New technique makes brain see-through to enable 3D analysis of fine structuresApr 11, 10:51 am
London, April 11 (ANI): Thanks to a breakthrough method developed by National Institutes of Health-funded researchers, scientists can now study the brain's finer workings, while preserving its 3-D structure and integrity of its circuitry and other biological machinery.Called CLARITY, the new technique opens the intact postmortem brain to chemical, genetic and optical analyses that previously could only be performed using thin slices of tissue. By replacing fat that normally holds the brain's working components in place with a clear gel, they made its normally opaque and impenetrable tissue see-through and permeable. This made it possible to image an intact mouse brain in high resolution down to the level of cells and molecules. The technique was even used successfully to study a human brain."CLARITY has the potential to unmask fine details of brains from people with brain disorders without losing larger-scale circuit perspective," said NIH Director Francis S. Collins, M.D., Ph.D., whose NIH Director's Transformative Research Award Program helped to fund the research, along with a grant from the National Institute of Mental Health NIMH."CLARITY will help support integrative understanding of large-scale, intact biological systems, explained Karl Deisseroth, M.D., Ph.D., of Stanford University in California. "It provides access to subcellular proteins and molecules, while preserving the continuity of intact neuronal structures such as long-range circuit projections, local circuit wiring, and cellular spatial relationships," he added.Until now, researchers seeking to understand the brain's fine structure and connections have been faced with tradeoffs. To gain access to deeply buried structures and achieve high enough resolution to study cells, molecules and genes, they had to cut brain tissue into extremely thin sections (each a fraction of a millimeter thick), deforming it. Loss of an intact brain also makes it difficult to relate such micro-level findings to more macro-level information about wiring and circuitry, which cuts across slices.In tackling this challenge, the researchers saw opportunity in the fact that the fats, or lipids, that physically support the brain's working components, such as neurons and their connections, also block chemical probes and the passage of light. So replacing the lipids with something clear and permeable - that would also hold everything else in place - might make it possible to perform the same tests in an intact brain that previously could only be done with brain tissue slices.Deisseroth's team infused into brain a high-tech cocktail, including a plastic-like material and formaldehyde. When heated, it formed a transparent, porous gel that biochemically integrated with, and physically supported, the brain's working tissue - while excluding the lipids, which were safely removed via an electrochemical process. The result was a brain transformed for optimal accessibility.They called the new method Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue Hydrogel - CLARITY, for short.Using CLARITY, the researchers imaged the entire brain of a mouse that had been genetically engineered to express a fluorescent protein. A conventional microscope revealed glowing details, such as proteins embedded in cell membranes and individual nerve fibers, while an electron microscope resolved even ultra-fine structures, such as synapses, the connections between neurons.In a series of experiments using CLARITY in mouse brain, the researchers demonstrated that, for the first time, standard immune- and genetics-based tests could be performed repeatedly in the same intact brain. Tracer molecules, such as antibodies, can be readily delivered for staining tissue - or removed - leaving brain tissue undisturbed.The researchers found that CLARITY outperformed conventional methods across a range of previously problematic technical challenges.When they used CLARITY to analyze a post-mortem human brain of a person who had autism, even though it had been hardening in formaldehyde for six years, they were able to trace individual nerve fibers, neuronal cell bodies and their extensions.Deisseroth, Kwanghun Chung, Ph.D., and other Stanford colleagues reported their findings in the journal Nature . (ANI)
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