For probing small distances in living cells, methods of super-resolution microscopy and molecular sensing are reported. A main requirement is low light exposure to maintain cell viability and to avoid photobleaching of relevant fluorophores. From this point of view, Structured Illumination Microscopy (SIM), Axial Tomography, Total Internal Reflection Fluorescence Microscopy (TIRFM) and often a combination of these methods are used. To show the high potential of these techniques, measurements on cell-substrate topology as well as on intracellular translocation of the glucose transporter GLUT4 are described. In addition, molecular parameters can be deduced from spectral data, fluorescence lifetimes or non-radiative energy transfer (FRET) between a donor and an acceptor molecule. As an example, FRET between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2 (Grb2) is described. Since this interaction, as well as further processes of cellular signaling (e.g., translocation of GLUT4) are sensitive to stimulation by pharmaceutical agents, methods (e.g., TIRFM) are transferred from a fluorescence microscope to a multi-well reader system for simultaneous detection of large cell populations.