AbstractBackground and ObjectiveThe 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 MethodsIn 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.ResultsMultiphoton 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.ConclusionMultiphoton 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. Lesers Surg. Med. 40:273–281, 2008. © 2008 Wiley‐Liss, Inc.