A method and system for determination and mapping the quantity of chromophores having a distinct spectrum attached to moving objects in an spectrally rich environment that may include multiple chromophores attached to stationary objects. An area of inters is imaged at different times and different wavelengths, and the spectral properties of the or more chromophores attached to the moving objects are separated from the stationary spectral properties of the background, followed by spectral analysis of the moving objects to determine their quantity. Application to the retinal vasculature is illustrated, showing the imaging, analyzing and quantifying of the oxygen saturation of retinal blood, resolved for the different vascular compartments—capillaries, arterioles, venules, arteries, and veins. Changes in the structure of the vascular environment are also determined, whether growth of new vessels or the elimination of existing ones, by the generation of path maps based on analysis of differential images taken at a single wavelength of the moving components in the blood flow.