The researchers were able to quickly obtain high-resolution images of blood vessels and neurons in the brain. –ScienceDaily

To create high-resolution 3D images of tissues such as the brain, researchers often use two-photon microscopy, which involves shining a high-intensity laser at the sample to induce fluorescence excitation. However, scanning deep into the brain can be difficult because light scatters from the tissues as it goes deeper, making images blurry.

Two-photon imaging is also time-consuming, as it typically requires scanning individual pixels one by one. A team of researchers from MIT and Harvard University has now developed a modified version of two-photon imaging that can image deeper into tissue and perform imaging much faster than previously possible.

This type of imaging could allow scientists to more quickly obtain high-resolution images of structures such as blood vessels and individual neurons in the brain, the researchers say.

“By modifying the laser beam entering the tissues, we have shown that we can go further and do finer imaging than previous techniques,” explains Murat Yildirim, a researcher at MIT and one of the authors of the new study.

MIT graduate student Cheng Zheng and former post-doctoral fellow Jong Kang Park are lead authors of the paper, which appears today in Scientists progress. Dushan N. Wadduwage, a former MIT postdoc who is now a John Harvard Distinguished Science Fellow in Imaging at Harvard University’s Center for Advanced Imaging, is the paper’s lead author. Other authors include MIT postdoctoral fellow Josiah Boivin; Yi Xue, a former graduate student of MIT; Mriganka Sur, Newton Professor of Neuroscience at MIT; and Peter So, professor of mechanical engineering and biological engineering at MIT.

Deep Imaging

Two-photon microscopy works by shining an intense beam of near-infrared light onto a single point on a sample, inducing the simultaneous absorption of two photons at the focal point, where the intensity is highest. This long-wavelength, low-energy light can penetrate deeper into tissue without damaging it, enabling subsurface imaging.

However, two-photon excitation generates fluorescence images, and the fluorescent signal is in the visible spectral region. When imaging deeper into tissue samples, the fluorescent light scatters more and the image becomes blurred. Imaging many tissue layers is also time consuming. Using wide-field imaging, in which an entire plane of tissue is illuminated at once, can speed up the process, but the resolution of this approach is not as high as that of point-to-point scanning.

The MIT team wanted to develop a method that would allow them to image a large tissue sample all at once, while maintaining the high resolution of point-to-point scanning. To achieve this, they found a way to manipulate the light they shine on the sample. They use a form of wide-field microscopy, projecting a plane of light onto tissue, but changing the amplitude of the light so they can turn each pixel on or off at different times. Some pixels are bright while neighboring pixels remain dark, and this predefined pattern can be detected in the light scattered by the fabric.

“We can turn each pixel on or off by this type of modulation,” Zheng explains. “If we turn off certain spots, it creates space around each pixel, so now we can know what’s going on in each of the individual spots.”

Once the researchers get the raw images, they reconstruct each pixel using a computer algorithm they created.

“We check the shape of the light and we get the response of the tissue. From these responses we try to determine the type of scattering of the tissue. When we do the reconstructions from our raw images, we can get a lot of insights. information that you can’t see in the raw footage,” says Yildirim.

Using this technique, the researchers showed they could image about 200 microns deep in slices of muscle and kidney tissue, and about 300 microns deep in mouse brains. It’s about twice as deep as was possible without that patterned excitement and computer reconstruction, Yildirim says. The technique can also generate images about 100 to 1,000 times faster than conventional two-photon microscopy.

Brain structure

This type of imaging should allow researchers to more quickly obtain high-resolution images of neurons in the brain, as well as other structures such as blood vessels. Imaging blood vessels in the brains of mice could be particularly useful for learning more about how blood flow is affected by neurodegenerative diseases such as Alzheimer’s disease, Yildirim says.

“All studies of blood flow or the morphology of blood vessel structures are based on two- or three-photon point scanning systems, so they are slow,” he says. “Using this technology, we can really do high-speed volumetric imaging of blood flow and blood vessel structure to understand changes in blood flow.”

The technique could also lend itself to measuring neuronal activity, by adding voltage-sensitive fluorescent dyes or fluorescent calcium probes that light up when neurons are fired. It could also be useful for analyzing other types of tissue, including tumors, where it could be used to help determine the edges of a tumor.

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