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Researchers could quickly obtain high-resolution images from blood vessels and neurons in the brain.
Two-photon microscopy is a technique that allows researchers to create 3D images of tissues, such as the brain. This involves using a high-intensity laser to aim at the specimen and induce fluorescence excitation. Scanning deep into the brain can be challenging because light scatters away from tissues as it moves deeper, blurring images.
Two-photon imaging can be time-consuming as each pixel must be scanned one at a. Researchers from Harvard University, and MIT have developed a modified form of two-photon imaging. This allows for imaging deeper into tissue and can be performed much faster than possible.
Researchers believe this imaging could enable scientists to quickly obtain high-resolution images, such as of blood vessels or individual neurons in the brain.
Murat Yildirim (MIT researcher and one of the researchers of the new study) says, “By modifying laser beam coming into the tissue, we showed we can go deeper”, and that finer imaging can be done than previous techniques.
Cheng Zheng, a former postdoc at MIT, and Jong Kang Park, a former postdoc at the Center for Advanced Imaging, co-authored the paper. It was published in Science Advances on July 7, 2021. The paper’s senior author is Dushan N. Walduwage, a former MIT postdoc and now a John Harvard Distinguished Science Fellowship in Imaging at Harvard University. Josiah Boivin is an MIT postdoc. Yi Xue is a former MIT graduate. Mriganka Sur at MIT is the Newton Professor in Neuroscience. Peter So is an MIT professor of mechanical and biological engineering.
Deep imaging
Two-photon microscopy is achieved by shining a beam of near-infrared light on a single spot within a sample. This causes the simultaneous absorption of two photons at that focal point. The intensity of the light is the highest. This low-energy, long-wavelength light can penetrate deeper into tissues without damaging them, which allows for imaging below the surface.
Two-photon excitation produces images by fluorescence. The fluorescent signal is located in the visible spectrum. The picture becomes blurry if the fluorescent light scatters further into the tissue samples. It can also be very time-consuming to image many layers of tissue. Wide-field imaging is a way to speed up the process. However, the resolution is lower than point-by-point.
The MIT team sought to create a method to image large tissue samples while maintaining high-resolution point-by-point scanning simultaneously. They developed a way to control the light they shine onto the piece. They employ a technique called wide-field microscopy. This involves shining a light plane onto the tissue and then changing the intensity of the light to turn each pixel on/off at different times. This predesigned pattern can be seen in the scattered light from the tissue. Some pixels are lit up, while others remain dark.
Zheng explains that each pixel can be turned on or off using this type of modulation. “If we turn off certain spots, it creates space around every pixel so that we can see what is happening in each spot.”
Researchers obtain raw images and then reconstruct each pixel with a computer algorithm they developed.
“We control the light’s shape and get the response from tissue. We can then determine the scattering in the tissue’s responses. Yildirim states that reconstructions are done from raw images. This allows us to get more information than you can see in the raw images.
The researchers could image slices of muscle and kidney tissue up to 200 microns deep and the brains of mice as far as 300 microns. Yildirim claims this is twice the depth possible without computational reconstruction and patterned excitation. This technique is also 100-1000 times faster than traditional two-photon microscopy.
Brain structure
This imaging technique should enable researchers to quickly obtain high-resolution images, including those of neurons and other structures like blood vessels. Yildirim believes that imaging blood vessels in mice’s brains could be especially useful in learning more about the effects of neurodegenerative diseases like Alzheimer’s on blood flow.
He says that all studies of blood flow and morphology of blood vessel structures are done using two-photon scans. This technology allows us to perform high-speed volumetric imaging and analysis of blood flow and blood vessel structures to understand changes in blood flow.
This technique can also measure neuronal activity using voltage-sensitive fluorescent dyes and calcium probes, which light up when neurons get excited. It can also analyze other tissue types, such as tumors.
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