Abstract
We performed dye-enhanced imaging of mouse brain microvessels using spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) microscopy. The resonant signals from C-H stretching in forward CARS usually show high background intensity in tissues, which makes CARS imaging of microvessels difficult. In this study, epi-detection of back-scattered SF-CARS signals showed a negligible background, but the overall intensity of resonant CARS signals was too low to observe the network of brain microvessels. Therefore, Evans blue (EB) dye was used as contrasting agent to enhance the back-scattered SF-CARS signals. Breakdown of brain microvessels by inducing hemorrhage in a mouse was clearly visualized using backward SF-CARS signals, following intravenous injection of EB. The improved visualization of brain microvessels with EB enhanced the sensitivity of SF-CARS, detecting not only the blood vessels themselves but their integrity as well in the brain vasculature.
Highlights
The imaging of microvessels in the animal brain is crucial for observing the integrity of the blood-brain barrier (BBB) in neurodegenerative diseases [1]
Raman spectra obtained for Evans blue (EB) powder showed that EB dye did not exhibit clear resonant Raman signals by CH2 or CH3 stretching between 2,800–3,100 cm-1 (Fig 1G). These results indicate that the SF-Coherent anti-Stokes Raman scattering (CARS) signal of the EB-containing tissue (Fig 1F) could not originate solely from the vibrational signal of EB itself distributed in the brain microvessels, since EB does not contain a C-H-rich chain (S3 Fig)
We report a new method to visualize the brain microvessels at a single-capillary level in mice using spectral-focusing CARS (SF-CARS)
Summary
The imaging of microvessels in the animal brain is crucial for observing the integrity of the blood-brain barrier (BBB) in neurodegenerative diseases [1]. To observe endothelial cell surface markers such as CD31, an intravenous infusion of fluorescent dyelabeled antibodies into the blood plasma is required [7,8]. The antibody-directed visualization of brain microvessels only provides an indirect estimate using the target protein distribution on brain endothelial cells. Exogenous dyes such as sodium fluorescein and Evans blue dye (EB) could be injected intravenously for observation of blood vessels [9, 10]. Exogenous dyes act as tracers, they contrast the blood vessels against the surrounding tissues
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