An integral role in the cross-talk that occurs between the human intestine and its resident microbiota is performed by innate bacterial sensors known as pattern recognition receptors. Two main classes of PRRs have been shown to regulate communication between the intestinal epithelium and the microbiota. Toll-like receptors and Nod-like receptors serve to alert the host to the presence of bacteria in the extracellular and intracellular spaces respectively. Using the azoxymethane /DSS model of CAC, Fukata and coworkers showed that TLR4 participates in the development of colorectal cancer. Although interesting, this study has not directly addressed the impact of the microbiota in CAC development. Additionally, the AOM/DSS model of CAC appears to show a dissociation between the severity of intestinal inflammation and cancer development. Many genetically encoded sensors have been developed based on fluorescence resonance energy transfer, the radiationless transfer of excited state energy from an excited donor to an acceptor, between fluorescent proteins. These biosensors provide a means to image the spatiotemporal dynamics of various intracellular signals including second messengers, protein-protein interactions and enzyme activities. Moreover, combined use of the sensors would be useful to correlate multiple signaling events for understanding complex signal transduction networks. However, to date, most FRET applications have used only a single sensor in a cell. The sensor has a broad spectral profile for two fluorescent proteins, hence,Gilteritinib when the several sensors are present at the same location, imaging without the significant spectral overlap is difficult. These dual FRET pairs are spectrally compatible, when the donors are excited alternately at two different wavelengths and the emissions are collected sequentially. But when used simultaneous excitation not sequential excitation for two donors, sensitized emission from the first acceptor YFP should still be detected in the second donor fluorescence channel, resulting in possible artifacts. Since alteration of excitation light between two different wavelengths necessitates a lag time, sequential acquisition in the previous strategies is not adequate to follow fast signal dynamics or signal changes in highly motile cells. Here we report a method for imaging of two FRET pairs excited simultaneously with a single excitation light. We constructed FRET sensors using Sapphire/RFP for combined use with CFP/YFP, and both donors were excited with a violet light. We detected the emissions from the two FRET pairs using a quad channel imager without a lag time, and then distinguished between four fluorescent proteins using a computational method, linear unmixing, that has been recently used to extract the individual contributions of fluorophores which are linearly summated on the spectral detection channels. First, we tried to image intracellular cAMP and cGMP in single cells, since various FRET sensors for these cyclic nucleotides have been Phosphatase Inhibitor Cocktail (EDTA-Free) developed but there is no report on simultaneous measurement of them. The result ensured that our dual FRET approach provides efficient detection which is comparable to conventional single FRET experiments. Next, we demonstrated to monitor both intracellular cAMP and Ca2+ in single cardiac myocytes showing periodic contraction. Our method enabled ratiometric measurements for two sensors to cancel out artifacts caused by contracting movement of the cell. Thus, we proposed an alternative approach for imaging of dual FRET sensors in a single cell, more suitable for highly motile cell samples.