Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.319400v1?rss=1 Authors: Hurley, M. E., Sheard, T. M. D., Norman, R., Kirton, H. M., Shah, S. S., Pervolaraki, E., Yang, Z., Gamper, N., White, E., Steele, D., Jayasinghe, I. Abstract: Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals. Copy rights belong to original authors. Visit the link for more info