A hydrogel process that renders a postmortem mouse brain transparent to light while keeping it intact has provided a highly detailed glimpse of its inner structure. The mound of convoluted gray matter and wiring that is the brain is complex and inscrutable. In their quest to comprehend how it works — and sometimes doesn’t — neuroscientists have struggled to fully understand its circuitry. Using a new method called CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel), optogeneticist Dr. Karl Deisseroth and a multidisciplinary team have imaged the neurological wiring in a mouse’s brain. The technique ushers in an entirely new era of whole-organ imaging that stands to fundamentally change our scientific understanding of the brain and, potentially, other organs as well. “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,” said Deisseroth, a bioengineer and psychiatrist at Stanford University. Traditionally, imaging organs like the brain involved a slicing or sectioning approach, which destroys long-distance neural connections. Techniques to treat the brain with organic molecules exist, but they facilitate only light penetration, not macromolecular probes. With CLARITY, Deisseroth’s team has taken a fundamentally 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 postdoctoral scholar Kwanghun Chung, the paper's first author. Intact adult mouse brain before and after the two-day CLARITY process developed in the lab of Dr. Karl Deisseroth of Stanford University. 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." Courtesy of the Deisseroth lab. The method works by replacing the brain’s fatty tissues called lipids with a hydrogel, which is built from within the brain itself in a process conceptually similar to petrification. The intact postmortem brain is immersed in a hydrogel solution and heated slightly to form a mesh that congeals everything in place except the fatty parts. These lipids are removed through a process called electrophoresis. What remains is 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 then goes an extra step. In preserving the full continuity of neuronal structures, it not only allows tracing of individual neural connections over long distances through the brain, but also provides a way to gather rich molecular information describing a cell’s function that is not possible with other methods. A 3-D rendering of clarified brain imaged from below (ventral half). Courtesy of the Deisseroth lab. “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. Fluorescent antibodies were used to target specific structures within the CLARITY-modified — or “clarified” — mouse brain to make only those structures light up under illumination. This enabled the investigators to trace neural circuits through the entire brain and explore deeply into the nuances of local circuit wiring. Relationships between cells and subcellular structures can also be investigated. “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, who is one of 15 experts on the "dream team" that will map out goals for the $100 million brain research initiative announced April 2 by President Obama. (See: Optics Community Hails Obama’s Brain Mapping Initiative) A 3-D view of stained hippocampus showing fluorescence-expressing neurons (green), connecting interneurons (red) and supporting glia (blue). Courtesy of the Deisseroth lab. The researchers are also able to destain 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 process can be repeated multiple times, the investigators said, and the different data sets aligned with one another. “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.” The process — performed primarily on a mouse brain, but also used to image zebra fish brains and preserved human brain samples — was described in Nature (doi: 10.1038/nature12107). For more on Deisseroth’s research, see: Light Workout: Optogenetics Stimulates Mouse Muscles. For more information, visit: www.stanford.edu