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

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.