FIGURE 3.

Advances in super-resolution imaging provide novel insight into RyR organization. Although previous work had suggested that RyRs are present in large clusters, dSTORM super-resolution imaging of RyRs has shown that the channels are rather present in multiple small, neighboring clusters. On the cell surface, these arrangements are present in double rows, on either side of z-lines [(A, left panel), (Baddeley et al., 2009)]. The increased resolution of RyR imaging by dSTORM (green) is noted in comparison with diffraction-limited imaging (red). Maximal occupancy of imaged clusters was calculated based on an assumed grid-link arrangement of channels (A, right panel). Quantitative assessment of more complex RyR arrangements within the cell interior requires high-resolution in 3D. Using 3D dSTORM, internal Ca²⁺ release units (CRUs) containing multiple RyR clusters with diverse configurations were observed [(B), enlargement of indicated region at right; (Shen et al., 2019)]. To gain insight into the orientation of RyRs within dyads, correlative 3D dSTORM imaging of RyRs was combined with confocal t-tubule imaging, and the channels were localized at the interfaces of the two signals [(C), enlargement of indicated region at right, (Shen et al., 2019)]. Even higher resolution imaging using the DNA-PAINT technique revealed that RyRs exhibit irregular packing within clusters [(D), (Jayasinghe et al., 2018)], in contrast to grid-based assumptions. Scale bars in left, right panels: A = 500 nm, 100 nm; B = 500 nm, 100 nm; C = 1 μm, 500 nm; D = 1 μm, 100 nm. Copyright permission was not required to reproduce the figures. (A) Copyright 2009, National Academy of Sciences.