Understanding the inner workings of the brain will provide useful information on the interconnections between neurons that control memories, thoughts, and other facets of our lives. A technique called CLARITY presents a transparent view of the brain tissue and, since its inception, has been widely used by scientists around the world who wish to have a peek at the protein and nucleic acid structure of the brain.
CLARITY (Clear, Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining compatible, Tissue hydrogel) is a process developed by Karl Deisseroth, a professor at Stanford University. The technique uses the chemical compound acrylamide and hydrogel electrophoresis to make the brain tissues transparent. But a few technicalities have prevented it from being more widely used. One is that most laboratories are not equipped to use CLARITY without damaging the brain tissue and the other is that conventional microscopes have not been designed to image the whole transparent brain. Solutions to both problems have been addressed by Deisseroth and his colleagues Raju Tomer and Li Ye and graduate student Brian Hsueh, in the June 19 issue of Nature Protocols .
CLARITY is used on brains that have been removed from the body. The human brain contains lipids which cover the nerve cells. This fatty covering is first removed by chemical treatments to make the brain transparent while keeping the original wirings intact. To do this the brain is immersed in a hydrogel solution where the hydrogel monomers infuse the tissue. On applying a little heat, the monomers are linked with all the cellular components except lipids. Electrophoresis is then used to remove all traces of lipids from the sample.
Unfortunately, most labs get the electrophoresis part of the procedure wrong. So Deisseroth's team devised an alternate way of pulling out the fat from the hydrogel-embedded brain – a technique they call passive CLARITY. Although this process is a little longer and involves immersing the tissue in clearing solution sodium dodecyl sulfate (SDS), it extracts the lipids and keeps the tissue intact. This method ensures that the sample remains undamaged--a risk that cannot be eliminated with electrophoretic CLARITY.
Once the lipids have been removed, imaging can be carried out. This is accomplished by using probes that bind to the target substance and then glow green, blue, yellow, or other colors in response to particular wavelengths of light. With CLARITY, the entire brain glows with different colors since the light-blocking lipids have been removed.
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. But by doing this the probes get bleached or stop working. This was the second problem faced by most labs as it was impossible to get a high resolution image of the entire brain without the probes getting bleached.
But the team worked around this problem by building their own microscope, which scanned the entire brain without bleaching the probes.
"We can now scan an entire plane at one time instead of a point," Deisseroth said, according to a press report . "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 a while, but previously didn't 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."
The components of the microscope are available commercially and Deisseroth's lab provides free training courses in CLARITY to help disseminate the techniques. CLARITY has been supported by the BRAIN Initiative, which is aimed at deeper understanding of the human brain. Its is also being funded by the Defense Advanced Research Projects Agency (DARPA) and the National Institute of Mental Health to provide clues about the origin of brain diseases like epilepsy or autism and help in developing potential therapies.