. Significance: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection. Aim: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients. Approach: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid ( ) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation. Results: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values ( to 14 ns, to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration. Conclusions: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo . Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Identifying isocitrate dehydrogenase (IDH)-mutation and glioma subtype during surgery instead of days later can aid in modifying tumor resection strategies for better survival outcomes. We report intraoperative identification of IDHmutant glioma (N = 12 patients) with a clinically compatible fluorescence lifetime imaging (FLIm) device (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm). The fluorescence-derived parameters were analyzed to study the optical contrast between IDH-mutant tumors and surrounding brain tissue. IDH-mutant oligodendrogliomas exhibited shorter lifetimes (3.3 ± 0.1 ns) than IDH-mutant astrocytomas (4.1 ± 0.1 ns). Both IDH-mutant glioma subtypes had shorter lifetimes than white matter (4.6 ± 0.4 ns) but had comparable lifetimes to cortex. Lifetimes also increased with malignancy grade within IDH-mutant oligodendrogliomas (grade 2: 2.96 ± 0.08 ns, grade 3: 3.4 ± 0.3 ns) but not within IDH-mutant astrocytomas. The current results support the feasibility of FLIm as a surgical adjuvant for identifying IDH-mutant glioma tissue.

Challenges in identifying a glioblastoma’s infiltrative edge during neurosurgical procedures result in rapid recurrence. A label-free fluorescence lifetime imaging (FLIm) device was used to evaluate glioblastoma’s infiltrative edge in vivo in 15 patients (89 samples). FLIm data were analyzed according to tumor cell density, infiltrating tissue type (gray and white matter), and diagnosis history (new or recurrent). Infiltrations in white matter from new glioblastomas showed decreasing lifetimes and a spectral red shift with increasing tumor cell density. Areas of high versus low tumor cell density were separated through a linear discriminant analysis with a ROC-AUC=0.74. Current results support the feasibility of intraoperative FLIm for real-time in vivo brain measurements and encourage refinement to predict glioblastoma infiltrative edge, underscoring the ability of FLIm to optimize neurosurgical outcomes.

INTRODUCTION Fluorescence-guided surgery can improve tumor identification and extent of surgical resection. 5-ALA is the standard for GBM, but is limited by lack of quantitative fluorescence, a need to work in the dark, and a lack of sensitivity for low grade tumors. We have developed a novel instrument for dye-free tissue autofluorescence lifetime imaging (FLIm) to identify glioma tissue during resection. This approach utilizes time-resolved autofluorescence measurements in narrow-band channels to assess markers of tissue metabolism. Compared to intensity-based imaging of exogenous fluorophores, FLIm has greater sensitivity without dependence on background lighting. The advantages of FLIm include quantitative tissue analysis, the ability to work under full light conditions, sensitivity for high and low grade gliomas, and the potential ability to identify IDH mutational status. In this study, we validated the use of FLIm for identification of glioma tissue at tumor resection margins. METHODS FLIm was used to image tissue margins during glioma resections and compared to microbiopsies from imaged regions to correlate fluorescence with histopathology. RESULTS FLIm was applied intraoperatively to 11 GBM and 5 LGG patients (7 imaged biopsies per patient). In GBM, tumor infiltration of cortex was associated with significantly decreased fluorescence lifetime (FL) in channels 2 (470/28nm;p<0.05) and 3 (542/50nm;p<0.002). In subcortical margins, FL was inversely proportional to the density of tumor in channels 2,3 (p<0.05). When IDH wild-type GBMs were compared to IDH1-mutant tumors, FL was noted to be significantly longer in channel 1 (390/40nm;p<0.05), and trended towards longer FL in channel 2, shorter FL in channel 3. In LGG, FL was significantly correlated with tumor density in channel 2 (p<0.01). CONCLUSIONS FLIm is a dye-free, quantitative alternative to 5-ALA for fluorescence guided glioma resections with sensitivity to high and low-grade tumors, and the ability to predict IDH mutations in GBM. Further validation studies are on-going.

In-situ identification of glioma subtype can enable modifications of clinical and surgical strategies. Particularly, astrocytoma benefit from more aggressive resection than oligodendroglioma, which have a more favorable response to post-surgical chemotherapy. Preoperative MRI and intraoperative histology cannot accurately determine glioma subtype. There is a need for real-time identification of adult-type diffuse glioma subtypes to aid the neurosurgeon's decisionmaking during resection surgery. Fluorescence lifetime imaging (FLIm) where tissue autofluorescence can be used as an indicator to distinguish among brain tumor tissue types in real-time could aid this process. Here, we report the use of label-free FLIm in distinguishing IDH-mutant glioma subtypes (astrocytoma and oligodendroglioma). The FLIm system (excitation: 355 nm; emission bands: 390/40 nm, 470/28 nm, 542/50 nm) was used to scan brain tissue from the resection margins of glioma patients during tumor resection. Fluorescence lifetimes were extracted and analyzed by constrained least-squares deconvolution with the Laguerre expansion method. FLIm data was validated with histopathology of collected biopsies. Current results show that FLIm provides optical contrast between tumor and healthy white matter, and between IDH-mutant astrocytoma (N=7 patients) and oligodendroglioma (N=5 patients). Tumors showed shorter lifetime values (470-nm: 3.6±0.6ns; 542-nm: 3.3±0.7ns) than healthy white matter (470-nm: 4.6±0.4ns; 542-nm: 4.3±0.5ns, p<0.01). Oligodendroglioma had shorter lifetimes in the 470-nm (3.3±0.1ns) and 542-nm (2.8±0.2ns) channels, which are associated with NAD(P)H and FAD fluorescence respectively, when compared with IDH-mutant astrocytoma (470-nm: 4.1±0.1ns; 542-nm: 3.9±0.2ns, p<0.01). Together, these results demonstrate the feasibility of using FLIm as an intraoperative tool in glioma diagnosis.