The era of slicing and sectioning brain tissue for study may be coming to an end. A new imaging technique known as CLARITY makes the brain transparent and provides 3-dimensional (3D) views of complete and intact neural networks, including fine wiring and molecular connections.
Scientists say CLARITY has ushered in a new era of neuroimaging that will play a big part in the US government's brain mapping initiative announced April 2 by President Barack Obama.
The CLARITY method is nothing less than "astounding," said William Newsome, PhD, Stanford University neurobiologist and co-chair of the working group charged with developing a roadmap for accomplishing the brain mapping initiative.
CLARITY stands for Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel. The technique, which involves chemical engineering integrated with bioengineering, was developed by a team at Stanford University led by Karl Deisseroth, MD, PhD, a bioengineer and psychiatrist, and member of the working group.
Intact Brain
Lipids in the brain hold all the components of the brain together but make the brain largely impermeable both to chemicals and light. The CLARITY technique replaces the brain's lipids with a clear hydrogel. The result is tissue that is transparent and permeable, making it possible to image intact brain with high resolution down to the level of cells and molecules without the need for slicing.
Three-dimensional view of stained hippocampus showing fluorescent-expressing neurons (green), connecting interneurons (red) and supporting glia (blue)
"CLARITY is a way of transforming biological tissue into a form that can be studied in the fully intact state," Dr. Deisseroth said in a Stanford University podcast. "What CLARITY provides is a way of not having to disassemble tissue. The process is somewhat akin to petrification or fossilization but the key important elements, the basic structure, the form and even the fine structure down to the level of the synapses are maintained in place," he said.
"This feat of chemical engineering promises to transform the way we study the brain's anatomy and how disease changes it," Thomas R. Insel, MD, director of the National Institute of Mental Health, commented in a statement.
"CLARITY has the potential to unmask fine details of brains from people with brain disorders without losing larger-scale circuit perspective," added National Institutes of Health Director Francis C. Collins, MD, PhD.
Unlike mechanical sectioning methods, CLARITY preserves continuity of structure, which allows tracing of neurites over distances and provides a class of distinct information about 3D and topologic morphology of traced neurons, the Stanford team explains in the journal Nature April 10.
Combining CLARITY with light microscopy could be used to see and trace fluorescent-labeled neurons and projections in the whole brain, followed by electron microscopy to probe and define connectivity patterns, they explain.
The Stanford team applied the technique to whole mouse brains and were able to view neurons in areas ranging from the outer layers of the cortex to deeper structures, such as the thalamus. They could trace neurons and neural circuits and see relationships between protein complexes, nucleic acids, and neurotransmitters.
Dr. Karl Deisseroth
The team also applied the technique to banked human brain and found that they could see and identify neurons and projections over large areas. "In 0.5-mm-thick blocks of frontal lobe from an autistic patient, stored in formalin for > 6 years, we were able to stain for axons with neurofilament protein and myelin basic protein and trace individual fibres," they report in Nature
Applications Beyond the Brain
"We are interested in the structural differences that may exist in the brain in healthy states or disease states. A widely held hypothesis but difficult to test is that certain classes of psychiatric diseases like autism that the abnormality in their brains may relate to 3-dimensional wiring or connectivity differences," Dr. Deisseroth said.
With CLARITY, it's also possible to destain brain specimens, flush out the fluorescent antibodies, and repeat the staining process anew using different antibodies to explore different molecular targets in the same brain, the researchers note in their article.
Dr. Deisseroth cautioned that CLARITY has leapfrogged the ability of scientists to deal with the data generated. "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.
CLARITY has applications beyond the brain; it may be applicable to any biological system.
"We've already been contacted by individuals studying cancers who want to apply CLARITY to biopsy samples and understand the exact volumetric or 3-dimensional arrangements of cells at different stages of human cancers. This is a natural application for CLARITY," Dr. Deisseroth said.
In addition, researchers studying normal or abnormal functioning and physiology of other tissues, such as lung or muscle or heart, both in terms of development and function of the mature tissue and repair strategies of the tissue are already beginning to apply CLARITY, he added. "It will be interesting to see how other branches of biology may put it to use," the researcher said.
The research was funded by the National Institute of Mental Health, the National Science Foundation, the Simons Foundation, and the President and Provost of Stanford University. The authors have declared competing financial interests; details are available in the online version of the paper.
A '"fly through" video from the Stanford researchers appears here.
Nature. Published online April 10, 2013. Abstract
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