Flavin fluorescence lifetime and autofluorescence optical redox ratio for improved visualization and classification of brain tumors

Abstract

PurposeModern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated.Experimental designFluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3).ResultsProtein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification.ConclusionsOur results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

Label-free spatially maintained measurements of metabolic phenotypes in cells

“…For example, a blue-shifted filter may increase the contribution of protein-bound FMN and reduce the sensitivity of the flavin autofluorescence measurements to FAD, confounding the interpretation of flavin autofluorescence lifetime data. Although FMN is typically only a small portion of the flavin autofluorescence for emission filters centered around 550 nm, protein-bound FMN has a longer fluorescence lifetime (around 5 ns) than FAD ( van den Berg et al, 2002 ), and changes in FMN concentration or binding dynamics could alter the measured lifetime values ( Reichert et al, 2023 ).…”

“…For example, a blue-shifted filter may increase the contribution of protein-bound FMN and reduce the sensitivity of the flavin autofluorescence measurements to FAD, confounding the interpretation of flavin autofluorescence lifetime data. Although FMN is typically only a small portion of the flavin autofluorescence for emission filters centered around 550 nm, protein-bound FMN has a longer fluorescence lifetime (around 5 ns) than FAD (van den Berg et al, 2002), and changes in FMN concentration or binding Frontiers in Bioengineering and Biotechnology frontiersin.org dynamics could alter the measured lifetime values (Reichert et al, 2023). The use of FAD as a primary electron carrier at multiple steps throughout the TCA and the electron transport chain suggests that FAD fluorescence lifetimes should be sensitive to alterations in cellular use of the oxidative metabolic pathway.…”

Meningiomas with CNS invasion

“…Advanced optical imaging techniques such as confocal microscopy, optical coherence tomography, and Raman spectroscopy have been used for “optical biopsies,” i.e., intraoperative identification of tumor tissue ( Shin et al, 2019 ). Reichert et al report an increased glycolytic activity of meningiomas as a possible explanation for their extremely high autofluorescence capacities during a modern visualization technique, namely the flavin mononucleotide fluorescence ( Reichert et al, 2023 ). Charalampaki et al have recently described confocal laser endomicroscopy which combined with multispectral fluorescence microscopy as a novel technique for intraoperative tumor visualization.…”