Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) underpins many genetically encoded fluorescent biosensors for monitoring biochemical activities in live cells. However, the FRET ratio (acceptor-to-donor signal ratio), commonly used as a proxy for FRET efficiency, is highly sensitive to imaging parameters, complicating data interpretation. Using FP-based barcodes, we introduced calibration standards into subsets of cells for normalization of fluorescence signals. Theoretical analysis indicated the need for both high- and low-FRET standards for calibration under different excitation intensities. We validated this prediction using engineered "FRET-ON" and "FRET-OFF" standards, demonstrating that calibrated FRET ratios are independent of imaging conditions. Including donor-only and acceptor-only cells enabled simultaneous determination of FRET efficiency for multiple biosensors. Calibration also restored expected reciprocal changes in donor and acceptor signals, often obscured by imaging fluctuations and photobleaching. Together, our studies introduce a simple strategy for robust, multiplexed FRET biosensor imaging, facilitating cross-experimental and long-term studies.