The aim of this study is the advancement of methods and applications for the two-photon laser microscopy for special biological questions. The implementation of a non-iterative biexponential evaluation method (new in this area) opens new possibilities on the imaging-sector of the two-photon microscopy. FLIM measurements with artificial skin constructs give information on the acid character of these skin equivalents and permit a comparison regarding the pH value. Therefore special fluorophors which sensitivly react to the pH value were employed. Furthermore, the effects on the energy metabolism of the insulin-producing b-cells have been investigated. The cell metabolism involves among other things the coenzymes NADH and NADPH. They belong to the pyridinenucleotide coenzymes, which are involved in numerous biological redox processes. Thus this nicotineamide-based coenzymes in oxidized form function as electron acceptors. These coenzymes can be simultaneously excited by two photons and fluoresce. Thereby they indicate the cellular metabolic state. The results are of particular biomedical significance, since function defects in the b-cells of the pancreatic islets lead to diabetes type II. Moreover, a special two-photon two-colour method is employed to extend the wavelength range of the Ti:Sapphire laser. An overlay of femtosecond pulses of different wavelengths could help to examine the fluorescence of the biological energy carrier molecule ATP with high temporal resolution.