Resonant Energy Transfer (RET) from an optically excited molecule to a non-excited molecule residing nearby has been used to detect molecular interactions in living cells. Information such as the number of proteins forming a molecular complex has been obtained so far for a handful of proteins, but only after exposing the samples sequentially to at least two different excitation wavelengths. Changes in the molecular makeup of a cellular region occurring during this lengthy process of measurement has limited the applicability of RET to determination of cellular averages. We developed a method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a spectrally-resolved two-photon microscope. The use of diffractive optics in a non-descanned configuration allows acquisition of a full set of spectrally-resolved images after only one complete scan of the excitation beam. This presentation will briefly describe our basic experimental setup and a simple theory of RET in oligomeric complexes, and it will review our recent results on determination of the geometry and size of oligomeric complexes of several proteins in yeast as well as in mammalian cells. This method basically transforms RET into a method for performing veritable structural determinations of protein complexes in vivo.© (2009) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.