A second corollary of the FRET-based rationalization for the heavy water florescence enhancement effect is a diminished effect if the fluorophore is engaged in a protein binding process that removes water molecules from the fluorophore surface. Although the FRET-based quenching process is inherently inefficient due to the small spectral overlap of the excited fluorophore with the high energy O-H vibrational transitions, there is considerable additivity due to the high number of independent energy-accepting water molecules that are close to the dye surface. Recently reported molecular dynamics simulations suggest that a free heptamethine cyanine dye molecule in pure aqueous solution is surrounded by a surface solvation shell comprised of 100 to 200 water molecules. Moreover, binding of the dye by albumin protein reduces the average number of water molecules surrounding the dye by about a factor of two (Scheme 2). Since FRET efficiency has a 1/r 6 dependence on the distance r between the excited state fluorophore as energy donor and nearby water molecule as energy acceptor, the number of surface water molecules should determine the amount of fluorescence quenching. This corollary was tested by acquiring ICG spectra in the presence and absence of BSA. A 1:500 ratio of dye (1 µM) to BSA (500 µM) was used to ensure that all the dye was bound by the BSA as a monomeric species (i.e., no dye aggregation in the BSA pocket). The spectral data in Figure and the imaging data in Figure show the same trend, that the presence of BSA greatly diminished the capacity of D2O to increase ICG florescence. Specifically, the ratio of integrated fluorescence intensities (I.F.D2O/I.F.H2O) for ICG was lowered from 2.94 when it was a free dye in solution to a ratio of 1.40 when ICG was bound to BSA. In the case of ICB, the presence of BSA has little impact on the heavy water florescence enhancement effect (i.e., the ratio of integrated fluorescence intensities (I.F.D2O/I.F.H2O) decreased from 1.56 for free ICB to 1.28 when ICB was bound to BSA). The fact that dye dehydration upon binding to BSA greatly diminishes the heavy water florescence enhancement effect for near-infrared ICG but has only a minor effect on the heavy water florescence enhancement effect for deep-red ICB is consistent with the proposed FRET-based quenching mechanism. It is worth emphasizing that non-radiative relaxation of excited state cyanine dyes, like ICG, can potentially occur by other pathways that are activated by protein binding. In this regard, fluorescence spectroscopy studies of long-wavelength dye-binding processes in D2O can be illuminating since they eliminate the dominating effect caused by a change in H2O quenching. For example, it is notable that ICB fluorescence in D2O is enhanced by 15% upon binding to BSA, but ICG fluorescence in D2O is decreased by 30% (Figure ). This trend was confirmed by independent microwell imaging experiments that compared image intensities for solutions of dye alone with dye + BSA in various ratios of H2O to D2O (Figure and Figure ). The fact that BSA quenches ICG fluorescence in D2O is extraordinary because enhancement of cyanine dye fluorescence upon binding to albumin in H2O is a universal phenomenon. Since ICG selfaggregation in the BSA binding pocket is very unlikely under the experimental conditions, there must be a photophysical process that quenches the electronically excited BSA/ICG complex more efficiently than the excited state of free ICG in D2O. A logical explanation is enhanced intersystem crossing of excited singlet state BSA/ICG to the excited triplet state (the triplet state quantum yield of ICG is in the range of 10-20% depending on microenvironment). However, other quenching processes such as photoisomerization or reversible nucleophilic addition by a BSA cysteine cannot be currently ruled out. To summarize this study of heavy water effects on fluorescence, the photophysical comparison of ICB and ICG in H2O and D2O has demonstrated three practically important features of the heavy water fluorescence enhancement effect. (a) The strategy of using D2O to increase the brightness of fluorescent cyanine dyes for microscopy or imaging is most effective when the dye emits at a wavelength above 650 nm.