A Japan-based research team led by Kanazawa University have found that bright spot areas have generally lower fluorescence in brain tumours than in normal tissues in images captured by irradiation with a 405 nm wavelength laser and 544.5–619.5 nm band-pass filter. This may facilitate discrimination of glioblastoma with or without 5-aminolevulinic acid fluorescence and could be applicable to other tumours.


Photodynamic diagnosis using 5-aminolevulinic acid (5-ALA) is now widely used for neurosurgical resection of brain tumours. Distinguishing a tumour from healthy tissue is based on greater 5-ALA-derived protoporphyrin IX accumulation in glioma cells than in non-cancerous cells, resulting in much greater red fluorescence (peak at 635 nm) when excited at 405 nm. However, it is still difficult to precisely distinguish the tumour margin and infiltrating regions from non-tumour tissue because the fluorescent boundary is usually vague. In our previous study, we noticed that bright spots in confocal microscopy images may be able to distinguish tumours from normal tissue.


Brain tumour tissues resected from 5-ALA-treated patients was sectioned to evaluate bright spots captured by a 544.5-619.5 nm wavelength band-pass filter that eliminated the fluorescence induced by 5-ALA under a confocal microscope. Border regions and adjacent normal tissues were observed. Pathological inspection was performed to confirm the locations of tumours, infiltrating tumour cells, and normal tissue regions by hematoxylin and eosin (H&E) staining of serial sections of the same samples. Bright spot areas were measured in the same region used for pathological inspection. This method was applied to brain tumours with and without red fluorescence as well as glioblastoma (GBM) and non-GBM brain tumours.


The bright spot area was substantially smaller in the GBM tumour than in normal brain tissues. It was also smaller in infiltrating tumours than in normal tissue at the margin. The same bright spot pattern was observed in tumours tissues without red fluorescence and in non-GBM tumours. Bright spot fluorescence has been suggested to be derived from lipofuscin based on emission spectra (mainly within 544.5?619.5 nm) and an optimal excitation wavelength (about 405 nm).

Significance and future prospects

Bright spot analysis is useful to facilitate discrimination of an infiltrating tumour from bordering normal tissue in photodynamic diagnosis using 5-ALA. This method is also potentially useful for tumours without 5-ALA-derived red fluorescence and non-GBM tumours. The mechanism of bright spot fluorescence reduction in tumours and its application for precise discrimination of brain tumours should be investigated further.



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Further information

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

Journal: Photodiagnosis and Photodynamic Therapy

Authors: Takeshi Yoneyama, Tetsuyo Watanabe, Sho Tamai, Katsuyoshi Miyashita, Mitsutoshi Nakada

DOI: 10.1016/j.pdpdt.2019.02.005

Funder: Japan Society for the Promotion of Science KAKENHI Grant Number 26242054