Macromolecular High-Affinity Binding Probed by Advanced Fluorescence Techniques.

Due to the extreme sensitivity and the intrinsic selectivity of fluorescence techniques, high-affinity binding can be measured even at extremely low molecule concentrations in the picomolar range. In particular, modern advanced techniques with fluorescence microscopes have provided considerable methodological advancements in recent years. Here, a brief description of the basic physical principles of fluorescence detection and its experimental measurement setups are provided. For interacting biomolecules in solution, confocal fluorescence microscopy enables some very effective approaches to characterize binding in complex sample environments and with small sample consumption. In addition to standard techniques with bulk samples in classical spectrometers, applications with single-molecule Förster resonance energy transfer, two-color coincidence detection, and fluorescence correlation spectroscopy are presented. The strength of the more advanced techniques lies in their broad applicability, ranging from fluorescence-based genetically encoded biosensors for use in living cells to the high controllability in the measurement of binding curves even at very low molecule concentrations. The advantages and limitations of the individual techniques are compared and recent state-of-the-art applications are discussed.