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High-Res Imaging Obtained with Conventional Microscopes

By LabMedica International staff writers
Posted on 24 Apr 2017
Researchers have developed a technique for obtaining high-resolution images of tissue samples with confocal microscopes, at a fraction of the cost of other techniques that offer similar resolution. More...
This level of resolution allowed them to see, for example, proteins that cluster together in complex patterns at brain synapses.

The new technique, developed by researchers of Massachusetts Institute of Technology (Cambridge, MA, USA), relies on expanding the tissue before imaging. The team previously showed it was possible to expand tissue volumes 100-fold, resulting in an image resolution of about 60 nanometers. This was “useful for many scientific questions but couldn’t come close the performance of the highest-resolution imaging methods,” said the study’s senior author Ed Boyden, associate professor at MIT.

Now they have shown that expanding a second time can boost the resolution to about 25 nanometers. This level of resolution could help them map neural circuits in more detail. “We want to be able to trace the wiring,” said Prof Boyden, “If you could reconstruct a complete brain circuit, maybe you could make a computational model of how it generates complex phenomena like decisions and emotions. Since you can map out the biomolecules that generate electrical pulses within cells and that exchange chemicals between cells, you could potentially model the dynamics of the brain.”

This approach could also be used to image many other phenomena. Resolution of ~25 nanometers is similar to that achieved by high-resolution techniques such as stochastic optical reconstruction microscopy (STORM). However, expansion microscopy is much cheaper and simpler to perform because no specialized equipment or chemicals are required. The method is also much faster and thus compatible with large-scale, 3D imaging.

The resolution does not yet match that of scanning electron microscopy (about 5 nanometers) or transmission electron microscopy (about 1 nanometer). However, electron microscopes are very expensive and not widely available, and with those microscopes it is difficult to label specific proteins.

To expand tissue samples, the researchers embed them in an expandable gel made of the very absorbent material polyacrylate. Before the gel is formed, they use antibodies to label the cell proteins they want to image. These antibodies bear DNA “barcodes”, attached to cross-linking molecules that bind the gel polymers. The researchers then break down the proteins that normally hold the tissue together, allowing the DNA barcodes to expand away from each other as the gel swells.

These enlarged samples can then be labeled with fluorescent probes that bind the DNA barcodes, and imaged with a confocal microscope, whose resolution is usually limited to hundreds of nanometers.

“My hope is that we can, in the coming years, really start to map out the organization of these scaffolding and signaling proteins at the synapse,” said Prof. Boyden. Combining iterative expansion microscopy with a new tool called temporal multiplexing should help to achieve that, he added. Currently, only a limited number of colored probes can be used to image different molecules in a tissue sample. With temporal multiplexing, researchers can label one molecule with a fluorescent probe, take an image, and then wash the probe away. This can then be repeated many times, each time using the same colors to label different molecules.

The researchers hope to achieve a third round of expansion, which could, in principle, enable resolution of about 5 nanometers. However, the resolution is also limited by the size of the labeling antibodies (~10-20 nanometers), which would need to be overcome.

The study, by Chang JB et al, was published in the April 17, 2017, issue of Nature Methods.


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