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

Significance: Indocyanine green (ICG) is the most widely used fluorophore in fluorescence-guided surgery (FGS), yet its spectral response depends on microenvironment, with implications for system design, inter-system comparisons, and phantom development. Aim: 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. Approach: EEMs and absorbance spectra were acquired with extracted excitation spectra, emission spectra, emission peaks, centroids, and integrated emission areas under the curve (AUCs). Concentration-dependent behavior was examined in DMSO, and albumin concentration dependence was assessed from 5-100 mg/mL. Data processing employed robust local regression to mitigate excitation scattering artifacts. Results: ICG in DMSO exhibited excitation-independent emission consistent with Kasha-Vavilov behavior. In contrast, ICG in BSA solution and 3DP resin displayed excitation-dependent emission with pronounced REES and additional non-linear 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 (~760-805 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. Conclusions: 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 inter-system comparisons and phantom standards, while the resulting datasets provide a practical reference for addressing excitation-dependent behavior in ICG sensing applications.