Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

Radiologically suspected low-grade gliomas (LGG) represent a special challenge for the neurosurgeon during surgery due to their histopathological heterogeneity and indefinite tumor margin. Therefore, new techniques are required to overcome these current surgical drawbacks. Intraoperative visualization of brain tumors with assistance of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence is one of the major advancements in the neurosurgical field in the last decades. Initially, this technique was exclusively applied for fluorescence-guided surgery of high-grade glioma (HGG). In the last years, the use of 5-ALA was also extended to other indications such as radiologically suspected LGG. Here, we discuss the current role of 5-ALA for intraoperative visualization of focal malignant transformation within suspected LGG. Furthermore, we discuss the current limitations of the 5-ALA technology in pure LGG which usually cannot be visualized by visible fluorescence. Finally, we introduce new approaches based on fluorescence technology for improved detection of pure LGG tissue such as spectroscopic PpIX quantification fluorescence lifetime imaging of PpIX and confocal microscopy to optimize surgery.

Precise intraoperative brain tumor visualization supports surgeons in achieving maximal safe resection. In this sense, improved prognosis in patients with high-grade gliomas undergoing protoporphyrin IX fluorescence-guided surgery has been demonstrated. Phase fluorescence lifetime imaging in the frequency-domain has shown promise to distinguish weak protoporphyrin IX fluorescence from competing endogenous tissue fluorophores, thus allowing for brain tumor detection with high sensitivity. In this work, we show that this technique can be further improved by minimizing the crosstalk of autofluorescence signal contributions when only detecting the fluorescence emission above 615 nm. Combining fluorescence lifetime and spectroscopic measurements on a set of 130 ex vivo brain tumor specimens (14 low- and 56 high-grade gliomas, 39 meningiomas and 21 metastases) coherently substantiated the resulting increase of the fluorescence lifetime with respect to the detection band employed in previous work. This is of major interest for obtaining a clear-cut distinction from the autofluorescence background of the physiological brain. In particular, the median fluorescence lifetime of low- and high-grade glioma specimens lacking visual fluorescence during surgical resection was increased from 4.7 ns to 5.4 ns and 2.9 ns to 3.3 ns, respectively. While more data are needed to create statistical evidence, the coherence of what was observed throughout all tumor groups emphasized that this optimization should be taken into account for future studies.

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.

OBJECTIVE Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is nowadays widely applied for improved resection of glioblastomas (GBMs). Initially, pretreatment with dexamethasone was considered to be essential for optimal fluorescence effect. However, recent studies reported comparably high rates of visible fluorescence in GBMs despite absence of dexamethasone pretreatment. Recently, the authors proposed fluorescence lifetime imaging (FLIM) for the quantitative analysis of 5-ALA–induced protoporphyrin IX (PpIX) accumulation. The aim of this study was thus to investigate the influence of dexamethasone on visible fluorescence and quantitative PpIX accumulation. METHODS The authors prospectively analyzed the presence of visible fluorescence during surgery in a cohort of patients with GBMs. In this study, patients received dexamethasone preoperatively only if clinically indicated. One representative tumor sample was collected from each GBM, and PpIX accumulation was analyzed ex vivo by FLIM. The visible fluorescence status and mean FLIM values were correlated with preoperative intake of dexamethasone. RESULTS In total, two subgroups with (n = 27) and without (n = 20) pretreatment with dexamethasone were analyzed. All patients showed visible fluorescence independent from preoperative dexamethasone intake. Furthermore, the authors did not find a statistically significant difference in the mean FLIM values between patients with and without dexamethasone pretreatment (p = 0.097). CONCLUSIONS In this first study to date, the authors found no significant influence of dexamethasone pretreatment on either visible 5-ALA fluorescence during GBM surgery or PpIX accumulation based on FLIM. According to these preliminary data, the authors recommend administering dexamethasone prior to fluorescence-guided surgery of GBMs only when clinically indicated.