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Conventional laser-induced fluorescence spectroscopy of endogenous chromophores like NADH and PP IX provides only uncertain information about the relative amounts of these metabolites in the observed cells due to varying optical tissue parameters. But for diagnostic applications the concentrations of these chromophores in many cases have to be determined quantitatively to establish a tissue- independent differentiation criterion. It is well-known that the individually and locally varying optical tissue parameters are major obstacles for the determination of the true chromophore concentrations by simple fluorescence spectroscopy. To overcome this problem, a fiber-based, 2- channel technique including a rescaled NADH-channel and a relative PP IX-channel was developed. The combined information of both channels can provide good tissue state separation. Ex-vivo studies with resected and frozen samples as well as in-vivo studies of squamous cells in the histologically confirmed states: normal, tumor margin, inflammation and hyperplasia were performed. At the identical tissue spot both, the rescaled NADH-fluorescence and the relative PP IX-fluorescence were determined. In the first case a nitrogen laser in the latter case a diode laser were used as excitation sources. In this study a good separation between the different tissue states was achieved. To obtain 2D quantitative fluorophore images of a certain tissue surface an even more sophisticated method is needed. At the moment a new rescaling method for VIS and IR light in the frequency domain is under construction. it can be applied within the validity range of the diffusion approximation and provides full (mu) a and (mu) s rescaling possibility in a 2D, non-contact mapping mode. The flying-spot device is planned to be used in combination with a standard operation microscope of ZEISS, Germany and will provide quantitative PP IX-fluorescence signals with a lateral resolution in the millimeter range.
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