simultaneous live cell imaging using dual fret sensors with a single excitation light同时使用双重烦恼活细胞成像传感器用单一激发光.pdfVIP

Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light Yusuke Niino, Kohji Hotta, Kotaro Oka* Center for Biosciences and Informatics, School of Fundamental Science and Technology, Keio University, Kohoku-ku, Yokohama, Japan Abstract 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 Ca2+ 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. Citation: Niino Y, Hotta K, Oka K (2009) Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light. PLoS ONE 4(6): e6036. doi:10.1371/journal.pone.0006036 Editor: Karl-Wilhelm Koch, University of Oldenburg, Germany Received March 23, 2009; Accepted May 29, 2009; Published June 24, 2009