Abstract
Resonant energy transfer from an optically excited donor molecule to a non-excited acceptor molecule residing nearby is widely used to detect molecular interactions in living cells. To date, resonant energy transfer has been used to obtain stoichiometric information, such as the number of proteins forming a complex, for a handful of proteins, but only after performing sequential scans of the emission wavelengths, excitation wavelengths, or sometimes both. During this lengthy process of measurement, the molecular makeup of a cellular region may change, limiting the applicability of resonant energy transfer to the determination of cellular averages. Here, we demonstrate a method for the determination of protein complex size, configuration, and spatial distribution in single living cells. It relies on a spectrally resolved two-photon microscope, a simple but competent theory, and a judicious selection of fluorescent tags. This approach eventually may lead to tracking the dynamics of individual molecular complexes inside living cells.
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Acknowledgements
This work was supported by a grant from the Wisconsin Institute for Biomedical and Health Technology (grant no. W620 to V.R.), seed funds from the UWM Research Growth Initiative (grant no. X014 to V.R.), and a grant from Canadian Institutes of Health Research (grant no. MOP43990 to J.W.W.). We thank D. Gillman for useful suggestions regarding the computer routines for instrument control, M. J. Woodside for his assistance with the lifetime imaging, and S. Angers for very useful discussions on BRET. We thank K. J. Blumer for providing YFP.
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V.R. coordinated the project, designed the TPM, built the optical set-up, developed the theory and the algorithms for image reconstruction and data analysis, analysed data, and wrote the paper. M.S., S.R. and M.F. carried out experiments using the TPM and analysed data. R.F. wrote computer codes for hardware control and for image reconstruction. M.M. interfaced the scanners with the computer and built an earlier version of the optical set-up. D.B.J. made the genetic constructs. L.P. and J.W.W. determined the RET efficiency using FLIM. D.K.S. participated in theoretical modelling. All authors contributed to manuscript editing.
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Raicu, V., Stoneman, M., Fung, R. et al. Determination of supramolecular structure and spatial distribution of protein complexes in living cells. Nature Photon 3, 107–113 (2009). https://doi.org/10.1038/nphoton.2008.291
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DOI: https://doi.org/10.1038/nphoton.2008.291
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