In situ attenuated total reflection (ATR) Fourier transform (FT) spectroscopy is presented as an adequate tool for studying molecular structure and function of biomembranes. In this article emphasis was directed to the production of suitable model bilayer membranes for optimum mimicking of natural biomembranes, and to special FTIR ATR techniques to achieve enhanced selectivity as well as time resolved information on complex membrane assemblies. In this context, the preparation of supported bilayers according to the LB/vesicle method is presented and the use of such model membranes to build more complex assemblies, e.g. with creatine kinase, a surface bound enzyme, and alkaline phosphatase, a membrane anchored enzyme. A comprehensive summary of equations used for quantitative ATR spectroscopy is given and applied to determine the surface concentration and orientation of membrane bound molecules. The use of supported bilayers for drug membrane interaction studies is demonstrated by the local anesthetic dibucaine. Besides of structural information’s, such studies result also thermodynamic date, such as adsorption isotherm and partition coefficient. A special ATR set-up for more precise background compensation is presented enabling the conversion of a single beam spectrometer into a pseudo double beam spectrometer. This optical component may be placed in the sample compartment of the spectrometer, and is referred to as single-beam-sample-reference (SBSR) attachment. Finally, a short theoretical introduction into time resolved modulation spectroscopy is given. Temperature modulated excitation of reversible conformational changes in the polypeptide poly-L-lysine and the enzyme RNase are shown as examples.

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