Microspectroscopic method for determination of size and distribution of protein complexes in vivo

S. Rath; A. P. Sullivan; M. R Stoneman; V. Raicu

doi:10.1117/12.821831

22 May 2009 Microspectroscopic method for determination of size and distribution of protein complexes in vivo

S. Rath, A. P. Sullivan, M. R Stoneman, V. Raicu

Proceedings Volume 7378, Scanning Microscopy 2009; 737829 (2009) https://doi.org/10.1117/12.821831
Event: SPIE Scanning Microscopy, 2009, Monterey, California, United States

Abstract

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.

Citation Download Citation

S. Rath, A. P. Sullivan, M. R Stoneman, and V. Raicu "Microspectroscopic method for determination of size and distribution of protein complexes in vivo", Proc. SPIE 7378, Scanning Microscopy 2009, 737829 (22 May 2009); https://doi.org/10.1117/12.821831

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