Revisiting Indocyanine Green: Effects of Serum and Physiological Temperature on Absorption and Fluorescence Characteristics

Indocyanine green (ICG) remains as the only near infrared dye approved by the FDA. Despite its long history of usage in clinical medicine, a systematic study of the effects of serum proteins at physiologically relevant levels and temperature on absorption and fluorescence characteristics of ICG has been missing. We incubated ICG at concentrations in the range of 0.6-25.8 μM in McCoy's 5a cell culture medium, without and with supplemental fetal bovine serum (FBS) at 5% and 10% levels. Our analyses of absorption and fluorescence spectra indicate that the peak absorbance of ICG associated with its monomeric form increases in the presence of FBS. For example, at ICG concentration of 25.8 μM, the monomer absorbance is increased by nearly 100% in the presence of 10% FBS. Similarly, there is an increase in the relative fluorescence quantum yield of ICG, by as much as nearly 3.5 times in the presence of FBS. When incubated at 37 °C, the presence of FBS in the cell culture medium helps maintain the monomeric absorption of ICG and sustain the increased fluorescence emission. We offer explanations to describe the possible photophysical mechanisms underlying the observed effects and discuss the importance of these results to in-vivo applications of ICG.