Abstract.SignificanceOptical imaging in the second near-infrared (NIR-II, 1000 to 1700 nm) region is capable of deep tumor vascular imaging due to low light scattering and low autofluorescence. Non-invasive real-time NIR-II fluorescence imaging is instrumental in monitoring tumor status.AimOur aim is to develop an NIR-II fluorescence rotational stereo imaging system for 360-deg three-dimensional (3D) imaging of whole-body blood vessels, tumor vessels, and 3D contour of mice.ApproachOur study combined an NIR-II camera with a 360-deg rotational stereovision technique for tumor vascular imaging and 3D surface contour for mice. Moreover, self-made NIR-II fluorescent polymer dots were applied in high-contrast NIR-II vascular imaging, along with a 3D blood vessel enhancement algorithm for acquiring high-resolution 3D blood vessel images. The system was validated with a custom-made 3D printing phantom andin vivoexperiments of 4T1 tumor-bearing mice.ResultsThe results showed that the NIR-II 3D 360-deg tumor blood vessels and mice contour could be reconstructed with 0.15 mm spatial resolution, 0.3 mm depth resolution, and 5 mm imaging depth in anex vivoexperiment.ConclusionsThe pioneering development of an NIR-II 3D 360-deg rotational stereo imaging system was first applied in small animal tumor blood vessel imaging and 3D surface contour imaging, demonstrating its capability of reconstructing tumor blood vessels and mice contour. Therefore, the 3D imaging system can be instrumental in monitoring tumor therapy effects.