Short communication: Fluorescence preservation of protoporphyrin IX in fresh frozen glioma tissue.

INTRODUCTION: 5-ALA-guided fluorescence surgery is widely used in glioma surgery to improve tumor visualization and extent of resection. However, it remains unknown whether Protoporphyrin IX (PpIX) fluorescence persists in fresh frozen tumor tissue and can be reliably detected after thawing. RESEARCH QUESTION: This study investigates whether PpIX fluorescence remains detectable in fresh frozen glioma tissue and compares the sensitivity of conventional surgical microscopy with a spectroscopic detection method. MATERIAL AND METHODS: In this prospective study, 5-ALA-positive and -negative glioma tissue samples were collected intraoperatively, snap-frozen, and stored in a biobank. After thawing, samples were analyzed using both a conventional surgical microscope and a novel spectroscopic prototype. Histopathological confirmation was performed using H&E staining. Fluorescence detection rates were compared between methods. RESULTS: A total of 46 samples were analyzed. Conventional microscopy failed to detect fluorescence in all thawed samples. In contrast, spectroscopic analysis identified PpIX fluorescence in all previously 5-ALA-positive samples, while negative controls showed no signal. Fluorescence detectability was independent of storage duration (5-14 months). These findings indicate that PpIX remains stable in frozen tissue. DISCUSSION AND CONCLUSION: PpIX fluorescence persists in fresh frozen glioma tissue and can be reliably detected using sensitive spectroscopic techniques, whereas conventional microscopy is insufficient post-thaw. These results validate the use of biobanked 5-ALA-positive samples for downstream molecular analyses and highlight the importance of advanced detection methods for studying subvisual fluorescence.