Big break for brain mapping
Image: Magnified analysis of proteome - MAP - allows researchers to study cells and also view long-range connections between neurons. [MIT]
MIT researchers have developed a new technique for imaging brain tissue at multiple scales, allowing them to image molecules within cells or take a wider view of the long-range connections between neurons.
So-called magnified analysis of proteome - MAP - linearly expands entire organs fourfold while preserving overall architecture and three-dimensional proteome organisation.
Crucially, the method is set to help researchers chart the connectivity and functions of neurons in the human brain and can be applied to other organs including the heart, lungs,liver and kidneys.
"We use a chemical process to make the whole brain size-adjustable, while preserving pretty much everything," says Professor Kwanghun Chung, Department of Chemical Engineering. "We preserve the proteome [proteins in a biological sample], we preserve nanoscopic details, and we also preserve brain-wide connectivity."
MAP builds on the tissue transformation method, CLARITY, pioneered by Chung, which preserves cells and molecules in brain tissue, rendering these structures transparent so molecules inside the cell can be imaged in 3D.
As Chung points out: "MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation."
"The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity," he adds.
However, to image the brain at multiple scales within the same tissue sample, Chung and colleagues developed the method to reversibly expand tissue samples while preserving nearly all of the proteins within the cells; these proteins can then be labeled with fluorescent molecules and imaged.
As Chung highlights, the technique relies on flooding the brain tissue with acrylamide polymers, which form a dense gel.
In this case, the gel is ten times denser than that used for CLARITY, providing the sample with more stability so researchers can denature and dissociate the proteins inside the cells without destroying the tissue structure.
Prior to denaturing, the researchers attach the proteins to the gel using formaldehyde, as takes place with CLARITY, and with these structures attached and denatured, the gel expands the tissue sample to around four times its original size.
"This is reversible and you can do it many times," highlights Chung. "You can then use off-the-shelf molecular markers, such as antibodies to label and visualise the distribution of all these preserved biomolecules."
In their studies, the researchers imaged neuronal structures such as axons and synapses by labeling proteins found in those structures, and they also labeled proteins that allow them to distinguish neurons from glial cells.
With the tissue expanded, researchers can use common microscopes to obtain images with a resolution as high as 60 nm - better than the usual 250 nm limit of light microscopes.
The approach also works with relatively large tissue samples, up to 2 mm thick.
"This is, as far as I know, the first demonstration of super-resolution proteomic imaging of millimeter-scale samples," says Chung.
Today's efforts to map the connections of the human brain rely on electron microscopy, but Chung and colleagues reckon the higher resolution MAP imaging technique can trace these connections more accurately.
Chung's lab is now working to speed up imaging and image processing, which is challenging because of the sheer volumes of data generated from imaging the expanded tissue samples.
"It's already easier than other techniques because the process is really simple and you can use off-the-shelf molecular markers, but we are trying to make it even simpler," concludes Chung.
Research is published in Nature Biotechnology.