A non-invasive brain imaging technique developed by researchers at ETH Zurich and the University of Zurich operates in the near infrared (NIR) spectrum to enable deep tissue fluorescence microscopy at superresolution to four times the limit depth imposed by the diffusion of light. According to the researchers, the technique, called diffuse optical localization imaging (DOLI), works in a resolution-depth regime previously inaccessible with optical methods.
DOLI could allow scientists to study neural processes at the level of individual cells and capillaries throughout the living brain without the need for highly invasive surgical methods.
DOLI uses a combination of individual techniques to enable high-resolution, non-invasive deep brain imaging. 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 has allowed us to significantly reduce the background scattering, absorption and intrinsic fluorescence of living tissue,” said Professor Daniel Razansky. The researchers also used an efficient shortwave infrared (SWIR) camera based on InGaAs sensors and a new quantum dot contrast agent that fluoresces strongly in the NIR-II window.
A new imaging method can capture images of the vascular system deep in the brains of mice. A conventional wide-field fluorescence image of mouse brain taken non-invasively in the visible light spectrum is shown at left, while the DOLI approach based on non-invasive localization operating in the NIR-II spectral window is shown. shown at right. Courtesy of Daniel Razansky, ETH Zurich, and the University of Zurich.
The researchers initially tested DOLI with models of synthetic tissue, called tissue phantoms, which simulate the properties of brain tissue. Tests have shown that DOLI can acquire microscopic resolution images at depths of up to 4mm in optically opaque tissue.
They then tested DOLI in live mice and were able to visualize brain microvasculature as well as the speed and direction of blood flow. They injected live mice with microdroplets encapsulating the fluorescent quantum dot at a concentration that created a sparse distribution in the bloodstream. They were able to locate these droplets individually in the brains of mice. By tracking these fluid targets, the researchers were able to build a high-resolution map of the brain microvasculature deep within the mouse brain.
“For the first time, we were able to clearly visualize the microvasculature and blood flow deep within the mouse brain in a completely non-invasive manner,” said Razansky.
Traditionally, the use of fluorescence microscopy to visualize biological processes at the cellular and molecular levels in the brains of animals has been limited by diffusion. Living tissues widely scatter and absorb light, blurring the images and making it difficult 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 scalp intact.
The researchers further observed that the size of the imaged microdroplets depended on how deep they were in the brain, indicating that the DOLI technique is capable of 3D imaging. “Interestingly, we also observed a strong dependence of the size of the spot recorded by the camera on the depth of the microdroplet in the brain, which enabled deep-resolved imaging,” said Razansky.
Researchers are working to improve DOLI’s resolution by optimizing its accuracy in all three dimensions. They also develop fluorescent agents which are smaller, have stronger fluorescence intensity and are more stable in vivo. This could significantly increase DOLI’s performance in terms of signal-to-noise ratio and imaging depth.
The DOLI technique takes advantage of the versatility and ease of use of established fluorescence imaging approaches. “What you basically need is a relatively simple and affordable camera setup without pulsed lasers or fancy optics. This facilitates dissemination in laboratories, ”said Razansky.
The researchers tested the new technique on tissue phantoms that mimic the average properties of brain tissue, demonstrating that they could acquire microscopic resolution images at depths of up to 4mm in optically opaque tissue. Courtesy of Daniel Razansky, University of Zurich and ETH Zurich.
The team said their study represents the first time that 3D fluorescence microscopy has been performed completely non-invasively at capillary-level resolution in an adult mouse brain, covering a field of view of approximately 1 cm.
“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 Razansky.
The method could also support the ease of use of other types of imaging and biomedical imaging methods.
“We expect DOLI to 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.”
The research was published in Optical (www.doi.org/10.1364/OPTICA.420378).