A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remain in a subject's blood stream for several minutes, allowing real-time imaging of the subject's circulatory system superimposed upon a conventional, visible light image of the subject. The system may provide an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide used to capture images. The system may be configured for use in open surgical procedures by providing an operating area that is closed to ambient light. The systems described herein provide two or more diagnostic imaging channels for capture of multiple, concurrent diagnostic images and may be used where a visible light image may be usefully supplemented by two or more images that are independently marked for functional interest.