Indocyanine green excitation-emission matrix characterization: excitation-dependent emission shifts and...

Indocyanine green (ICG) is the most widely used fluorophore in fluorescence-guided surgery (FGS), and its microenvironment-dependent spectral response is relevant for system design, intersystem comparisons, and phantom development.

To characterize ICG with excitation-emission matrices (EEMs) in the microenvironments of dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) solutions, and 3D-printed (3DP) resin and assess excitation-dependent emission, including red-edge excitation shifts (REES) and departures from Kasha's rule of excitation-independent emission.

EEMs and absorbance spectra were acquired with extracted excitation spectra, emission spectra, emission peaks, centroids, and integrated emission areas under the curves (AUCs). Concentration-dependent behavior was examined in DMSO, and albumin concentration dependence was assessed from 5 to $100\mathrm{mg}/\mathrm{mL}$ . Data processing employed robust local regression to mitigate excitation scattering artifacts.

ICG in DMSO exhibited excitation-independent emission consistent with Kasha-Vavilov behavior. By contrast, ICG in BSA solution and 3DP resin displayed excitation-dependent emission with pronounced REES and additional nonlinear departures from Kasha's rule. To our knowledge, this represents the first documentation of REES and broader anti-Kasha effects for ICG or any FGS fluorophore. Within the excitation range most relevant to ICG-FGS ( $\sim 760$ to 805 nm), emission spectra of the BSA solution and 3DP resin overlapped closely, with similar AUC-based comparisons, suggesting that ICG in 3DP resin can serve as a suitable surrogate reference for albumin-bound ICG.

The EEM characterization shows that excitation-dependent behavior is a defining feature of ICG in biologically relevant environments, demonstrating that emission cannot be assumed to follow classical Kasha-Vavilov behavior. Reliable comparisons and imaging system design therefore require spectra acquired at defined excitation wavelengths with AUC integration within the emission detection band. Excitation-specific spectra from EEMs establish a consistent framework for intersystem comparisons and phantom standards, whereas the resulting datasets provide a practical spectroscopic reference for addressing excitation-dependent behavior in ICG sensing applications.