Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light
Figure 2
cGMP sensor using FRET with Sapphire/RFP red cGES-DE5.
(A) Domain structure of red cGES-DE5. (B) In vitro emission spectra of red cGES-DE5 expressed and isolated from HEK293T cells. Black and red lines represent the spectra at zero and saturated cGMP (200 µM), respectively. (C) Concentration response curves of red cGES-DE5 for cGMP and cAMP. Curves were determined in vitro from the change in emission ratio at 510 nm (Sapphire) to 580 nm (RFP) (n = 4). Half-maximal effective concentration (EC50) value for cGMP was 40±8 nM (means±s.e.m.). (D) cGMP imaging by using red cGES-DE5 in PC12 cells. Representative fluorescence image in G. ch. (left) and traces (right) are shown. Typical response to stimulation with 5 µM adenosine and 100 µM IBMX for cAMP and subsequent stimulation with 2 µM SNAP for cGMP (n = 9). (E) SNAP washout experiments in PC12 cells expressing red cGES-DE5. Representative fluorescence image in G. ch. (left) and time trace of the FRET signal when stimulated with 5 µM SNAP (right) are shown (n = 8). Applied SNAP was washed out by continuous superfusion within 1 min. Scale bar, 10 µm.