Label-free two-photon fluorescence lifetime imaging of FADH2 for rapid diagnosis and grading of glioma

Abstract

Abstract. Significance Glioma diagnosis currently relies on histopathology, which provides essential morphological information but fails to capture the dynamic metabolic heterogeneity underlying tumor progression. Aim This study seeks to establish a label-free fluorescence lifetime imaging strategy to characterize the metabolic microenvironment of gliomas, with the goal of achieving accurate tumor grading and real-time malignancy assessment. Approach We developed a phasor plot-based two-photon fluorescence lifetime imaging microscopy (TP-FLIM) system. This imaging approach was utilized to evaluate tumor tissues by mapping microenvironmental phenotypes across four distinct glioma subtypes. In addition, we performed longitudinal in vivo tracking within an orthotopic glioma mouse model to monitor metabolic changes over time. Results This method successfully identified distinct tissue regions defined as reduced flavin adenine dinucleotide (FADH2) hotspots. These areas serve as specific metabolic niches that are strongly associated with collagen IV deposition. Furthermore, the longitudinal in vivo tracking validated the feasibility of utilizing these FADH2 signatures to accurately discriminate varying degrees of tumor malignancy. Conclusions These findings establish FADH2 hotspots as a promising and reliable optical biomarker. The study highlights TP-FLIM as a rapid and label-free platform capable of providing intraoperative metabolic navigation, precise diagnosis, and molecular stratification for glioma.