Microscopy Method Enables Deep In Vivo Brain Imaging

2021.10.11 / By admin

HEIDELBERG, Germany, Oct. 4, 2021 — 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. Courtesy of Isabel Romero Calvo, EMBL.
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.

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