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The primo vascular system is a novel circulatory system forming a network throughout an animal's body. Primo vessels were recently observed in the fourth ventricle of the brain and in the spinal cord of a rat by using fluorescent nanoparticles. In order to quantify the nanoparticles in the primo vessels, we measured the florescence of the nanoparticles and calibrated the measurements by using a reference suspension. We removed the noise due to autofluorescence with the technique of multispectral imaging. The line densities of nanoparticles and the contrast values of their images were, respectively, 0.5 ± 0.5 ng/mm and 0.7 ± 0.5 for primo vessels in the fourth ventricle, and 1.3 ± 0.6 ng/mm and 1.4 ± 0.2 for primo vessels in the spinal cord. The data obtained from and the procedures used in this work could be useful in evaluating the feasibility of using nanoparticles as a contrast agent during MRI or CT imaging of …