Analysis of receptor-receptor interaction by combined application of FRET and microscopy

G protein-coupled receptors (GPCRs) participate in the regulation of many cellular processes and, therefore, represent key targets for pharmacological treatment. The existence of GPCR homo- and heterodimers has become generally accepted, and a growing body of evidence points to the functional importance of oligomeric complexes for the receptor trafficking, receptor activation, and G protein coupling in native tissues. Quantitative molecular microscopy is becoming more and more important to investigate such receptor-receptor interaction in their native environments. Förster resonance energy transfer (FRET) is thereby utilized to aim at investigating the interaction of molecules at distances beyond diffraction-limited spatial resolution. The exact determination of the FRET signals, which are often only fractions of the fluorescence signals, requires extensive experimental effort. Moreover, the correct interpretation of FRET measurements as well as FRET data-based modeling represents an essential challenge in microscopy and biophysics. In this chapter, we present and discuss variety of acquisition protocols and models based on "linear unmixing FRET" (lux-FRET) to investigate receptor-receptor interaction in living cells with high spatial and temporal resolution. Here, we show how to apply lux-FRET in spectroscopic and different imaging devices, based either on spectral detection or on filter cubes. We focus on detailed description for FRET measurements and analyses based on sophisticated acquisition procedures according to different experimental setups and also provide several examples of biological applications.