Extended Data Fig. 4: Representative examples illustrating steps in image processing.

(a) Raw fluorescence image. (b) Pixel-wise extrapolated background. (c) Background-subtracted image, with a putative Ca²⁺ spark indicated. (d) Temporally-averaged data for initial coarse identification of Ca²⁺ spark. (e) Extracted spark time course, with frames separated by 20 ms. Raw and diffusion-subtracted outputs are presented, blurred by a 2-pixel Gaussian filter to reduce false detections. (f) Averaged background revealing SR structure. (g) PALM RyR image used to align the signal with SR structure, accounting for chromatic shift. This alignment enabled direct correlation of confirmed Ca²⁺ sparks (h) to RyR positions (i, merged image). The described analysis steps were performed for all recordings of Ca²⁺ sparks and RyRs. ~80% of Ca²⁺ release events were successfully aligned with RyRs, i.e. situated within 300 nm of a cluster (see Fig. 3c).