Indocyanine green: physicochemical factors affecting its fluorescence in vivo

This study reinvestigates the spectral properties of ICG (Indocyanine green) in vivo, the role of quenching, and the possibility of an interaction of ICG with blood components and/or vessel walls. ICG quenching as a function of concentration was studied by spectrophotometry on whole blood samples from golden hamsters. Fluorescence ICG characteristics were evaluated by front-face fluorometry. In vivo, fluorescence measurements were performed on the femoral artery of golden hamsters. In vitro, on whole blood samples, fluorescence intensity is modified by ICG quenching as concentration increases above 80 microgram/ml. The maximum fluorescence peak is not affected and remains centered at 832 nm. The in vivo measurements display a similar fluorescence intensity shape, which is affected only by ICG concentrations. However, the maximum fluorescence emission peak is modified significantly with time. Between 0 and 120 min, four phases can be distinguished in which a wavelength shift from 826 to 835 nm is observed. The wavelength shift with change in fluorescence intensity observed in vivo could be due to a localization of ICG molecules in sites more hydrophobic than serum proteins. It is possible to hypothesize the presence of an endothelium-bound form with a specific fluorescence spectrum. The amphiphilic properties of ICG are consistent with fixation of some ICG molecules on sites other than plasmatic proteins after injection. The process of fixation of ICG molecules on surface components or within the vascular endothelium could be due to a change in the microenvironment of some ICG molecules.