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Abstract
The spatial organization of protein-protein interactions inside of cells is an important component of cellular signaling. These interactions can be imaged using fluorescent resonance energy transfer (FRET) microscopy methods. We have described a generalized mathematical solution and method for any number of interacting FRET pairs (N-Way FRET). Here, we combine N-Way FRET with our three-dimensional FRET stoichiometry reconstruction (3D-FSR) to allow 3D-3Way-FRET image deconvolution. By developing a multi-camera instrument, we are able to acquire the 6 images needed per z-plane with sub-second time resolution, allowing observation of live cell dynamics. Furthermore, we implemented improved fast Fourier transforms and parallelized calculations to achieve a 10-fold decrease in the computational time required to reconstruct 3D-3Way-FRET data. This reduction has made it feasible to analyze dynamic 2Way and 3Way FRET data with 3D reconstructions, increase the number of reconstruction iterations, as well as explore stopping criteria for the algorithm.
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