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futurity_medicine 00356.txt

#Fly-through brain images could unravel how we think Scientists have improved on a new imaging technology that provides spectacular fly-through views of the brain and how it wired. CLARITY was introduced last year and has been used by laboratories around the world to better understand the brain wiring. However two technological fixes could make it even more broadly adopted, says Karl Deisseroth, professor of bioengineering and of psychiatry and behavioral sciences at Stanford university. The first problem was that laboratories were not set up to reliably carry out the CLARITY process. Second, the most commonly available microscopy methods were designed not to image the whole transparent brain. here have been a number of remarkable results described using CLARITY Deisseroth says, ut we needed to address these two distinct challenges to make the technology easier to use. In a paper published in Nature Protocols, Deisseroth presented solutions to both of those bottlenecks. hese transform CLARITY, making the overall process much easier and the data collection much faster, he says. He and coauthors, including postdoctoral fellows Raju Tomer and Li Ye and graduate student Brian Hsueh, 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 probe the origins of brain diseases. CLEARING OUT THE FAT When you look at the brain what you see is the fatty outer covering of the nerve cells within, which blocks microscopes from taking images of the intricate connections between deep brain cells. The idea behind CLARITY was to eliminate that fatty covering while keeping the brain intact, complete with all its intricate inner wiring. The way Deisseroth and his team 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 structures embedded in the firm water-based gel, or hydrogel. This is called electrophoretic CLARITY. The electric field aspect was a challenge for some labs. bout half the people who tried it got it working right away, Deisseroth says, ut others had problems with the voltage damaging tissue. 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 team devised an alternate way of pulling out the fat from the hydrogel-embedded brain 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. CHEMICALS, A WARM BATH, AND TIME lectrophoretic CLARITY is important for cases where speed is critical, and for some tissues, Deisseroth says. ut 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 eventually treat the disease. But scientists, including Deisseroth, had been wary of trying electrophoretic CLARTY on these valuable clinical samples with even a very low risk of damage. t a rare and precious donated sample, you don want to have a chance of damage or error, he says. ow the risk issue is addressed, and on top of that you can get the data very rapidly. SEE FINE WIRING STRUCTURES 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 produces the colorful cellular images that are so common in biology research. Using CLARITY, these colorful structures become visible throughout the entire brain, since no fat remains to block the light. But here the hitch. Those probes stop working, or get bleached, after theye been exposed to too much light. That fine if a scientist is just taking a picture of a small cellular structure which takes little time. But to get a high-resolution image of an entire brain, the whole tissue is bathed in light throughout the time it takes to image it point by point. This approach bleaches out the probes before the entire brain can be imaged at high resolution. The second advance of the new paper addresses this issue, making it easier to image the entire brain without bleaching the probes. e can now scan an entire plane at one time instead of a point, Deisseroth says. hat 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 a while, but previously didn have high enough resolution to see the fine details of cellular structures. e advanced traditional light sheet microscopy for CLARITY, and can now see fine wiring structures deep within an intact adult brain, Deisseroth says. His lab built their own microscope, but the procedures are described in the paper, and the key components are commercially available. Additionally, Deisseroth lab provides free training courses in CLARITY, modeled after his optogenetics courses, to help disseminate the techniques. The work is funded by the Defense Advanced Research Projects Agency (DARPA), National institute of mental health, National Science Foundation, the National Instituteon Drug abuse, the Simons Foundation, and the Wiegers Family Fund A

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