Systems and methods for imaging are presented. The method includes producing excitation light configured to induce fluorescence in an imaging agent that selectively binds to a target species in a region of interest (ROI) of a subject that also includes a background species. A first and a second spectral region are selected such that a determined difference between fluorescence corresponding to the target and the background species in the first spectral region differs from a corresponding difference in the second spectral region. First and second fluorescence images are generated from the fluorescence corresponding to the first and second spectral regions. Additionally, a fluorescence ratio for the background species in the first and second fluorescence image is determined. The first fluorescence image is then multiplied or divided with the determined ratio to generate an intermediate image that is subtracted from the second fluorescent image to reconstruct a background-subtracted image.