1. An imaging system, comprising: one or more illumination sources configured to produce excitation light that induces fluorescence emissions in an imaging agent configured to bind to a target species in a region of interest of a subject, wherein the region of interest comprises the target species and a background species; at least one optical element configured to separate the fluorescence emissions into a first spectral region and a second spectral region, wherein a determined difference between fluorescence corresponding to the target species and the background species in the first spectral region differs from a corresponding difference in the second spectral region; one or more signal detectors configured to collect fluorescence emissions corresponding to the first spectral region and the second spectral region; and a processing unit operatively coupled to the one or more signal detectors, wherein the processing unit is configured to: generate a first fluorescence image from the fluorescence emissions corresponding to the first spectral region and a second fluorescence image from the fluorescence emissions corresponding to the second spectral region; determine a ratio of fluorescence corresponding to the background species in the first fluorescence image and the second fluorescence image; multiply or divide the first fluorescence image with the determined ratio to generate an intermediate image; and reconstruct a background-subtracted image by subtracting the intermediate image from the second fluorescence image.