Unraveling the blue shift in porphyrin fluorescence in glioma: The 620 nm peak and its potential significance in tumor biology

In glioma surgery, the low-density infiltration zone of tumors is difficult to detect by any means. While, for instance, 5-aminolevulinic acid (5-ALA)-induced fluorescence is a well-established surgical procedure for maximizing resection of malignant gliomas, a cell density in tumor tissue of 20–30% is needed to observe visual fluorescence. Hyperspectral imaging is a powerful technique for the optical characterization of brain tissue, which accommodates the complex spectral properties of gliomas. Thereby, knowledge about the signal source is essential to generate specific separation (unmixing) procedures for the different spectral characteristics of analytes and estimate compound abundances. It was stated that protoporphyrin IX (PpIX) fluorescence consists mainly of emission peaks at 634 nm (PpIX(634)) and 620 nm (PpIX(620)). However, other members of the substance group of porphyrins fluoresce similarly to PpIX due to their common tetrapyrrole core structure. While the PpIX(634) signal has reliably been assigned to PpIX, it has not yet been analyzed if PpIX(620) might result from a different porphyrin rather than being a second photo state of PpIX. We thus reviewed more than 200,000 spectra from various tumors measured in almost 600 biopsies of 130 patients. Insufficient consideration of autofluorescence led to artificial inflation of the PpIX(620) peak in the past. Recently, five basis spectra (PpIX(634), PpIX(620), flavin, lipofuscin, and NADH) were described and incorporated into the analysis algorithm, which allowed more accurate unmixing of spectral abundances. We used the improved algorithm to investigate the PpIX(620) signal more precisely and investigated coproporphyrin III (CpIII) fluorescence phantoms for spectral unmixing. Our findings show that the PpIX(634) peak was the primary source of the 5-ALA-induced fluorescence. CpIII had a similar spectral characteristic to PpIX(620). The supplementation of 5-ALA may trigger the increased production of porphyrins other than PpIX within the heme biosynthesis pathway, including that of CpIII. It is essential to correctly separate autofluorescence from the main PpIX(634) peak to analyze the fluorescence signal. This article highlights the need for a comprehensive understanding of the spectral complexity in gliomas and suggests less significance of the 620 nm fluorescence peak for PpIX analysis and visualization.