23 February 2006 Three-dimensional FRET microscopy
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Abstract
A complete understanding of cellular behavior will require precise temporal and spatial measurement of protein-protein interactions inside living cells. FRET Stoichiometry (Hoppe, A.D. et al., 2002 Biophys. J. 83:3652) has been used to measure the timing and spatial organization of protein-protein interactions in cells expressing yellow fluorescent protein (YFP)-labeled proteins and cyan fluorescent protein (CFP)-labeled proteins. However, all FRET data collected in a single plane of a widefield microscope is a distorted 2D representation of a 3D object. Here we show that image blurring in the widefield microscope dramatically reduces sensitivity and spatial discrimination of FRET-based measurements of protein interactions. We present an algorithm for 3D restoration and calculation of FRET data that greatly increases signal-to-noise ratio and accuracy. The approach uses maximum likelihood deconvolution to quantitatively reassign out-of-focus light in 3D-FRET data sets. FRET Stoichiometry calculations performed on test constructs of linked YFP-CFP produced images that displayed uniform apparent FRET efficiencies (both EA and ED) and molar ratio of 1. 3D images of cells expressing free YFP and free CFP indicated apparent FRET efficiencies of 0%. Furthermore, 3D-FRET Stoichiometry imaging of the interaction of activated YFP-Rac1 with CFP-PBD in living cells produced superior detail with maximal apparent FRET efficiencies that were consistent with in vitro data. Together, these data demonstrated 3D-FRET Stoichiometry could accurately measure the fractions of interacting molecules and their molar ratios with high 3D spatial resolution.
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Adam D. Hoppe, Adam D. Hoppe, Joel A. Swanson, Joel A. Swanson, Spencer L. Shorte, Spencer L. Shorte, } "Three-dimensional FRET microscopy", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608904 (23 February 2006); doi: 10.1117/12.654819; https://doi.org/10.1117/12.654819
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