Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs

Fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are powerful tools for dynamically measuring cellular molecular events because they offer high spatial and temporal resolution in living cells. Despite the broad use of FP-based FRET biosensors in cell biology, imaging of multiple molecular events (multi-parameter molecular imaging) in single cells using current FRET pairs remains difficult because it usually requires a control group for additional data calibration. Hence, spectrally compatible FRET pairs that do not require complex image calibration are the key to widespread applications of FP-based FRET biosensors in multi-parameter molecular imaging. Here, we report a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKOκ (yellow and orange FP, Y/O). We demonstrate that additional image correction is not necessary for these dual FRET pairs. Using these dual FRET pairs, we achieve simultaneous imaging of Src and Ca2+ signaling in single living cells stimulated with epithelial growth factor (EGF). By converting traditional FRET biosensors into B/G and Y/O-based biosensors, additional applications are available to elucidate the dynamic relationships of multiple molecular events within a single living cell.

► This paper develops a new combination of fluorescent protein-based pairs for dual FRET imaging.
► These FRET pairs are spectrally compatible without the need of additional image correction.
► Simultaneous imaging of Src kinase activity and Ca2+ signaling is achieved by dual FRET imaging.