Many will remember the colorless colas that came and went in the early 90s. While they were nothing more than a gimmick, Stanford University researchers have developed a clear technology that should prove a little more beneficial to humanity. They have developed a process called CLARITY that turns a normally opaque brain transparent, allowing postmortem examinations to be done without slicing and dicing and opening the doors to a wealth of information about our least understood organ.

The main culprits responsible for the brain’s inscrutability are lipids, fatty molecules that occur naturally throughout the body and act as structural components of cell membranes. These molecules give the brain much of its structure, but they also make it largely impenetrable to both chemicals and light.

While automated slicing and sectioning, or treating the brain with organic molecules to allow the penetration of light, (but not macromolecular probes), do allow a peek inside the brain, it would be far better if it was possible to remove the lipids altogether. That’s exactly what bioengineer and psychiatrist Karl Deisseroth, MD, PhD, and his team has done.

Clear thinking

Because simply removing the lipids would cause the remaining tissue to fall apart, the research team found a way to replace them with an optically transparent hydrogel. This is done by first immersing the intact, postmortem brain in a hydrogel solution, which contains short individual molecules known as hydrogel monomers. These hydrogel monomers infuse the tissue and, when the brain is heated to about body temperature, begin to congeal into polymers that form a mesh throughout the brain and hold everything together. Everything, that is, except the lipids.

This then allows the lipids to be extracted using an electric field in a process called electrophoresis. The researchers say the whole process preserves the biochemistry of the brain so that what remains is a 3D, transparent brain with all of its neurons, axons, dendrites, synapses, proteins, nucleic acids and other important structures right where they should be.

"This feat of chemical engineering promises to transform the way we study the brain's anatomy and how disease changes it," says Thomas Insel, MD, director of the National Institute of Mental Health. "No longer will the in-depth study of our most important three-dimensional organ be constrained by two-dimensional methods."

Molecular information

As well as allowing individual neural connections to be traced through the brain, CLARITY also gives researchers a way to gather molecular information describing a cell’s function by using fluorescent antibodies that seek out and attach to specific proteins. In this way, Deisseroth's team was able to get specifically targeted structures to light up under illumination, giving them the ability to trace neural circuits through the brain or delve into the intricacies of local circuit wiring. Additionally, they can see the relationships between cells and look at the chemical relationships of protein complexes, nucleic acids and neurotransmitters.

And by flushing the fluorescent antibodies out of the brain so the process can be repeated, the researchers are able to explore different molecular targets in the same brain. The researchers say this staining/destaining process can be repeated multiple times.

"Being able to determine the molecular structure of various cells and their contacts through antibody staining is a core capability of CLARITY, separate from the optical transparency, which enables us to visualize relationships among brain components in fundamentally new ways," says Deisseroth.

The technology offers such a wealth of new information about the brain Deisseroth says CLARITY has leapfrogged the ability to deal with the data it will provide.

"Turning massive amounts of data into useful insight poses immense computational challenges that will have to be addressed," he says. "We will have to develop improved computational approaches to image segmentation, 3D image registration, automated tracing and image acquisition."

While most of the research was performed on a mouse brain, similar results were also achieved on zebrafish and preserved human brain samples. Deisseroth says there’s also the potential for CLARITY to be used not only in the mammalian brain, but also in other tissues or diseases to study intrasystem relationships.

"CLARITY may be applicable to any biological system, and it will be interesting to see how other branches of biology may put it to use.” But for the immediate future, CLARITY’s focus will be the brain. It is likely to play an important part in the BRAIN project recently announced by U.S. President Barack Obama, with Deisseroth serving on the BRAIN project working group.

The CLARITY process is described in a paper published online April 10 in the journal Nature.

Deisseroth explains the CLARITY technology and its applications in the following video.

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