The spatiotemporal properties of the Ca 2+ release process in skeletal muscle fibers were determined using an improved confocal spot detection system. Muscle fibers were loaded with the low affinity fluorescent Ca 2+ indicator OGB-5N and localized action potential-induced fluorescence signals were recorded from consecutive locations separated by 200 nm within a single sarcomere. Three-dimensional reconstructions of the Ca 2+ transients illustrate the existence of domains of increased

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... e around Ca 2+ release sites in the neighborhood of the T-tubules. We estimated the dimensions of these domains by drawing isochronal curves (∆F/F vs. spot position) and fitting Gaussian profiles to them. It was found that the earliest detectable full-width-at-half-maximum of these profiles was 0.77 ± 0.25 µm and increased rapidly with time to 1.4 ± 0.2 µm at their peak (18 o C). A brief, but statistically significant, delay of 0.8 ± 0.42 ms was observed between the onset of the fluorescence transients at the Z-and M-lines. Our results are compatible with the possibility that, in response to AP stimulation, Ca 2+ is not released exclusively from the junctional region of the sarcoplasmic reticulum, but from a broader expanse of the triadic region.