New microscopy technique penetrates deeper into living brain
The imaging method is poised to bring new insights into how the brain functions in terms of health and disease.
Researchers have developed a new technique that allows fluorescence microscopic imaging at four times the depth limit imposed by light scattering. Fluorescence microscopy is often used to image molecular and cellular details of the brain in animal models of various diseases but, until now, has been limited to small volumes and highly invasive procedures due to the intense diffusion of light through the skin and the skull.
“Visualization of biological dynamics in an undisturbed environment, deep within a living organism, is essential for understanding the complex biology of living organisms and the progression of disease,” said Daniel Razansky, team leader at research at the University of Zurich and ETH Zurich, both in Switzerland. . “Our study represents the first time that 3D fluorescence microscopy has been performed completely non-invasively at capillary-level resolution in an adult mouse brain, effectively covering a field of view of approximately 1 centimeter.
In OpticaThe Optical Society (OSA) review for High Impact Research, the researchers describe their new technique, called diffuse optical localization imaging (DOLI). It takes advantage of what is known as the second near infrared (NIR-II) spectral window from 1000 to 1700 nanometers, which has less scattering.
“Enabling high-resolution optical observations in deep living tissue is a long-standing goal in biomedical imaging,” said Razansky. “DOLI’s superb resolution for optical deep tissue observations can provide functional information about the brain, making it a promising platform for the study of neuronal activity, microcirculation, neurovascular coupling and neurodegeneration. ”
Reach greater depth
For the new technique, the researchers inject a living mouse intravenously with fluorescent microdroplets at a concentration that creates a sparse distribution in the bloodstream. Tracking these fluid targets allows reconstruction of a high resolution map of brain microvasculature deep within the mouse brain.
“The method eliminates background light scattering and is performed with the scalp and skull intact,” Razansky said. “Interestingly, we also observed a strong dependence of the size of the point recorded by the camera on the depth of the microdroplets in the brain, which enabled deep-resolved imaging.
The new approach benefits from the recent introduction of highly efficient shortwave infrared cameras based on InGaAs sensors. Another key feature was the use of new contrast agents exhibiting strong fluorescence responses in the NIR-II window, such as lead sulfide based quantum dots (PbS).
Crisp and clear imagery
The researchers first tested the new technique in synthetic models of tissue known as tissue phantoms that mimic the average properties of brain tissue, demonstrating that they could acquire microscopic resolution images at depths of up to 4 millimeters in optically opaque fabrics. They then performed DOLI in live mice where brain microvascularization as well as the speed and direction of blood flow could be visualized in a completely non-invasive manner.
Researchers are working to optimize the precision in all three dimensions to improve the resolution of DOLI. They also develop improved fluorescent agents which are smaller, have stronger fluorescence intensity and are more stable in vivo. This will dramatically increase DOLI’s performance in terms of achievable signal-to-noise ratio and image depth.
“We anticipate that DOLI will emerge as a powerful approach for fluorescence imaging of living organisms at previously inaccessible depth and resolution regimes,” said Razansky. “This will dramatically improve the in vivo applicability of fluorescence microscopy and tomography techniques.”
Reference: “Diffuse optical localization imaging for non-invasive deep brain microangiography in the NIR-II window” by Quanyu Zhou, Zhenyue Chen, Justine Robin, Xosé-Luís Deán-Ben and Daniel Razansky, May 27, 2021, Optica.
DOI: 10.1364 / OPTICA.420378