Objective: Studying cellular physiology often requires measuring of both intermolecular (e.g. in protein-protein interaction analysis) and intramolecular distances (e.g. detecting conformational changes of a protein during activation of signalling cascades). Microscopic methods based on the Fluorescence Resonance Energy Transfer (FRET) are well established solutions for these tasks. However, a number of methodological problems can impede the retrieval of reliable results. Spectral crosstalk, chromatic aberration, focal plane shifts and especially false positive FRET signals caused by random dimerization of the fluorescent dyes can easily lead to misinterpretation of the experimental data. Methods: In order to overcome these problems, we developed a Nipkow Disc based FRET microscopy system that is easy to operate without expert knowledge of FRET and the underlying optical difficulties. The system automatically accounts for all relevant sources of errors, produces various statistics and provides visualization of the acquired FRET data in two, three and four (i.e. time resolved 3D) dimensions. Results: Our new system was used to study two questions of basic physiologic interest: The interaction analysis of the two subunits of the hypoxia-inducible transcription factor 1 demonstrates protein-protein interaction analysis to better understand the assembly process of the transcription factor complex. As an example for time lapse observations, the conformational change of the fluorophore labelled heat shock protein 33 as a sensor for oxidant stress was studied. Conclusion: Our system can efficiently circumvent the described methodological problems in FRET measurements. The acquisition process becomes significantly less susceptible to user errors and therefore yields more precise and better comparable results.