Older methods of assaying for protein:protein interactions in situ relied upon chemical cross-linking and antibodies. The obvious problem with these methods is that they alter the physiological conditions within the cell. Plus, they offer limited information on spatial distribution of the proteins in question. These problems can be solved by instead employing Fluorescence Resonance Energy Transfer (FRET) based techniques.

FRET, basically occurs when an excited donor fluorophore comes into close proximity to an acceptor fluorophore. Energy is transferred between the two, altering the emission of the acceptor and quenching the donor. By modifying proteins so they are tagged with the relevant fluorophores, interactions can be visualized. Interaction is inferred because of the close physical proximity necessary for FRET (10 nM).

Peter Verveer, Oliver Rocks, Ailsa Harpur and Philippe Bastiaens provide a set of protocols and information panels describing the use of FRET for imaging protein interactions. These include Measuring FRET by Sensitized Emission, Measuring FRET by Acceptor Photobleaching, Imaging Protein Interactions by FRET Microscopy: FRET Measurements by Sensitized Emission, Imaging Protein Interactions by FRET Microscopy: FRET Measurements by Acceptor Photobleaching, Imaging Protein Interactions by FRET Microscopy: FLIM Measurements, Imaging Protein Interactions by FRET Microscopy: Labeling Proteins with Fluorescent Dyes, and Imaging Protein Interactions by FRET Microscopy: Cell Preparation for FRET Analysis.