Fluorescence Imaging Using Deep-Red Indocyanine Blue, a Complementary Partner for Near-Infrared Indocyanine Green

Indocyanine Blue (ICB) is the deep-red pentamethine analogue of the widely used clinical near-infrared heptamethine cyanine dye Indocyanine Green (ICG). The two fluorophores have the same number of functional groups and molecular charge and vary only by a single vinylene unit in the polymethine chain, which produces a predictable difference in spectral and physicochemical properties. We find that the two dyes can be employed as a complementary pair in diverse types of fundamental and applied fluorescence imaging experiments. A fundamental fluorescence spectroscopy study used ICB and ICG to test a recently proposed Förster Resonance Energy Transfer (FRET) mechanism for enhanced fluorescence brightness in heavy water (D₂O). The results support two important corollaries of the proposal: (a) the strategy of using heavy water to increase the brightness of fluorescent dyes for microscopy or imaging is most effective when the dye emission band is above 650 nm, and (b) the magnitude of the heavy water florescence enhancement effect for near-infrared ICG is substantially diminished when the ICG surface is dehydrated due to binding by albumin protein. Two applied fluorescence imaging studies demonstrated how deep-red ICB can be combined with a near-infrared fluorophore for paired agent imaging in the same living subject. One study used dual-channel mouse imaging to visualize increased blood flow in a model of inflamed tissue, and a second mouse tumor imaging study simultaneously visualized the vasculature and cancerous tissue in separate fluorescence channels. The results suggest that ICB and ICG can be incorporated within multicolor fluorescence imaging methods for perfusion imaging and hemodynamic characterization of a wide range of diseases.