Brightness Dye amphiphilicity was assessed by three separate experimental criteria. The first was association with albumin protein which is well known to have a hydrophobic pocket that accepts amphiphilic dyes. An albumin tryptophan quenching assay was employed to measure dissociation constants (Kd) for dye binding to bovine serum albumin (BSA) in water (Figure ). 17 There was similarly high affinity with measured Kd values of (1.77 ± 0.19) µM for ICG and (2.40 ± 0.32 µM) for ICB. The second comparison of amphiphilicity determined the propensity to form dye self-aggregates in water. As shown by the absorption spectra in Figure , low micromolar solutions of ICG exhibited two major peaks, one corresponding to the free monomer dye in solution, and a blue-shifted band corresponding to a dye H-aggregate. In both cases the fraction of H-aggregate increased with higher dye concentration and at any one dye concentration the ICG sample exhibited a higher fraction of H-aggregate than the ICB sample as judged by the ratio of peaks. Addition of BSA to these dye samples produced two effects on the absorption spectra that indicate binding of monomeric dye by the BSA, the fraction of H-aggregate peak decreased, and the absorption band for the monomeric form of each dye was red-shifted along with an increase in fluorescence brightness (Figure ). The third comparison of amphiphilicity measured LogP for dye partitioning between a layer of 1-octanol and an aqueous layer comprised of HEPES buffer (50 mM HEPES, 100 mM NaCl, pH 7.4). Notably, there was >1000-fold difference in the partitioning ratio as illustrated vividly by the photographs in Figure . That is, ICG preferred the 1-octanol layer with a measured LogP value of 2.05, whereas ICB preferred the aqueous layer with a measured LogP value of -1.16. Partitioning of each dye into the aqueous layer increased if it included BSA to bind the dye (Figure ). Together, the three sets of experiments reveal quantitative differences in amphiphilic behavior; ICB is much more hydrophilic than ICG and less likely to self-aggregate in aqueous solution, but it has a similar high affinity for albumin protein. solution of ICG was quite degraded after storage for 6 days. Repeating this chemical stability measurement with an equivalent sample of ICB (1 mM in pure water) revealed much greater chemical stability with no measurable degradation after 30 days (Figure ). A second set of stability experiments monitored the dye over a 2 hour period in mouse serum or human serum.