Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

Orange fluorescent proteins (FPs) are attractive candidates as Förster resonance energy transfer (FRET) partners, bridging the gap between green and red∕far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red∕far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair

Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living Cells

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/EBPα (expressing mTFP or mVenus) with the heterochromatin protein 1α (HP1α, 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