A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types

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.

Keywords: Calcium imaging; Correlative imaging; DNA-PAINT; Live cell imaging; Myocytes.