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

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

Methods (San Diego, Calif.) (2020-10-16)
Miriam E Hurley, Thomas M D Sheard, Ruth Norman, Hannah M Kirton, Shihab S Shah, Eleftheria Pervolaraki, Zhaokang Yang, Nikita Gamper, Ed White, Derek Steele, Izzy Jayasinghe
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.

MATERIALS
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Sigma-Aldrich
Anti-RYR2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution