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

Significance: Indocyanine Green (ICG) is the most widely used fluorophore in fluorescence-guided surgery (FGS), yet its spectral characteristics can significantly vary in different microenvironments. This variability impacts the design and effectiveness of fluorescence sensing systems used in medical imaging and diagnostics. Aim: Provide the first excitation-emission matrix (EEM) characterization of ICG in different microenvironments to comprehensively understand spectral shifts, including dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) solutions, and 3D-printed (3DP) resin. Approach: EEMs and absorbance spectra of ICG in DMSO, BSA solutions, and 3DP resin were acquired using a CCD-based fluorescence and absorbance spectrometer. The study investigated the impact of these microenvironments and varying concentrations on ICG′s fluorescence behavior. Results: ICG in DMSO exhibited symmetric spectra across varying excitation wavelengths, confirming conventional fluorophore behavior. In contrast, ICG in BSA solution and 3DP resin showed a notable ′rotation′ of central spectral features, indicative of red-edge excitation shifts (REES), such that fluorescence emission varied with excitation wavelength. Furthermore, varying ICG concentration measurements showed fluorescence quenching, concentration-dependent red shifts (CDRS), and disparities between absorbance and emission spectra due to inner filter effects (IFE). Conclusions: This study provides the first EEM characterization of ICG alongside the first report of REES in FGS fluorophores. These results highlight the importance of considering excitation wavelengths in spectral comparisons, illustrating that EEM data offers more comprehensive analysis than conventional fluorescence spectra collected at a single excitation wavelength. This study lays the groundwork for improved fluorophore characterizations for advancing fluorescence sensing in clinical applications, including FGS imaging.