Measurement of two-photon properties of indocyanine green in water and human plasma excited at the 1700-nm window

Indocyanine green (ICG) is a human compatible dye and is ideal for deep-tissue two-photon fluorescence (2PF) microscopy excited at the 1700-nm window in vivo. However, the two-photon excitation and emission properties of this dye remain unknown. Here we demonstrate measurement of the two-photon excitation and emission properties of ICG in both water and human plasma, using home-built two-photon action cross-sectional measurement and two-photon emission spectrum measurement systems. Our results show that excited from 1600 to 1800 nm, 2PF can be generated from ICG dissolved in both water and human plasma. The measured two-photon action cross-sectional ησ₂ of ICG dissolved in human plasma is an order-of-magnitude larger than that dissolved in water. The measured two-photon emission spectrum overlaps with the one-photon emission spectrum for ICG dissolved in both human plasma and water. Our results will provide key two-photon parameters for the clinical use of ICG.