In a feat that almost sounds like science fiction, researchers at Stanford University have found a way to render a mouse brain transparent, making it easier to study the complex organ.
On Wednesday Stanford announced the news about the process it calls CLARITY, which it claimed “ushers in an entirely new era of whole-organ imaging that stands to fundamentally change our scientific understanding of the most-important-but-least-understood of organs, the brain, and potentially other organs, as well.”
By combining neuroscience and chemical engineering, with CLARITY “the postmortem brain remains whole — not sliced or sectioned in any way — with its three-dimensional complexity of fine wiring and molecular structures completely intact and able to be measured and probed at will with visible light and chemicals,” Stanford said in a press release.
CLARITY was described in an article published online in the journal Nature by bioengineer and psychiatrist Dr. Karl Deisseroth, leading a multidisciplinary team that included postdoctoral scholar Kwanghun Chung.
“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 in a prepared statement.
“This feat of chemical engineering promises to transform the way we study the brain’s anatomy and how disease changes it,” Dr. Thomas Insel, director of the National Institute of Mental Health, said in a prepared statement. “No longer will the in-depth study of our most important three-dimensional organ be constrained by two-dimensional methods.”
The research was performed primarily on a mouse brain, but the researchers also used CLARITY on zebrafish and on preserved human brain samples with similar results.
Neuroscientists have struggled to fully understand the brain’s circuitry in order to understand how the brain works.
CLARITY is the result of a research effort in Deisseroth’s lab to extract the opaque elements — in particular the lipids — from a brain and yet keep the important features fully intact.
Lipids are fatty molecules, and in the brain they help form cell membranes, giving the organ its structure. But Lipids pose a double challenge for biological study “because they make the brain largely impermeable both to chemicals and to light,” Stanford said.
Neuroscientists wanted to extract the lipids in order to reveal the brain’s fine structure without slicing or sectioning, but for one major hitch: removing these structurally important molecules causes the remaining tissue to fall apart.
Prior investigations have focused instead on automating the slicing/sectioning approach, or in treating the brain with organic molecules that facilitate the penetration of light only, but not macromolecular probes. With CLARITY, Deisseroth’s team took a different 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 Chung, the paper’s first author.
This new form is created by replacing the brain’s lipids with a hydrogel.
“The hydrogel is built from within the brain itself in a process conceptually similar to petrification, using what is initially a watery suspension of short, individual molecules known as hydrogel monomers,” Stanford said.
“The intact, postmortem brain is immersed in the hydrogel solution and the monomers infuse the tissue. Then, when ‘thermally triggered,’ or heated slightly to about body temperature, the monomers begin to congeal into long molecular chains known as polymers, forming a mesh throughout the brain. This mesh holds everything together, but, importantly, it does not bind to the lipids.”
With the brain tissue shored up in this manner, researchers were able to extract lipids through a process called electrophoresis. What they had left was a 3-D, transparent brain with all of its important structures — neurons, axons, dendrites, synapses, proteins, nucleic acids and so forth — intact and in place.
CLARITY not only preserves neuronal structures, it allows the tracing of individual neural connections over long distances through the brain, but also provides a way to gather molecular information describing a cell’s function is that isn’t possible with 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,” Deisseroth said.
Using fluorescent antibodies that seek out and attach themselves only to specific proteins, researchers were able to target specific structures within the CLARITY-modified — or “clarified” — mouse brain and make those structures and only those structures light up under illumination.
Researchers were able to trace neural circuits through the entire brain and study the nuances of local circuit wiring. They could also examine the relationships between cells and investigate subcellular structures. They can even look at 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.
He knows what he’s talking about. Deisseroth is one of 15 experts on the “dream team” that will map out goals for the $100 million brain research initiative announced earlier this month by President Obama.
With CLARIFY, researchers can now also de-stain the clarified brain, flushing out the fluorescent antibodies and repeating the staining process anew using different antibodies to explore different molecular targets in the same brain. This staining/de-staining process can be repeated multiple times, the authors showed, and the different data sets aligned with one another.
CLARITY has also made it possible to perform highly detailed, fine-structural analysis on intact brains — even human tissues that have been preserved for many years, the team showed.
“Transforming human brains into transparent-but-stable specimens with accessible wiring and molecular detail may yield improved understanding of the structural underpinnings of brain function and disease,” Stanford said.
According to Deisseroth, cautioned that CLARITY has surpassed our ability to deal with the data.
“Turning massive amounts of data into useful insight poses immense computational challenges that will have to be addressed.” he said. “We will have to develop improved computational approaches to image segmentation, 3-D image registration, automated tracing and image acquisition,” he said.
Indeed, such pressures will increase as CLARITY could begin 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.
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