Fluorescent images were obtained using fluo-3 as the Ca indicator and a custom-built wide-field, high speed digital imaging system (ZhuGe et al., 1999). Rapid imaging was made possible by using a cooled high sensitivity charge-coupled device camera (128 × 128 pixels) developed in conjunction with the Massachusetts Institute of Technology Lincoln Laboratory. The camera was interfaced to a custom-made inverted microscope equipped with a 60× oil immersion lens (numerical aperture of 1.3), with each pixel covering a 333 × 333–nm area of the cell. The 488-nm line of a multiline argon laser provided fluorescence excitation for the indicator fluo-3, and a laser shutter controlled the exposure duration. Emission of the Ca2+ indicator was monitored at wavelengths >500 nm. To obtain a constant concentration of Ca2+ indicator, 50 µM fluo-3 was delivered through the patch pipette, and measurements were not commenced until 10–15 min after disruption of the patch. After this time, no significant change in background fluorescence was detected. Subsequent image processing and analysis were performed off line using a custom-designed software package running on a PC.
