However, there is a notable caveat, the heavy water florescence enhancement effect for an amphiphilic near-infrared cyanine dye, like ICG, is substantially diminished if the dye is bound by albumin protein. In this scenario, the surface of the albumin-bound dye is partially dehydrated which lowers the efficiency of fluorescence quenching by FRET to H2O and diminishes the magnitude of the heavy water florescence enhancement effect. Thus, to ensure a large heavy water florescence enhancement effect, it is crucial to employ a hydrophilic near-infrared dye that has emission > 650 nm and negligible affinity for proteins or membranes. During these studies we observed that ICG fluorescence in D2O is decreased by 30% upon binding to BSA. This finding is extraordinary because enhancement of dye fluorescence upon binding to albumin in H2O is a universal phenomenon. We suggest that researchers consider dye-binding experiments in D2O, as a simple way to mask the extensive fluorescence quenching by H2O and uncover more subtle alternative dye excited state relaxation pathways.