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Proceedings Paper

In vivo stoichiometry monitoring of G protein coupled receptor oligomers using spectrally resolved two-photon microscopy
Author(s): M. R. Stoneman; D. R. Singh; V. Raicu
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Paper Abstract

Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the cell under study is scanned a number of times in order to accumulate enough spectral information to accurately determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor, Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.

Paper Details

Date Published: 26 February 2010
PDF: 6 pages
Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 756913 (26 February 2010); doi: 10.1117/12.843916
Show Author Affiliations
M. R. Stoneman, Univ. of Wisconsin-Milwaukee (United States)
D. R. Singh, Univ. of Wisconsin-Milwaukee (United States)
V. Raicu, Univ. of Wisconsin-Milwaukee (United States)


Published in SPIE Proceedings Vol. 7569:
Multiphoton Microscopy in the Biomedical Sciences X
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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