Background: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence shows high sensitivity in detecting the tumor core of high-grade gliomas (HGG) but poor sensitivity for tissue of low-grade gliomas (LGG) and the margins of HGG. The characteristic emission peak for PpIX is known to be located at 635 nm. Recently, a second emission peak was described at 620 nm wavelength in LGG and the tumor infiltration zone of HGG. Methods: During surgery, samples from the tumor core and tumor infiltration zone of 43 WHO grade 2–4 gliomas were collected after preoperative 5-ALA administration, and their PpIX emission spectra, as well as fluorescence lifetimes, were determined by ex vivo analysis. Subsequently, the relative PpIX peak contribution (RPPC) was retrieved by calculating the integral of the two bands corresponding to the two emission peaks of PpIX (615–625 nm, 625–635 nm) and correlated with fluorescence lifetimes. Results: The mean RPPC decreased in samples with descending order of WHO grades, non-fluorescing samples, and infiltrative tumor regions, indicating a shift toward the 620 nm peak in porphyrin fluorescence. The porphyrin peak shift across all specimens correlated with lower fluorescence lifetimes (R: 0.854, R-squared: 0.729). Conclusions: The observed peak shift has important implications for fluorescence lifetime analyses since the lifetimes of other porphyrins contribute to the overall decay dynamics. Based on these initial data using fluorescence lifetime, this knowledge is of major importance, especially for detecting tissue from LGG that lack visible fluorescence, to further optimize the visualization of these tumor tissue using this promising imaging modality.

Background: High hypericin-loaded polyvinylpyrrolidone (HHL-PVP) constitutes a novel approach to utilize the promising characteristics of hypericin for photodynamic diagnosis (PDD) and therapy (PDT) of brain tumors in an orally bioavailable formulation. The aim of this study was to investigate the ability of a Complementary Metal-Oxide-Semiconductor (CMOS) camera-based fluorescence imaging system to selectively visualize HHL-PVP in glioblastoma tissue even in the presence of 5-Aminolvevulinic acid (5-ALA) induced fluorescence, which is widely utilized in brain tumor surgery. Methods: We applied a previously established system with a non-hypericin specific filter for 5-ALA fluorescence visualization and a newly introduced hypericin-specific filter at 575–615 nm that transmits the spectrum of hypericin, but not 5-ALA fluorescence. Glioblastoma specimens obtained from 12 patients (11 with preoperative 5-ALA intake) were ex vivo incubated with HHL-PVP. Subsequently, fluorescence intensity and lifetime changes using both the non-hypericin specific filter and hypericin-specific filter were measured before and after HHL-PVP incubation and after subsequent rinsing. Results: While no significant differences in fluorescence signal were observed using the non-hypericin specific filter, statistically significant increases in fluorescence intensity (p = 0.001) and lifetime (p = 0.028) after HHL-PVP incubation were demonstrated using the hypericin-specific filter. In consequence, specimens treated with HHL-PVP could be identified according to the fluorescence signal with high diagnostic sensitivity (87.5%) and specificity (100%). Conclusions: Our CMOS camera-based system with a hypericin-specific filter is capable of selectively visualizing hypericin fluorescence in glioblastoma tissue after ex vivo HHL-PVP incubation. In the future, this technique could facilitate clinical investigations of HHL-PVP for PDD and PDT while maintaining the current standard of care with 5-ALA guidance.