Label-free wide-field imaging of brain tumors using near-infrared endogenous fluorescence and reflectance normalization.

SIGNIFICANCE: Maximizing safe tumor resection remains a major challenge in brain tumor surgery due to the lack of reliable real-time intraoperative contrast between tumor and healthy brain tissue. Label-free optical methods based on endogenous tissue fluorescence could provide simple, noninvasive feedback to guide resection while avoiding the logistical and regulatory limitations of exogenous fluorophores. AIM: The study aims to investigate whether endogenous near-infrared (NIR) fluorescence can differentiate tumor from normal brain tissue in vivo and to develop a wide-field fluorescence and reflectance imaging system for label-free visualization of brain tumor specimens ex vivo. APPROACH: We analyzed in vivo data acquired with a hand-held near-infrared spectroscopy probe from 26 patients (430 measurements) to quantify endogenous fluorescence differences between glioblastoma or astrocytoma and normal brain. Based on these findings, we built a wide-field imaging prototype using 808-nm laser excitation, red-shifted fluorescence detection with an InGaAs short-wave infrared camera, and co-registered reflectance imaging for ratiometric normalization. The system was validated with ex vivo human surgical specimens (14 samples from five patients). RESULTS: Endogenous fluorescence intensity measured in vivo was significantly higher in normal brain tissue compared with tumor, yielding positive predictive values of 85 to 88% for tumor and a negative predictive value of 96% for normal tissue. Ex vivo wide-field imaging reproduced this contrast: normalized fluorescence was lower in tumor regions than in histologically confirmed normal areas, consistent with metabolic differences observed in vivo. CONCLUSIONS: Endogenous NIR fluorescence provides intrinsic contrast between normal and tumoral brain tissue. The demonstrated corrected wide-field fluorescence imaging approach could offer a feasible, label-free method for real-time visualization of tumor margins, supporting its future clinical translation as an intraoperative guidance tool.