Two-photon excited fluorescence microscopy combined with spectral and time-resolved measurements for fluorophore identification

Two-photon excited fluorescence microscopy was used to study unstained tissue and paper samples. As an excitation source a mode-locked Ti:Sapphire laser was utilized. In the experiments we used a conventional fluorescence microscope with a scanning board. The incoming laser pulses were focused onto the sample and the epifluorescence observed. In the spectroscopic measurements the fluorescence light was projected either on the slit of an polychromator with a CCD camera or, in some experiments, on a streak camera connected to the polychromator. The signal was then detected by a 2D-CCD camera. Fluorescence images were scanned by recording the fluorescence light pixel by pixel with a photomultiplier tube. Signal filtering and image processing were performed on a personal computer. Tissue samples from animals treated with photodynamic therapy were examined. The tissue contained protoporphyrin IX as a photosensitizer.