Imaging protein interactions in vivo with sub-cellular resolution

TL;DR

This paper introduces a novel spectrally-resolved two-photon microscopy method for imaging protein interactions in living cells with sub-cellular resolution, enabling real-time tracking of protein complex dynamics.

Contribution

The authors develop a new imaging technique that allows in vivo visualization of protein interactions at sub-cellular resolution without sequential excitation, improving upon existing RET methods.

Findings

Achieved sub-cellular resolution imaging of protein complexes in living cells.

Demonstrated real-time tracking of protein complex formation and dissociation.

Provided a simple theoretical framework for spectrally-resolved RET imaging.

Abstract

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.