  Resonant Energy Transfer (RET) from an optically excited donor molecule (D)
to a non-excited acceptor molecule (A) residing nearby is widely used to detect
molecular interactions in living cells. Stoichiometric information, such as the
number of proteins forming a complex, has been obtained so far for a handful of
proteins, but only after exposing the sample sequentially to at least two
different excitation wavelengths. During this lengthy process of measurement,
the molecular makeup of a cellular region may change, and this has so far
limited the applicability of RET to determination of cellular averages. Here we
demonstrate a method for imaging protein complex distribution in living cells
with sub-cellular spatial resolution, which relies on a spectrally-resolved
two-photon microscope, a simple but competent theory, and a keen selection of
fluorescent tags. This technology may eventually lead to tracking dynamics of
macromolecular complex formation and dissociation with spatial resolution
inside living cells.
