Super-resolution microscopies are commonly utilised to obtain nanometre-scale information regarding the structures within a cell. This information is rarely accompanied by any functional detail, a key requirement when observing a cellular system. The most recent publication from the applied biophotonics group describes the development of a correlative super-resolution protocol. This protocol enables the fast second-messenger signalling of primary cell types to be visualised in regard to a cell’s underlying structure.
Application of the correlative super-resolution protocol has been undertaken across various cell types and disease models throughout Miriam Hurley’s PhD studies. Specifically, the group has applied the protocol to study the movement of calcium (Ca2+) against the nanoscale patterns of the ryanodine receptor, a Ca2+ release channel which gives rise to these Ca2+ signals within wildtype primary cells.
The protocol relies upon the combination of total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals with subsequent DNA-PAINT imaging of the RyRs. The publication outlines a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets to demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels which are locally activated during the genesis of cytoplasmic Ca2+ signals.
This work has formed the basis of Miriam’s PhD studies, funded by the Leeds Anniversary Research Scholarship and the Royal Society Research Grant (first research grant awarded to PI, Izzy Jayasinghe).
Access the full article at: https://doi.org/10.1016/j.ymeth.2020.10.005.