title: Ratiometric fluorescence imaging of dual bio-molecular events in single living cells using a new FRET pair mVenus/mKOκ-based biosensor and a single fluorescent protein biosensor.
abstract: Genetically coded fluorescent protein (FP)-based biosensors are powerful tools for the non-invasive tracking of molecular events in living cells. Although a variety of FP biosensors are available, the simultaneous imaging of multiple biosensors (multi-parameter imaging) in single living cells remains a challenge and is far from routinely used to elucidate the intricate networks of molecular events. In this study, we established a novel combination of FP biosensors for dual-parameter ratiometric imaging, consisting of a new fluorescence resonance energy transfer (FRET) pair mVenus (yellow FP)/mKOκ (orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor Grx1-roGFP2. Under our imaging condition, 1.4±0.05% of Grx1-roGFP2 signal contributes to the mVenus channel and 5.2±0.12% of the mVenus signal contributes to the Grx1-roGFP2 channel. We demonstrate that such low degree of cross-talk causes negligible distortion of the ratiometric signal of the YO-based FRET biosensor and Grx1-roGFP2. By using this dual-parameter ratiometric imaging approach, we achieved simultaneous imaging of Src/Ca(2+) signaling and glutathione (GSH) redox potential in a single cell, which was previously unattainable. Furthermore, we provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H(2)O(2) via its effect on GSH-based redox system, demonstrating the power of this dual-parameter imaging approach for elucidating new connections between different molecular events that occur in a single cell. More importantly, the dual-parameter imaging approach described in this study is highly extendable.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Su
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Qingming
authors.initials: Q
authors.lastname: Luo
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D040081:Protein Array Analysis / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes