Photonics Spectra BioPhotonics Vision Spectra Photonics Showcase Photonics Buyers' Guide Photonics Handbook Photonics Dictionary Newsletters Bookstore
Latest News Latest Products Features All Things Photonics Podcast
Marketplace Supplier Search ProdSpec Product Search Career Center
Webinars Photonics Media Virtual Events Industry Events Calendar
White Papers Videos Contribute an Article Suggest a Webinar Submit a Press Release Subscribe Advertise Become a Member


Microscopy Method Enables Deep In Vivo Brain Imaging

A method developed by the Prevedel Group at the European Molecular Biology Laboratory (EMBL) allows neuroscientists to observe live neurons deep within the brain — or any other cell hidden within an opaque tissue. The method is based on three-photon microscopy and adaptive optics.

The method increases the ability of scientists to observe astrocytes generating calcium waved in deep layers of the cortex, and to visualize any other neural cells in the hippocampus, the region of the brain responsible for spatial memory and navigation. The phenomenon takes place regularly in the brains of all live mammals. Lina Streich from the Prevedel Group and her collaborators were able to use the technique to capture the fine details of these versatile cells at unprecedented high resolution.

A deformable mirror used in microscopy to focus light within live tissues. An EMBL team combined adaptive optics and three-photon microscopy to support the ability of medical personnel to image deep in the hippocampus. Courtesy of Isabel Romero Calvo, EMBL.

In neurosciences, brain tissues are usually observed in small model organisms or in ex vivo samples that need to be sliced to be observed — both of which represent nonphysiological conditions. Normal brain cell activity takes place only in live animals. The mouse brain, however, is a highly scattering tissue, said Robert Prevedel. “In these brains, light cannot be focused very easily, because it interacts with the cellular components,” he said. “This limits how deep you can generate a crisp image, and it makes it very difficult to focus on small structures deep inside the brain with traditional techniques.

“With traditional fluorescence brain microscopy techniques, two photons are absorbed by the fluorescence molecule each time, and you can make sure that the excitement caused by the radiation is confined to a small volume. But the farther the photons travel, the more likely they are lost due to scattering.”

One way to overcome this is to increase the wavelength of the exciting photons toward the infrared, which ensures enough radiation energy to be absorbed by the fluorophore. Additionally, using three photons instead of two enables crisper images to be obtained deep inside the brain. Another challenge remained, however: making sure that the photons are focused, so that the whole image is not blurry.

Streich and her team used adaptive optics, which is often applied to astronomy; astrophysicists use deformable, computer controlled mirrors to correct in real time for the distortion in the lightwave front caused by atmospheric turbulence. In Prevedel’s lab, the distortion is caused by the scattering inhomogenous tissue, though the principle and the technology are very similar.

“We also use an actively controlled deformable mirror, which is capable of optimizing the wave fronts to allow the light to converge and focus, even deep inside the brain,” Prevedel said. “We developed a custom approach to make it fast enough to use on live cells in the brain.”

To reduce the invasiveness of the technique, the team also minimized the number of measurements needed to obtain high-quality images.

“This is the first time these techniques have been combined, and thanks to them, we were able to show the deepest in vivo images of live neurons at high resolution,” Streich said.

The scientists, who worked in collaboration with colleagues from EMBL Rome and the University of Heidelberg, even visualized the dendrites and axons that connect the neurons in the hippocampus, while leaving the brain completely intact.

“This is a leap toward developing more advanced noninvasive techniques to study live tissues,” Streich said.

Although the technique was developed for use on a mouse brain, it is easily applicable to any opaque tissue, the researchers said.

“Besides the obvious advantage of being able to study biological tissues without the need to sacrifice the animals or to remove overlaying tissue, this new technique opens the way to study an animal longitudinally, that is, from the onset of a disease to the end,” Streich said. “This will give scientists a powerful instrument to better understand how diseases develop in tissues and organs.”

The research was published in Nature Methods (www.doi.org/10.1038/s41592-021-01257-6).

Explore related content from Photonics Media




LATEST NEWS

Terms & Conditions Privacy Policy About Us Contact Us

©2024 Photonics Media