The calcium (Ca2+) signals underlying the contractile function of cardiomyocytes originate from ryanodine receptors (RyR) located within intracellular signalling sites known as ‘couplons’. In the healthy heart, the spatiotemporal properties of the unitary Ca2+ signals (sparks) require rapid co-activation and subsequent closing of RyRs as a result of (a) the close packing of RyRs into clusters and (b) the structural organisation of the couplon by its primary molecular tether, Junctophilin-2 (JPH2). Recent super-resolution imaging has been unable to fully resolve single RyRs and partner proteins within the clusters due to resolution still limited to ~30 nm. We have used greatly enhanced super-resolution imaging based on the new DNA-PAINT’ method (1) to visualise the nanoscale molecular arrangement of RyR and JPH2 in peripheral couplons of ventricular myocytes at a resolution of 5-10 nm. Male Wistar rats weighing ~300 g (n = 8) were euthanized using a Schedule 1 non-recovery procedure approved by the University of Exeter animal ethics committee. Hearts were then dissected; ventricular myocytes were enzymatically isolated, fixed and immunolabelled against RyR2 and JPH2 as described previously (2). DNA-PAINT imaging (1) was performed using a modified total internal reflection fluorescence (TIRF) microscope. In regions identified as RyR clusters in dSTORM images, DNA-PAINT revealed arrays of punctate labelling, each reporting single RyRs within a couplon. Analysis of these receptor locations revealed that RyRs are spaced at distances of 43.2 ± 0.4 nm (n = 1802 clusters), similar to recent measurements on limited electron tomograms (3) but greater than the in vitro RyR packing distances observed in artificial bilayers (4). This loose arrangement was not explained by the remaining RyR localisation errors. It manifested as (a) observable gaps within RyR clusters accounting for 35.5 ± 0.5% (n = 2062) of the cluster area and (b) fewer RyRs present within peripheral clusters (7.3 ± 0.2 RyRs per cluster; n = 2062) than previously estimated with super-resolution dSTORM (2). These new measurements need to be incorporated into simulations of Ca2+ spark genesis (5) and termination to understand the functional consequences of the non-uniform RyR distribution within the cardiac couplon. Dual-colour DNA-PAINT revealed that most of these gaps are occupied by 62.4 ± 2.2 % (n=11 cells) of the JPH2s which are intimately organised and/or bound to RyR. The data also discover subpopulations of JPH2 external to the couplon. These are likely biomarker of the cells’ turnover of JPH2 which is now known to be altered in heart failure. The improved, molecular-scale resolution was critical to our new observations; DNA-PAINT is a unique tool that can provide a new window into the molecular remodelling that occurs within the couplon in cardiac pathologies.