抄録
Fluorescence imaging is a powerful method to visualize and analyze spatiotemporal dynamics of molecules in a living cell. Here we have developed a prism-based total internal reflection fluorescence microscopy (TIRFM ) system to simultaneously visualize and analyze three 2nd messengers, Ca2+, cAMP and diacylglycerol (DAG), that involve many cellular functions. Employed were three fluorescent indicators: 1) fura-2 AM for Ca2+, 2) Epac1-camp, a CFP-YFP FRET-based cAMP indicator, for cAMP and 3) C1-mRFP, a tandem DAG binding domain of PKC gamma, for DAG. These indicators were transfected into Cos 7 cells. DAG signal was taken by TIRFM, and Ca2+ and cAMP signals were taken by epifluorescence microscopy. When three 2nd messengers were evoked in Cos7 cell stimulated with 100uM ATP, each signal was successfully dissected without significant overlap of emission wave length among three signals. Unmixing of overlapping signals was also discussed in this study. [J Physiol Sci. 2008;58 Suppl:S61]
