Calcium Imaging and cGMP Sensing

cGMP and Calcium imaging was performed as described elsewhere [15 (link), 40 (link)]. Briefly, a single adult animal that expressed genetically encoded cGMP indicator cGi-500 [25 (link)] or that express calcium indicator GCaMP3 [48 (link)] and tagRFP in AFD was placed on a 10% agar pad on a cover slip with 0.1 μm polystyrene beads (Polysciences, Warrington, PA, USA) and covered by another cover slip for immobilization [49 (link)]. The immobilized animals were placed on a Peltier-based temperature controller (Tokai Hit, Fujinomiya, Japan) on a stage of BX61WI microscope (Olympus, Tokyo, Japan). The cyan and yellow, or red and green fluorescence was separated by the Dual-View optics system (Teledyne photometrics, AZ, USA), and the images were captured by an EM-CCD camera C9100-13 ImageEM (Hamamatsu Photonics, Hamamatsu, Japan) at 1 Hz frame rate. Excitation pulses were generated by SPECTRA light engine (Lumencor, Beaverton, OR, USA). The fluorescence intensities were measured by the MetaMorph imaging system (Molecular Devices). Change of fluorescence ratio R (CFP/YFP for cGMP imaging with cGi-500 and GFP/RFP for Ca²⁺ imaging with GCaMP3 and tagRFP), (R–R₀) / R₀, was plotted, where R₀ is average of R from t = 1 to t = 31.
Expression of PDE-5::GFP was observed with LSM880 confocal microscope (Zeiss, Oberkochen, Germany).