Researchers have developed a new technique that allows micro­scopic fluorescence imaging at four times the depth limit imposed by light diffusion. 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 intense light scat­tering by the skin and skull.

“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,” said research team leader Daniel Razansky from the University of Zurich and ETH Zurich, both in Switzerland. “Our study represents the first time that 3D fluorescence micro­scopy has been performed fully non­invasively at capillary level resolution in an adult mouse brain, effectively covering a field of view of about 1 centimeter.” Now, the researchers describe their new technique, which is called diffuse optical locali­zation imaging (DOLI). It takes advantage of the second near-infrared (NIR-II) spectral window from 1000 to 1700 nanometers, which exhibits less scattering.

“Enabling high-resolution optical obser­vations in deep living tissues represents a long-standing goal in the biomedical imaging field,” said Razansky. “DOLI's superb resolution for deep-tissue optical observations can provide functional insights into the brain, making it a promising platform for studying neural activity, microcirculation, neuro­vascular coupling and neuro­degeneration.” For the new technique, the researchers intravenously inject a living mouse with fluorescent micro­droplets at a concen­tration that creates a sparse distribution in the blood stream. Tracking these flowing targets enables recon­struction of a high-resolution map of the deep cerebral micro­vasculature in the mouse brain.

“The method eliminates background light scattering and is performed with the scalp and skull intact,” said Razansky. “Interes­tingly, we also observed strong dependence of the spot size recorded by the camera on microdroplet's depth in the brain, which enabled depth-resolved imaging.” The new approach benefits from the recent intro­duction of highly efficient short-wave infrared cameras based on InGaAs sensors. Another key building block was the use of novel contrast agents exhi­biting strong fluores­cence responses in the NIR-II window, such as lead sulfide (PbS)-based quantum dots.

The researchers first tested the new technique in synthetic models of tissue that mimic average brain tissue properties, demonstrating that they could acquire micro­scopic resolution images at depths of up to 4 milli­meters in optically opaque tissues. They then performed DOLI in living mice where cerebral micro­vasculature as well as blood flow velocity and direction could be visualized entirely non­invasively. 

The researchers are working to optimize precision in all three dimensions to improve DOLI's resolution. They are also deve­loping improved fluorescent agents that are smaller, have stronger fluores­cence intensity and are more stable in vivo. This will signi­ficantly boost DOLI's perfor­mance in terms of the achievable signal to noise and imaging depth. “We expect 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 greatly enhance the in vivo applica­bility of fluorescence micro­scopy and tomo­graphy techniques.” (Source: OSA)

Reference: Q. Zhou et al.: Diffuse optical localization imaging for noninvasive deep brain microangiography in the NIR-II window, Optica 8, 796 (2021); DOI: 10.1364/OPTICA.420378

Link: Dept. of Information Technology and Electrical Engineering, Institute for Biomedical Engineering, ETH Zurich, Switzerland