Resetting of Ca2+ signals in single moving sperm. A sperm cell is loaded with the Ca2+ indicator Fluo-4 AM and caged cGMP. The cell is repeatedly stimulated by releasing cGMP from its caged derivative with six consecutive UV flashes. After each flash, the [Ca2+]i rapidly decreases and rises anew. The recording was performed using an epifluorescent microscope (IX71). Frames were acquired at 30 frames/s using a back-illuminated EM CCD camera (DU-897D). Photolysis of caged compounds was achieved using a mercury lamp (U-RFL-T). The irradiation time was controlled by a mechanical shutter (VS25). Laser stroboscopic illumination (488-nm wavelength and 2-ms pulse) was achieved using an acousto-optical tunable filter (AA Opto-Electronic Company). The fluorescence was filtered by a 500-nm long pass filter (500 ALP; Omega Optical, Inc.). Superposition of the scale bar and flash number to the original video was performed using MATLAB, and editing was finalized using VideoStudio Pro X4 (Corel Corporation).
