First 3D super-resolution image of dendritic spines captured by Yale researchers

Deep tissue imaging is possible with the new STED technology, which displays the internal movements of neurons.

Researchers have created a novel microscopy technique that can see subcellular structures in 3D super-resolution from roughly 100 microns deep into living tissue, including the brain. The approach might help show small changes in neurons that occur over time, during learning, or as a result of disease by offering scientists a better understanding into the brain.

The new method is a further development of stimulated emission depletion (STED) microscopy, a ground-breaking technique for achieving nanoscale resolution by bypassing optical microscopes’ typical diffraction limit. For discovering this super-resolution imaging technology, Stefan Hell received the Nobel Prize in Chemistry in 2014.

The researchers report how they utilized their novel STED microscope to study the 3D structure of dendritic spines deep inside the brain of a living mouse in super-resolution in Optica, The Optical Society’s (OSA) magazine for high impact research. Dendritic spines are little protrusions on neurons’ dendritic branches that receive synaptic input from nearby neurons and they play an important role in neuronal activity.

“Our microscope is the first instrument in the world to achieve 3D STED super-resolution deep inside a living animal,” said leader of the research team Joerg Bewersdorf from Yale School of Medicine. “Such advances in deep-tissue imaging technology will allow researchers to directly visualize subcellular structures and dynamics in their native tissue environment,” said Bewersdorf. “The ability to study cellular behavior in this way is critical to gaining a comprehensive understanding of biological phenomena for biomedical research as well as for pharmaceutical development.”

The most common method for imaging cultivated cell specimens is conventional STED microscopy. Imaging dense tissue or living creatures with the technology is much more difficult, especially when the super-resolution features of STED are extended to the third dimension for 3D-STED. This restriction arises because thick, optically dense tissue inhibits light from reaching deeply and focusing properly, limiting the STED microscope’s super-resolution capabilities.

STED microscopy was used with two-photon excitation (2PE) and adaptive optics to address this difficulty. “2PE enables imaging deeper in tissue by using near-infrared wavelengths rather than visible light,” said Mary Grace M. Velasco, first author of the paper. “Infrared light is less susceptible to scattering and, therefore, is better able to penetrate deep into the tissue.”

Adaptive optics were also integrated into the researchers’ system. “The use of adaptive optics corrects distortions to the shape of light, i.e., the optical aberrations, that arise when imaging in and through tissue,” said Velasco. “During imaging, the adaptive element modifies the light wavefront in the exact opposite way that the tissue in the specimen does. The aberrations from the adaptive element, therefore, cancel out the aberrations from the tissue, creating ideal imaging conditions that allow the STED super-resolution capabilities to be recovered in all three dimensions.”

The scientists put their 3D-2PE-STED approach to the test on a cover slip by photographing well-characterized structures in grown cells. 3D-2PE-STED resolved volumes that were more than ten times smaller than when utilizing 2PE alone. They also demonstrated that their microscope was considerably superior than a standard two-photon microscope in resolving the distribution of DNA in the nucleus of mouse skin cells.

The researchers utilized their 3D-2PE-STED microscope to study the brain of a live mouse after these experiments.
They determined the 3D structure of individual spines by zooming in on a section of a dendrite. They examined the identical location two days later and found that the spine structure had been altered. The researchers found no evidence of harm from the imaging in the structure of the neurons in their pictures or in the behavior of the mice. They do, however, want to investigate this further.

“Dendritic spines are so small that without super-resolution it is difficult to visualize their exact 3D shape, let alone any changes to this shape over time,” said Velasco. “3D-2PE-STED now provides the means to observe these changes and to do so not only in the superficial layers of the brain, but also deeper inside, where more of the interesting connections happen.”

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