Expanded Tissues Show Confocal Microscopes More Detail
CAMBRIDGE, Mass. — Nobel Prize-winning superresolution microscopy techniques circumvent the diffraction limit of light to image the smallest details of cells. But there is another way.
Researchers at MIT have developed a method for making biological tissue samples physically larger, rendering their nanoscale features visible to conventional confocal microscopes.
Using inexpensive, commercially available chemicals and microscopes commonly found in research labs, the technique could give more scientists access to 3-D superresolution imaging.
“Instead of acquiring a new microscope to take images with nanoscale resolution, you can take the images on a regular microscope,” said MIT professor Dr. Edward Boyden. “You physically make the sample bigger, rather than trying to magnify the rays of light that are emitted by the sample.”
The process involves meshes of sodium polyacrylate, the superabsorbent chemical used in disposable diapers. When exposed to water, these meshes expand, and the cellular structures around them expand too.
Cells in rodent brain slices, as well as cells grown in vitro, are first fixed in formaldehyde and then gently stripped of their fatty membranes before being labeled with fluorescent markers.
A precursor is then added and heated to form the polyacrylate gel. Proteins that hold the specimen together are digested, allowing it to expand uniformly. Finally, the sample is washed in salt-free water to trigger the expansion.
Even though the proteins have been broken apart, the original location of each fluorescent label stays the same relative to the overall structure of the tissue because it is anchored to the polyacrylate gel by antibodies.
Scientists modified the superabsorbant diaper compound sodium polyacrylate to enlarge brain tissue and image it in 3-D using fluorescent tags and confocal microscopes. Courtesy of the Boyden Lab/MIT.
Samples were imaged before expansion using superresolution microscopes, and imaged afterward using confocal microscopes. Expansion gave the confocal microscopes an effective 70-nm lateral resolution — sharp enough to resolve details of the cell protein complexes, the spaces between rows of skeletal microtubule filaments and the two sides of synapses.
The diffraction limit means standard microscopes can’t resolve objects smaller than about 250 nm. “Unfortunately, in biology that’s right where things get interesting,” Boyden said.
Three inventors of superresolution microscopy won the 2014 Nobel Prize in chemistry. Superresolution techniques, however, have their own limitation: They work best with small, thin samples, and take a long time to image large samples. They can also be hampered by optical scattering in thick samples.
“If you want to map the brain, or understand how cancer cells are organized in a metastasizing tumor, or how immune cells are configured in an autoimmune attack, you have to look at a large piece of tissue with nanoscale precision,” Boyden said.
The MIT technique allowed imaging of samples approximately 500 × 200 × 100 µm in volume.
“The other methods currently have better resolution, but are harder to use, or slower,” said graduate student Paul Tillberg. “The benefits of our method are the ease of use and, more importantly, compatibility with large volumes, which is challenging with existing technologies.”
Funding came from the National Institutes of Health, National Science Foundation, New York Stem Cell Foundation, Jeremy and Joyce Wertheimer and the Fannie and John Hertz Foundation.
The research was published in Science (doi: 10.1126/science.1260088).
For more information, visit www.mit.edu.
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