FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough. The approach quantifies FRET signals and calculates the apparent energy transfer efficiencies (Es). The method was validated by measurement of a genetic (FRET standard) construct consisting of three different fluorescent proteins (FPs), mTFP, mVenus, and tdTomato, linked sequentially to one another. In addition, three 2-FP reference constructs, tethered in the same way as the 3-FP construct, were used to characterize the energy transfer pathways. Fluorescence lifetime measurements were employed to compare the relative relationships between the FPs in cells producing the 3-FP and 2-FP fusion proteins. The 3sFRET microscopy method was then applied to study the interactions of the dimeric transcription factor C/EBPa (expressing mTFP or mVenus) with the heterochromatin protein 1a (HP1a, expressing tdTomato) in live-mouse pituitary cells. We show how the 3sFRET microscopy method represents a promising live-cell imaging technique to monitor the interactions between three labeled cellular components.
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