The heptamethine cyanine dye, ICG, is by far the most used near-infrared fluorescent dye in biomedicine. ICB, the homologous pentamethine cyanine dye with a deleted vinylidene unit, exhibits the expected spectral differences (Table ) including absorption/emission maxima peaks at 100 nm lower wavelength. Compared to ICG, ICB is >1000-fold more hydrophilic but still has high affinity for albumin protein. These specific spectral and physicochemical differences enable the design of comparative experiments that test fundamental hypotheses in fluorescence-based research. The strategy was incorporated into a spectroscopy study that tested a recently proposed FRET mechanism for enhanced dye fluorescence brightness in heavy water (D2O). A switch from H2O to D2O increased the fluorescence brightness for near-infrared ICG by a factor of 2.94, which was significantly more that the increase of 1.56 determined for deep-red ICB. This trend is consistent with a FRET-based quenching pathway and increased spectral overlap of the ICG emission band with high energy O-H absorption bands in the region 700-900 nm. It is increasingly clear that the heavy water fluorescence enhancement effect will be most useful for microscopy or imaging when the fluorophore emits near-infrared wavelengths above 650 nm.