Intracellular protein-mediated molecular events are organized in a complicated network that governs how a cell responds to environmental change. To study this complex network, it is imperative to develop techniques that empower researchers to simultaneously monitor multiple molecular events in single living cells. Fluorescent protein (FP)-based fluorescence resonance energy transfer  (FRET)  microscopy is a powerful tool for investigating protein-protein interaction in living cells. Since the first application of GFP for bio-labeling, FPs with different colors have been discovered, making multi-color fluorescence imaging in living cells possible. However, the simultaneous imaging of molecular events in a single living cell with multiple pairs of FP biosensors (multi-parameter imaging) is still in its infancy and is far from being a regular tool for elucidating complex signal networks. The major obstacle in implementing multi-parameter fluorescence imaging experiments is that the spectrum of GFP-like FPs is confined to the narrow visible wavelength (∼400–650 nm).  Moreover, the excitation and emission spectra of FP are broad and usually cover a range of approximately 100 nm. These intrinsic limitations cause spectral crosstalk between different biosensors, which requires extra correction in order to obtain unbiased biological information.

   Zhihong Zhang’s research group from Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology focuses on development of FP-based biosensors and their application in living cells. Recently, substantive progress has been made in dual-molecular fluorescence imaging. Through in-depth spectral analysis of current FPs, a new FRET pair, which is composed of mVenus (yellow FP)/mKOκ (orange FP)-based (abbreviated as YO), has been developed. By combining Src biosensor based on this FRET pair with calcium biosensor based on mTagBFP /sfGFP FRET pair, simultaneous imaging of Src and Ca2+ signaling is achieved in single living cells. Furthermore, by combining mVenus/mKOκ-based Src biosensor with single fluorescent Grx-roGFP2 biosensor, they achieved simultaneous imaging of Src signaling and glutathione (GSH) redox potential in single living cells, which was previously unattainable. Furthermore, they provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H2O2 via its effect on GSH-based redox system. The ability of revealing dynamic interaction between dual molecular events in a single cell strongly demonstrates the power of dual-molecular fluorescence imaging approaches developed by Zhang’s group.
   The above results have been published at Biosensor and Bioelectronics (SCI, IF=5.602) in 2012 and 2013, respectively (Ratiometric fluorescence imaging of dual bio-molecular events using mVenus/mKOκ-based FRET biosensors and single fluorescent protein biosensor in single living cells. Biosensors and Bioelectronics, 31 (1): 292-298, 2012; Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP /sfGFP and mVenus/mKOκ fluorescent protein pairs. Biosensors and Bioelectronics. 46: 97-101, 2013). Furthermore, the results are cited by Chemical Reviews (IF 40.197), commenting that “the red-shifted FP FRET pair, mVenus/mKOκ, was used in combination with a single FP sensor, Grx1-roGFP2. These spectrally compatible probes had minimal cross-talk and were shown to exhibit great spatiotemporal precision.”
            This work was supported by the National Basic Research Program of China (Grant no. 2011CB910401), Science Fund for Creative Research Group of China (Grant no. 61121004), National Natural Science Foundation of China (Grant no. 81172153), National Science and Technology Support Program of China (Grant no. 2012BAI23B02) and Program for New Century Excellent Talents in University of China(NCET-08-0220 to Z.H. Zhang).