April 11, 2013
All About CLARITY: Scientists Develop See-Through Brain
WATCH VIDEO: [3D Analysis Of Intact Mouse Hippocampus]
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The new process is called CLARITY, and it ushers in an entirely new era of whole-organ imaging that could fundamentally change our understanding of the brain and other organs. Bioengineer and psychiatrist Karl Deisseroth, MD, PhD, led the team whose findings are published online in the journal Nature.
"Studying intact systems with this sort of molecular resolution and global scope – to be able to see the fine detail and the big picture at the same time – has been a major unmet goal in biology, and a goal that CLARITY begins to address," Deisseroth said.
"This feat of chemical engineering promises to transform the way we study the brain's anatomy and how disease changes it," said Thomas Insel, MD, director of the National Institute of Mental Health (NIMH). "No longer will the in-depth study of our most important three-dimensional organ be constrained by two-dimensional methods."
Primarily, the research in this study was conducted on a mouse brain. However, CLARITY has been tested on zebrafish and on preserved human brain samples. The results were similar to the mouse brain, establishing a potential path for studies of human samples and other organisms.
"CLARITY promises to revolutionize our understanding of how local and global changes in brain structure and activity translate into behavior," said Paul Frankland, PhD, a senior scientist in neurosciences and mental health at the Hospital for Sick Children Research Institute (SickKids) in Toronto.
THROUGH A GLASS DARKLY
The brain is a complex and inscrutable mass of convoluted grey matter and wiring, which neuroscientists have struggled to understand in their quest to comprehend how the brain works, and why it sometimes doesn't.
Prior to the Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue Hydrogel (CLARITY) process, brain matter was sliced into thin sections to be studied, which interrupts the structure and integrity of its circuitry. CLARITY is the result of an effort to extract the brain's opaque elements — particularly the lipids — while keeping the important features intact.
Found throughout the brain and body, lipids are fatty molecules that help form cell membranes and give the brain much of its structure. For biological study, however, lipids pose a double challenge because they make the brain largely impermeable to both chemicals and light.
To reveal the fine structure without slicing and sectioning the brain, neuroscientists have tried removing the lipids. There is just one small problem, however. Removing these structurally important molecules causes the remaining tissue to fall apart.
Previous efforts have focused on automating the slicing or sectioning approach to brain research, or have attempted to treat the brain with organic molecules that allowed the penetration of light but not macromolecular probes. CLARITY takes a fundamentally different, and novel, approach.
"We drew upon chemical engineering to transform biological tissue into a new state that is intact but optically transparent and permeable to macromolecules," said postdoctoral scholar Kwanghun Chung, PhD, the paper's first author.
This new state is achieved by replacing the brain's lipids with a hydrogel built from within the brain itself in a process that is akin to petrification. The process uses what is initially a watery suspension of short, individual molecules known as hydrogel monomers. The monomers infuse the tissues after the intact postmortem brain is immersed in the hydrogel solution. The brain is then "thermally triggered," or heated slightly to approximate body temperature, causing the monomers to congeal into long molecular chains known as polymers, which form a mesh throughout the brain. The mesh holds the surrounding tissues together but does not bind to the lipids.
The researchers are then able to rapidly and vigorously extract lipids through a process called electrophoresis, leaving behind a 3D, transparent brain with all the important structures such as neurons, axons, dendrites, synapses, proteins and nucleic acids intact.
"CLARITY has the potential to unmask fine details of brains from people with brain disorders without losing larger-scale circuit perspective," NIH Director Francis S. Collins, MD, PhD, said in a statement. The NIH Director's Transformative Research Award Program helped to fund the research, along with a grant from the NIMH.
GOING THE EXTRA MILE
CLARITY isn't finished there, however. Because it preserves the full continuity of neuronal structure, CLARITY not only allows researchers to trace individual neural connections over long distances through the brain, but also provides a way to gather rich, molecular information describing a cell's function impossible through other methods.
"We thought that if we could remove the lipids nondestructively, we might be able to get both light and macromolecules to penetrate deep into tissue, allowing not only 3-D imaging, but also 3-D molecular analysis of the intact brain," said Deisseroth, who holds the D.H. Chen Professorship. Deisseroth is a member of the "dream team" of experts who will map out goals for the $100 million brain research initiative announced April 2 by President Obama.
The research team demonstrated that it can target specific structures within the CLARITY-modified mouse brain using fluorescent antibodies that are known to seek out and attach themselves only to specific proteins. This targeting allows them to make those specific structures, and only those structures, light up under illumination. The scientists can then trace neural circuits through the entire brain or explore deeply into the nuances of local circuit wiring. The relationships between cells become clear and they are able to investigate subcellular structures as well. The researchers are even able to examine chemical relationships of protein complexes, nucleic acids and neurotransmitters.
"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," said Deisseroth.
Even more astounding, the researchers are able to "flush out" the fluorescent antibodies, destaining the clarified brain. They can then repeat the process using different antibodies to examine different molecular targets within the same brain. The team showed that this destaining process could be repeated several times and that the data sets aligned with one another.
A WHOLE NEW WORLD
According to the research team, CLARITY has made it possible to perform highly detailed, fine-structural analysis on intact brains – even human tissues that have been preserved for many years. For example, the team used CLARITY to examine the brain of a person who had autism, even though the brain had been stored in formaldehyde for six years. They were able to trace individual nerve fibers, neuronal cell bodies and their extensions.
The ability to transform human brains into stable, transparent specimens with accessible circuitry and molecular detail may improve our understanding of the structural underpinnings of brain function and disease.
Deisseroth cautions, however, that CLARITY has leapfrogged the ability to deal with the data.
"Turning massive amounts of data into useful insight poses immense computational challenges that will have to be addressed. We will have to develop improved computational approaches to image segmentation, 3-D image registration, automated tracing and image acquisition," he said.
In other words, our ability to decipher the data now needs to catch up with our ability to generate it. The pressure to develop the supporting technology will only increase as CLARITY begins to support a deeper understanding of large-scale intact biological systems and organs, perhaps even entire organisms.
"Of particular interest for future study are intrasystem relationships, not only in the mammalian brain but also in other tissues or diseases for which full understanding is only possible when thorough analysis of single, intact systems can be conducted," Deisseroth said. "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."
Image Below: Intact adult mouse brain before and after the two-day CLARITY process. In the image on the right, the fine brain structures can be seen faintly as the areas of blurriness above the words "number," "unexplored," "continent" and "stretches." Credit: Karl Deisseroth Lab / Stanford University Medical Center