Figuring out how cellular molecules work in real time and space has been hampered by a kind of uncertainty principle. Tissues can be imaged while intact—but with preparations that prevent molecular phenotyping. Or they can be labeled—but then imaged only in sections.
Mouse brains have been made transparent before, most notably by using the SCALE method developed in 2011 by Japanese researchers. But CLARITY is different, said Deisseroth, because it allows “not just transparency but labeling within the transparent organ in a very flexible, versatile way. You can use any molecular label to paint different neurons different colors and see which are excitatory and which are inhibitory. CLARITY can add a lot of information that was not available before.”
CLARITY replaces the structural function of the cell membrane’s lipid bilayer—which effectively keeps useful reagents like antibodies, fluorescent dyes, and photons out of cells—with a formaldehyde crosslinked hydrogel mesh. The hydrogel is permeable to light and macromolecules, but physically supports the tissue structure by chemically incorporating native biomolecules. Lipids and other molecules that remain unbound are removed to reduce light scattering.
Using this method, it took the researchers about a week to clear the tissue in an adult mouse brain. They then imaged the entire 5-6-mm thick translucent mouse brain at cellular resolution using single-photon microscopy. Structural features like synapses and dendritic spines remained in place; labeled fibers were easily seen in the neocortex, nucleus accumbens, caudate-putamen, and amygdala.
In contrast to other fixing methods, CLARITY retains about 92% of native proteins, so the brain can be subjected to repeated labeling; the group demonstrated that at least three rounds of labeling can be performed successfully. And since CLARITY’s hydrogel both increases tissue permeability and decreases light scattering relative to cell membranes, molecular probes can diffuse deep into, and be detected deep inside, intact tissue. Diesseroth and colleagues believe that the technique might one day be compatible with electron microscopy, allowing more detailed imaging.
In addition to the mouse brain, the group clarified 0.5-mm blocks of a human brain that had been stored in formalin for over six years. The blocks were from the frontal lobe of an autistic patient, and staining revealed structural abnormalities in deep layers of the tissue that were absent in normal age- and sex- matched controls.
“The brain is the most difficult organ, because it has the most dense areas of lipid interfaces and that is what blocks light,” said Deisseroth. “We don’t anticipate serious problems with other organs, although the methodology might have to be modified somewhat.” Already, cancer researchers have contacted him about clarifying tumor biopsies.
Deisseroth was one of the scientists who proposed and supports the recently announced BRAIN Initiative. “CLARITY arose because of interactions between neuroscientists and chemical engineers—groups that do not normally speak to each other,” he noted. “This new project will also get people talking. Imagine the opportunities and new ideas it will bring to light.”
- Chung, K., J. Wallace, S.-Y. Kim, S. Kalyanasundaram, A. S. Andalman, T. J. Davidson, J. J. Mirzabekov, K. A. Zalocusky, J. Mattis, A. K. Denisin, S. Pak, H. Bernstein, C. Ramakrishnan, L. Grosenick, V. Gradinaru, and K. Deisseroth. 2013. Structural and molecular interrogation of intact biological systems. Nature advance online publication(April).