Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching

Creative Commons License

Belykh E., Miller E. J., Patel A. A., Bozkurt B., Yagmurlu K., Robinson T. R., ...More

SCIENTIFIC REPORTS, vol.8, 2018 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 8
  • Publication Date: 2018
  • Doi Number: 10.1038/s41598-018-30247-6
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Acibadem Mehmet Ali Aydinlar University Affiliated: No


Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples.