A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types
| Autor: | Thomas M. D. Sheard, Nikita Gamper, Shihab Shah, Ed White, Eleftheria Pervolaraki, Derek S. Steele, Zhaokang Yang, Ruth Norman, Miriam E. Hurley, Izzy Jayasinghe, Hannah M. Kirton |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Physics
Correlative 0303 health sciences Cell type Total internal reflection fluorescence microscope Time Factors Ryanodine receptor 030302 biochemistry & molecular biology Feature extraction Ryanodine Receptor Calcium Release Channel General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Calcium imaging Cytosol Live cell imaging Second messenger system Biophysics Calcium Myocytes Cardiac Calcium Signaling Molecular Biology 030304 developmental biology |
| Zdroj: | Methods (San Diego, Calif.). 193 |
| ISSN: | 1095-9130 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect