Indocyanine Green (ICG) is a heptamethine cyanine dye (Scheme 1) with near-infrared absorption and fluorescence emission wavelengths. For more than 60 years, ICG has been used clinically for various types of diagnostic and imaging procedures. This includes diagnostic methods that assess liver function, cardiac output, or blood volume, and imaging methods for visualizing choroidal blood flow, revascularized tissue, or exposed tumors during surgery. Although ICG is a water-soluble dye, its amphiphilic molecular structure promotes dye selfaggregation at concentrations > 5 µM or reversible association with hydrophobic sites on proteins or membranes. Upon intravenous dosing, ICG rapidly binds to blood proteins, especially albumin, and within a few minutes it is cleared through the liver as an unchanged molecule and excreted into the intestines as a component of the bile juice. The rapid clearance is a major reason why the clinical safety record of ICG is quite favorable. The long-wavelength near-infrared light used for ICG fluorescence excitation and detection can penetrate relatively deeply through skin and tissue because there is negligible absorption by endogenous pigments and reduced scattering of the light. However, ICG is known for several clinical performance limitations including chemical instability in aqueous solution, which limits its clinical use to six hours after dose reconstitution, and moderate susceptibility to photobleaching. In addition, ICG selfaggregation or albumin association means the spectral properties, including fluorescence quantum yield and electronic excited state lifetime are concentration dependent. Efforts to mitigate these drawbacks have included strategies that encapsulate ICG within protective nanocapsules such as liposomes, proteins, or hyperbranched polymers. There are also preclinical and clinical protocols that combine ICG with other fluorescent dyes with different wavelengths for multicolor imaging and sensing applications. The feasibility of this approach is enhanced by ongoing advances in camera technology that enables advanced methods of multicolor and lifetime imaging. As part of an ongoing project to develop structural analogues of ICG with customized imaging properties, we decided to evaluate the pentamethine version of ICG which we call Indocyanine Blue (ICB, see Scheme 1). The chemical synthesis of ICB has been reported, but it also can be purchased from several different commercial vendors at an affordable price and in relatively large amount. Despite its ready availability, there are very few published studies of ICB chemical and spectral properties, and only one report noting its propensity to self-aggregate at high concentration in aqueous solution. In terms of biomedical imaging, a short communication in 1979 suggested a potential use of ICB for choroidal angiography but the idea has not been pursued further in any subsequent publication. Here we describe a detailed comparison of ICG and ICB and we present our results as three sets of distinct findings. First, we measure and compare the physicochemical, and spectral properties of these two homologous dyes which differ by a single vinylene unit in the polymethine chain. Second, we employ the two fluorophores as a complementary pair in a fluorescence spectroscopy study that supports a recently proposed photophysical explanation for enhanced dye fluorescence brightness in heavy water and we test two important corollaries of the proposed mechanism. Third, we demonstrate the high potential of these dyes as a complementary pair of fluorescence imaging agents in a related collection of dual-channel, cell microscopy and mouse tumor imaging studies.