Imaging neuronal calcium fluorescence at high spatio-temporal resolution

A rapid fluorescence imaging system was developed and utilised to investiga te the time-course of intracellular calcium concentration ([Ca2+](i)) gradi ents generated by action potentials in CA1-CA3 pyramidal cells within brain slices of the rat hippocampus. The system, which is based on a fast commer cial CCD camera, can acquire hundreds of 128 x 128 pixel images in sequence , with minimal inter-frame interval of 2.5 ms (400 frames/s) and 12 bit/pix el accuracy. By synchronising patch clamp recordings with image capture, th e timing of transmembrane potential variation, ionic Ca2+ current and Ca2diffusion were resolved at the limit of the relaxation time for the dye-Ca2 + binding reaction (approximately 5 ms at room temperature). Numerical simu lations were used to relate measured fluorescence transients to the spatio- temporal distribution of intracellular Ca2+ gradients. The results obtained indicate that dye reaction-diffusion contributes critically to shaping int racellular ion gradients. (C) 1999 Elsevier Science B.V. All rights reserve d.