Ruiz Alberto J, Lyon Sophie A, LaRochelle Ethan P M, Samkoe Kimberley S
QUEL Imaging, White River Junction, VT, USA.
Dartmouth Engineering, Hanover, NH, USA.
bioRxiv. 2025 Aug 13:2025.08.12.667954. doi: 10.1101/2025.08.12.667954.
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.
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.
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.
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).
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.
吲哚菁绿（ICG）是荧光引导手术（FGS）中使用最广泛的荧光团，但其光谱特性在不同微环境中可能有显著差异。这种变异性会影响医学成像和诊断中使用的荧光传感系统的设计和有效性。
首次对ICG在不同微环境中的激发-发射矩阵（EEM）进行表征，以全面了解光谱位移，包括二甲基亚砜（DMSO）、牛血清白蛋白（BSA）溶液和3D打印（3DP）树脂。
使用基于电荷耦合器件（CCD）的荧光和吸收光谱仪获取ICG在DMSO、BSA溶液和3DP树脂中的EEM和吸收光谱。该研究调查了这些微环境和不同浓度对ICG荧光行为的影响。
ICG在DMSO中，在不同激发波长下呈现对称光谱，证实了传统荧光团的行为。相比之下，ICG在BSA溶液和3DP树脂中显示出中心光谱特征的显著“旋转”，这表明存在红边激发位移（REES），即荧光发射随激发波长而变化。此外，不同ICG浓度测量显示出荧光猝灭、浓度依赖性红移（CDRS）以及由于内滤效应（IFE）导致的吸收光谱和发射光谱之间的差异。
本研究首次对ICG进行了EEM表征，同时也是FGS荧光团中首次报道REES。这些结果突出了在光谱比较中考虑激发波长的重要性，表明EEM数据比在单一激发波长下收集的传统荧光光谱提供了更全面的分析。本研究为改进荧光团表征奠定了基础，以推进包括FGS成像在内的临床应用中的荧光传感。
