Bright spot analysis for photodynamic diagnosis of brain tumors using confocal microscopy

In a previous study of photodynamic tumor diagnosis using 5-aminolevulinic acid (5-ALA), the authors proposed using fluorescence intensity and bright spot analyses under confocal microscopy for the precise discrimination of tumorous brain tissue (such as glioblastoma, GBM) from normal tissue. However, it remains unclear if bright spot analysis can discriminate infiltrating tumor in the boundary zone and whether this method is suitable for GBM with no 5-ALA fluorescence or for other tumor types.

Brain tumor tissue resected from 5-ALA-treated patients was sectioned to evaluate bright spots under confocal microscopy with a 544.5 - 619.5 nm band-pass filter, which eliminated the fluorescence induced by 5-ALA. Border regions and adjacent normal tissues were observed for differences in bright spot distribution. Histopathology was also conducted by hematoxylin and eosin (H&E) staining of serial slices from the same samples to confirm the locations of tumorous, infiltrating, and normal regions. Bright spot areas were then calculated for the same regions evaluated by histopathology. This method was applied for GBM with and without 5-ALA-induced fluorescence as well as for lower-grade gliomas and other brain tumor types.

The bright spot area was substantially smaller in the GBM body than in normal brain tissues. Bright spot area was also smaller in infiltrating tumors than in normal tissue at the margin. The same bright spot pattern was observed in tumorous tissues with no 5-ALA-induced fluorescence and in non-GBM tumors. The bright spot fluorescence is suggested to arise from lipofuscin based on emission spectra (mainly within 544.5 - 619.5 nm) and optimum excitation wavelength (about 405 nm).

Bright spot analysis is useful for discriminating infiltrating tumor from bordering normal tissue as an alternative or complement to photodynamic diagnosis with 5-ALA. This method is also potentially useful for tumors with no 5-ALA-derived red fluorescence and other nervous system tumors.