New Technique Expands Depth and Resolution of Fluorescence Microscopy

Swiss researchers have developed a new technique that allows microscopic fluorescence imaging at four times the depth limit imposed by light diffusion. In a paper published today in Optica, the team describes 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.

"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. "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."

"Enabling high-resolution optical observations in deep living tissues represents a long-standing goal in the biomedical imaging field," added 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."

To demonstrate the 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.