Multiphoton excitation fluorescence microscopy of 5-aminolevulinic acid induced fluorescence in experimental gliomas

Background and Objective: The clinical usefulness of 5-ALA guided detection of tumor tissue has been demonstrated for a number of malignancies. However, current techniques of intraoperative detection of protoporphyrin IX fluorescence in situ do not offer subcellular resolution. Therefore, discrimination of non-specific 5-ALA induced fluorescence remains difficult. Materials and Methods: In this study we have used an orthotopic glioma model to analyze PpIX fluorescence in tumor tissue and normal brain by multiphoton excitation microscopy after intraperitoneal administration of 5-ALA. A DermaInspect in vivo imaging system was used for autofluorescence measurements at 750 nm excitation and detection in the green channel of a standard photomultiplier module. For detection of PpIX fluorescence at different excitation wavelengths a red sensitive version of the photomultiplier and a filter combination of short pass filters and a color glass long pass filter was used restricting the sensitivity in the red channel to a range of 580-700 nm. Results: Multiphoton microscopy allowed a higher structural definition of tumor tissue based on the excitation of 5-ALA induced PpIX fluorescence compared to autofluorescence imaging. The high resolution of multiphoton microscopy allowed discrimination of fluorescence from the cytoplasm of tumor cells and 5-ALA induced PpIX fluorescence of normal brain parenchyma adjacent to tumor. Fluorescence lifetime imaging showed significantly longer fluorescence lifetimes of 5-ALA induced PpIX fluorescence in tumor tissue compared to normal brain. This allowed definition and visualization of the tumor/brain interface based on this parameter alone. Conclusion: Multiphoton microscopy of 5-ALA induced PpIX fluorescence in brain tumor tissue conceptually provides a high resolution diagnostic tool, which in addition to structural information may also provide photochemical/functional information.