A noninvasive brain imaging technique developed by researchers at ETH Zurich and the University of Zurich works in the near-infrared (NIR) spectrum to enable superresolution deep-tissue fluorescence microscopy at four times the depth limit imposed by light diffusion. According to the researchers, the technique, called diffuse optical localization imaging (DOLI), operates in a resolution-depth regime previously inaccessible with optical methods. DOLI could allow scientists to study neuronal processes at the level of single cells and capillaries across the entire living brain without needing to use highly invasive surgical methods. DOLI uses a combination of individual techniques to enable noninvasive deep brain imaging at high resolution. To reduce scattering, the researchers used a specific spectral region, the second near-infrared (NIR-II) spectral window from 1000 to 1700 nm, for imaging. “This allowed us to greatly reduce the background scattering, absorption, and intrinsic fluorescence of the living tissues,” professor Daniel Razansky said. The researchers also used an efficient shortwave infrared (SWIR) camera based on InGaAs sensors and a new quantum dot contrast agent that fluoresces strongly within the NIR-II window. A new imaging method can capture images of vasculature deep in the brains of mice. A conventional widefield fluorescence image of the mouse brain taken noninvasively in the visible light spectrum is shown on the left, while the noninvasive localization-based DOLI approach operating in the NIR-II spectral window is shown on the right. Courtesy of Daniel Razansky, ETH Zurich, and the University of Zurich. The researchers initially tested DOLI with synthetic tissue models, called tissue phantoms, that simulate the properties of brain tissue. The tests showed that DOLI could acquire microscopic resolution images at depths of up to 4 mm in optically opaque tissues. They then tested DOLI in living mice and were able to visualize cerebral microvasculature as well as blood flow velocity and direction. They injected live mice with microdroplets encapsulating the fluorescent quantum dot at a concentration that created a sparse distribution in the blood stream. They were able to localize these droplets individually in the mice brains. By tracking these flowing targets, researchers were able to construct a high-resolution map of the deep cerebral microvasculature in the mouse brain. “For the first time, we were able to clearly visualize the microvasculature and blood circulation deep in the mouse brain entirely noninvasively,” Razansky said. Traditionally, the use of fluorescence microscopy to visualize biological processes at the cellular and molecular levels in animal brains has been limited by scattering. Living tissue scatters and absorbs light extensively, blurring images and making it hard to identify the exact location of the fluorescent agent inside the brain. The DOLI method eliminates background light scattering and is performed with the scalp and skull intact. The researchers further observed that the size of the imaged microdroplets depended on how deep in the brain they were located, indicating that the DOLI technique is capable of 3D imaging. “Interestingly, we also observed strong dependence of the spot size recorded by the camera on the microdroplet’s depth in the brain, which enabled depth-resolved imaging,” Razansky said. The researchers are working to improve DOLI’s resolution by optimizing its precision in all three dimensions. They are also developing fluorescent agents that are smaller, have stronger fluorescence intensity, and are more stable in vivo. This could significantly boost DOLI’s performance in terms of achievable signal-to-noise and imaging depth. The DOLI technique takes advantage of the versatility and ease-of-use of established fluorescence imaging approaches. “You basically need a relatively simple and affordable camera setup without any pulsed lasers or sophisticated optics. This facilitates the dissemination in labs,” Razansky said. The researchers tested the new technique in tissue phantoms that mimic average brain tissue properties, demonstrating that they could acquire microscopic resolution images at depths of up to 4 mm in optically opaque tissues. Courtesy of Daniel Razansky, University of Zurich, and ETH Zurich. The team said that its study represents the first time that 3D fluorescence microscopy has been performed fully noninvasively at capillary-level resolution in an adult mouse brain, covering a field of view of about 1 cm. “Visualization of biological dynamics in an unperturbed environment, deep in a living organism, is essential for understanding the complex biology of living organisms and progression of diseases,” Razansky said. The method could also support the usability of other types of imaging and biomedical imaging methods. “We expect that DOLI will emerge as a powerful approach for fluorescence imaging of living organisms at previously inaccessible depth and resolution regimes,” Razansky said. “This will greatly enhance the in vivo applicability of fluorescence microscopy and tomography techniques.” The research was published in Optica (www.doi.org/10.1364/OPTICA.420378).