Hurley, ME orcid.org/0000-0003-0553-3079, Sheard, TMD orcid.org/0000-0003-4940-3188, Norman, R et al. (8 more authors) (2020) A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types. Methods. ISSN 1046-2023
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Keywords: | Correlative imaging; DNA-PAINT; Live cell imaging; Calcium imaging; Myocytes |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 30 Oct 2020 16:53 |
Last Modified: | 30 Oct 2020 16:53 |
Status: | Published online |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.ymeth.2020.10.005 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:167216 |