N-Way FRET Microscopy for Imaging Multiple Protein Interactions Within a Single Living Cell

Abstract

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool for probing nanoscale protein interactions while capturing the microscale organization of these interactions. However, current FRET microscopy approaches are limited to the analysis two interacting fluorescently labeled proteins at a time. This limitation precludes the use of FRET microscopy for simultaneous measurement of multiple biochemical activities. Here we present a new FRET microscopy method that generalizes quantitative FRET microscopy to any number of fluorophores interacting in any combination. This approach makes use of Parallel Factor Analysis (PARAFAC) to define excitation/emission spectral fingerprints for FRET between any number of interacting fluorophores from observations of reference samples on any instrument. The resultant spectral fingerprints are then used in a simple linear unmixing model recover the distributions of free and interacting fluorophores as well as their apparent FRET efficiencies. Input data can consist of either complete spectral data or filter-based methods. The approach was validated using control constructs consisting of fluorescent protein fusions inside living cells. This method will enable intracellular analysis of sequential biochemical interactions that could not previously be observed.