The F-Techniques: Using Fluorescence Microscopy to Measure and Visualize Interactions and Mobilities of Biomolecules

Fluorescence is the property of atoms and molecules to absorb a photon of a certain wavelength and to emit a photon of a longer wavelength after a characteristic delay. The well-developed chemical and genetic strategies to attach fluorophores to biomolecules of interest make fluorescence a very sensitive, highly specific and thus widely applied marker for in vitro and in vivo studies and especially for light microscopy. Although mainly used for localization studies, fluorescence also allows to characterize quantitatively biological processes like molecular interactions and transport/diffusion. Here we will introduce different spectral and photophysical properties as well as fluorescence microscopy-based methods that exploit them, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM), often referred to as F-techniques.