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

Abstract

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 (S ) state in aqueous solution. Furthermore, in this work, we demonstrate the two-photon cellular imaging application of ICG using direct fluorescence emission from S state for the first time. Our results show that two-photon excitation to S 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.

The effect of nanoencapsulation of ICG on two-photon bioimaging

“…Previously, our group had reported the nonlinear excitation of free ICG to the S 2 state followed by its direct relaxation to S 0 state causing an emission and used for the multiphoton imaging applications. 21 However, the use of free form of ICG has limitations such as poor photostability and cellular uptake. Herein, ICG-PLA NPs were used to overcome the limitations of free ICG and its nonlinear excitation to the S 2 state followed by emission was reported for 2P bioimaging.…”

“…19,20 Additionally, this property was used for two-photon (2P) bioimaging. 21 Despite its application as a 2P imaging chromophore, the conventional delivery of ICG has limitations such as poor cellular uptake and aqueous photostability, which limits its application. Incorporating ICG within a nanocarrier is a promising way to overcome the limitations of free ICG.…”

Two-Photon Absorption: An Open Door to the NIR-II Biological Window?

“…This is evidenced by the commercially available and FDAapproved dye indocyanine green (ICG, 27) whose σ 2 is more than doubled compared to 26. Interestingly, ICG (27) was also used as a contrast agent for 2P fluorescence imaging at 790 nm (Kumari and Gupta, 2019), which leads to excitation in its blue-shifted S 0 →S 2 band. The fluorophore thus presented an Anti-Kasha fluorescence at 570 nm, emitting directly from the S 2 excited state, which provided it with an excitation-dependent 2PEF.…”

Fusogenic Viral Protein-Based Near-Infrared Active Nanocarriers for Biomedical Imaging

“…Near-infrared fluorescence (NIRF) has the potential for noninvasive deep tissue imaging, particularly for early-stage cancer diagnosis. − NIR imaging is capable of higher tissue penetration and better visualization of cellular microstructure due to reduced photon scattering and negligible tissue autofluorescence in the optical window (650 to 950 nm). − Additionally, the use of NIR-active exogenous contrast agents could further improve the signal-to-noise ratio (SNR) of fluorescence imaging. , ICG is the only Food and Drug Administration (FDA)-approved cyanine dye and has been actively used for the past six decades for NIR imaging. − However, the free form of ICG has several limitations such as concentration-dependent aggregation, low thermal and optical stability, lack of targeting ability, and short circulation time in the body . Due to these limitations, the broader application of ICG as a potential contrast agent for bioimaging is not achieved yet.…”