Fast imaging in two dimensions resolves extensive sources of Ca2+ sparks in frog skeletal muscle

1. Ca2+ sparks were monitored by confocal laser-scanning microscopy of fluo -3 at video rates, in fast twitch muscle fibres, stimulated by exposure to caffeine and/or low [Mg2+]. Scanning was in two spatial dimensions ('2D') o r 2D plus time, at 4 ms per image frame. Sparks were identified in 2D image s of normalized fluorescence by an automatic procedure, which also evaluate d the event's location and morphometric parameters. 2. Most sparks were circular, but some were elongated, especially in caffei ne. Separation of the spark from circular symmetry was quantified by its ec centricity (length/width - 1). 3. In an internal solution with 0.4 mM [Mg2+]; sparks (989 events in 4 cell s) had amplitude 0.73, width 1.94 mum, length 2.12 mum and eccentricity not significantly different from 0. Upon application of 1 mM caffeine, length (of 2578 events in the same cells) increased significantly (by 0.41 mum, or 19%), width increased by 0.18 mum (9 %) and amplitude decreased slightly. The eccentricity became significantly different from 0, and the sparks' lon g axis predominantly oriented parallel to the plane of the Z disks. 4. More than 10% of the events in caffeine had length greater than 4 mum, a relatively: flat top, and a sharp termination at both ends of the major ax is. Additionally, there was only a weak correlation between eccentricity an d amplitude. These properties suggest that the elongated events are produce d by simultaneous opening of multiple channels within a junction, rather th an anisotropic diffusion of Ca2+ or random overlap of round sparks. 5. Elongated events often increased in eccentricity early in their evolutio n. Then, most remained elongated during their rise and decay, while others spread spatially in the plane of the Z disks. In 1-2% of the events, the ce ntre of mass migrated in space, over time, at similar to0.1 mum ms(-1). 6. These spatio-temporal features require the involvement of multiple relea se channels, at spatially resolvable locations. Because sources often sprea d over distances greater than 1 or 2 mum, and arrays of junctional elements (couplons) are at most 1 2 mum long, it must be possible for activation of release to propagate between neighbouring couplons, especially under the i nfluence of caffeine and/or low [Mg2+]..