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.