Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+) and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.
Niino
Yusuke
Y
Center for Biosciences and Informatics, School of Fundamental Science and Technology, Keio University, Yokohama, Japan.
Hotta
Kohji
K
Oka
Kotaro
K
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D032383:Myocytes, Cardiac / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D015398:Signal Transduction; D000097573:Red Fluorescent Protein
D001426:Bacterial Proteins; D002118:Calcium; D000242:Cyclic AMP; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria