these colorful structures become visible throughout the complete brain, because no fat remains to block the light.

The difficulty is that those probes stop working, or get bleached, after they have been exposed to too much light. That is acceptable if a scientist is just taking a picture of a small cellular structure, which takes little time. However, to get a high-resolution image of an entire brain, the whole tissue is immersed in light throughout the time it takes to image it piece by piece. This approach bleaches out the probes before the entire brain can be imaged at high resolution.

The second development of the new study tackles this problem, making it easier to image the entire brain without bleaching the probes. “We can now scan an entire plane at one time instead of a point,” Prof. Deisseroth said. “That buys you a couple orders of magnitude of time, and also efficiently delivers light only to where the imaging is happening.”

The technique is called light sheet microscopy and has been around for some time, but earlier did not have high enough resolution to see the fine details of cellular structures. “We advanced traditional light sheet microscopy for CLARITY, and can now see fine wiring structures deep within an intact adult brain,” Prof. Deisseroth commented.

Prof. Deisseroth’s lab constructed their own microscope, but the procedures are described in the paper, and the key components are commercially available. Furthermore, the lab provides free training courses in CLARITY, modeled after his optogenetics courses, to help disseminate the techniques.

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