The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas.