Pituitary adenomas count among the most common intracranial tumors. During pituitary oncogenesis structural, textural, metabolic and molecular changes occur which can be revealed with our integrated ultrahigh-resolution multimodal imaging approach including optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM) on an unprecedented cellular level in a label-free manner. We investigated 5 pituitary gland and 25 adenoma biopsies, including lactotroph, null cell, gonadotroph, somatotroph and mammosomatotroph as well as corticotroph. First-level binary classification for discrimination of pituitary gland and adenomas was performed by feature extraction via radiomic analysis on OCT and MPM images and achieved an accuracy of 88%. Second-level multi-class classification was performed based on molecular analysis of the specimen via LSRM to discriminate pituitary adenomas subtypes with accuracies of up to 99%. Chemical compounds such as lipids, proteins, collagen, DNA and carotenoids and their relation could be identified as relevant biomarkers, and their spatial distribution visualized to provide deeper insight into the chemical properties of pituitary adenomas. Thereby, the aim of the current work was to assess a unique label-free and non-invasive multimodal optical imaging platform for pituitary tissue imaging and to perform a multiparametric morpho-molecular metabolic analysis and classification.

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.