title: Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs.
abstract: 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 Ca(2+) 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.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Su
authors.forename: Shaotao
authors.initials: S
authors.lastname: Pan
authors.forename: Qingming
authors.initials: Q
authors.lastname: Luo
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D049408:Luminescent Agents / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D019061:src-Family Kinases / Q000032:analysis* / Q000378:metabolism
chemical_list: D049408:Luminescent Agents; D008164:Luminescent Proteins; D019061:src-Family Kinases; D002118:Calcium