Two-photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near-infrared dye: Application of anti-Kasha's rule for two-photon fluorescence imaging

In recent years, two-photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near-infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two-photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two-photon excitation property of ICG to the second excited singlet (S2 ) state in aqueous solution. Furthermore, in this work, we demonstrate the two-photon cellular imaging application of ICG using direct fluorescence emission from S2 state for the first time. Our results show that two-photon excitation to S2 state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well-known "tissue-optical window." This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two-photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.