Red-to-Near-Infrared Fluorescence Resonance Energy Transfer Reporters for Dual Imaging Applications.

Fluorescence resonance energy transfer (FRET) biosensors enable quantitative visualization of protein interactions and signaling dynamics in living cells. However, most fluorescent protein (FP) FRET pairs occupy overlapping spectral regions, limiting multiplexing and simultaneous imaging of multiple pathways. In particular, the limited availability of well-characterized near-infrared (NIR) acceptors has constrained robust dual-FRET measurements within single cells. Here, we characterize a red-to-NIR FP FRET pair comprising mRuby2 and miRFP670nano3. Fluorescence lifetime imaging microscopy (FLIM) demonstrates efficient energy transfer with minimal spectral bleed-through and compatibility with established CFP/YFP-based reporters. In comparison with alternative dual-FRET strategies, including dark acceptors and long Stokes shift FPs, this pair enables multiplexed imaging without extensive spectral unmixing or correction. Structural modeling suggests that favorable donor-acceptor geometry may contribute to its FRET efficiency. We demonstrate the utility of this red-to-NIR pair by simultaneous FLIM-FRET imaging of Akt and S6K activity in single cells, providing parallel readouts of distinct branches of PI3K-mTOR signaling. In three-dimensional invasion assays, dual imaging of Src and ROCK reporters reveals spatially coordinated signaling dynamics not evident in single-reporter measurements. Together, these results establish mRuby2/miRFP670nano3 as a practical addition to the FP-FRET toolbox for multiplexed interrogation of signaling networks in living cells.