The Gift of Being Crystal Clear

The clear view: A three-dimensional rendering of clarified brain imaged from below (ventral half). The clear view: A three-dimensional rendering of clarified brain imaged from below (ventral half). Courtesy of the Deisseroth lab, Stanford University

Do we understand crystal clear thinking? Perhaps, when someone is expressing clear thoughts we understand the concept, but in reality, we really do not understand what happens inside the brain when one is thinking clearly. But now in a Nature paper, scientists at Stanford University have unveiled CLARITY, which is an acronym for a technique to render a post-mortem brain completely clear, that is, optically transparent and permeable to macromolecules. One day CLARITY may help us understand clear thinking and much more. Importantly, it could give us a handle to better understand the extremely complex neuronal circuitry in the brain, with its 1014-1015 synapses. It could help us better understand the abnormal circuit wiring that occurs in humans affected by developmental pathologies, such as many psychiatric disorders. What an exciting moment for us to be witnessing this discovery!

One of the hurdles in imaging the brain is that a fundamental component of the cell plasma membrane are lipid bilayers whose function is to create a barrier against the free diffusion of molecules. The lipid bilayers also have the inconvenient property (at least for imaging) of scattering light. In life and in the post-mortem state, these lipids create a barrier to molecular probes used in experiments and photons of light, making study and observation difficult. Extracting lipids from cells with organic solvents causes damages to other parts of the cells, mostly allowing proteins to “leak out.” Subsequently the structure loses consistency and falls apart, making study impossible. Many attempts to solving this problem have been made with some success but CLARITY seems now to have sunk in.

cyrstal-clearA closer look: Three-dimensional view of stained hippocampus showing fluorescent-expressing
neurons (green), connecting interneurons (red), and supporting glia (blue).
Courtesy of the Deisseroth lab, Stanford University
Karl Deisseroth, Kwanghun Chung, and colleagues1, developed a three-step process that overcomes this thorny problem while preserving the properties of the tissue. First, CLARITY requires infusing the brain with an acrylamide hydrogel and a fixative (formaldehyde), which in this first step does not permanently crosslink the tissue. Yet it does bind covalently (a type of chemical bond) proteins, nucleic acids, small molecules, but not, most critically, lipids. In step two, by raising the temperature, the hydrogel and the tissue hybridizes, becoming a sort of Jell-O, giving support to the cells in the tissue. In step three, an electric field is applied so that in the presence of a common lab detergent called SDS, lipids move from the negative pole toward the positive outside of the brain tissue, et voilà! We have a crystal clear brain.

The most impressive aspect of this scientific advance is that brain tissue, now lipid-free and supported by the hydrogel, is fully permeable to various nucleic acid probes, antibodies, and other molecules that scientists use for visualizing neurons and neural circuits. Being able to carry out these experiments on an intact brain or large chunks of one, is a huge advantage. Up to now, we could image in whole mount only brains that were already (relatively) transparent such as early embryonic brains where the circuitry is being assembled but not yet complete. The standard technique until now was to section the tissue and eventually reconstruct images by layers. There was always a great deal of error margin and labor involved. CLARITY might revolutionize the way we process tissues and greatly facilitate our understanding of important concepts in neurobiology. Further, there is no reason why this technology cannot be applied to other tissues or organs besides the brain.

One limit of CLARITY is that it can only work in post-mortem tissue. That’s a significant limit since in the field of integrative biology—thanks to super resolution technology—it is now possible to perform experiments in living cells; super resolution is already changing our view of biological process, but the two approaches are not mutually exclusive and should be seen instead as synergetic, serving different and complementary functions.

I now have a confession to make to the readers of the Activation Energy blog. Since the day I closed my lab to work in science policy and management, I have been having a ball. Shockingly, I never missed the lab for a moment, despite having enjoyed every moment of it when I was there. I simply moved on to other exciting and important things, and never regretted my decision. In the lab, I spent countless hours on molecular neuroanatomy, cutting, chopping, slicing, and dicing in whichever stereotactical direction the brain tissue ran. Then I would stain and interpret, assembling maps, discussing results, and redoing it all over again. This is why the process is called research and not just search.

But when I read the CLARITY paper published in Nature, I felt, for the first time since leaving the bench, obsolete. I felt like someone who still writes with a feathered quill, dipped in ink. Most of all, I felt professional envy. In my lab days, I would have loved to have taken this technology into my hands. So I will miss not being able to play with Jell-O brains. Perhaps soon I will be able to visit a lab that uses this technique and perhaps there I will be invited to spend a few hours puttering around with a perfectly clear brain. And yet it is in moments like this, when a new technique or a conceptual breakthrough throws open a view to unimagined horizons, that I realize scientists in the 21st century are in the most fortunate profession on earth. Every day, we hope to see more clearly.

  1. Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K. (2013). Structural and molecular interrogation of intact biological systems. Nature. Apr 10. doi: 10.1038/nature12107. [Epub ahead of print]
Stefano Bertuzzi

Dr. Stefano Bertuzzi is the Executive Director of the American Society for Cell Biology. In this position he is responsible, with the ASCB Board, for strategic planning and all operations at the Society to serve the needs of its ~9,000 members and to promote the field of cellular biology and basic science.

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