For the project, we had to measure the local calcium signals within heart muscle cells and measure redox signals. This required extensive methodological development. The calcium signals occur in very small places within the cells (microdomains smaller than 2 micrometer) and are very brief (< 50 ms upstroke). We used different approaches and followed up on opportunities.
(1) Calcium signaling. To enable tracking of this fast and local calcium signaling we developed and optimized a dual-camera based confocal microscopy device. By simultaneously tracking the cell motion, we can map cell-signaling parameters during the full contraction cycle. A collaboration within the host institution led to development of original methods for unbiased analysis. The combination of fast confocal imaging and the novel advanced analysis algorithms allowed us to study localized calcium events in cardiac myocytes, in relation to the cell structure and the organization of phosphorylation. The fellow is preparing a manuscript to report on the latter findings. Importantly, she could apply her novel imaging algorithms for cell identification in the study of tissues from heart disease. Her approach and work with the team contributed to major advances in unbiased image analysis in several studies (Sci Rep, 2019; J Am Coll Cardiol, 2019)
(2) ROS. We used rare-earth doped nanoparticles, which the fellow had developed in previous works, as quantitative ROS sensors. We could show a proof of concept of their membrane localization on the cardiac cells. These ROS sensors have a very narrow absorption band (at 396±5 nm) and require a specific laser light illumination, which we implemented. The optical design of the available confocal system was however not fully compatible with this new light path, preventing the desired combined imaging of the calcium and ROS signals. As a contingency measure, the fellow eventually tested a 2-photon illumination strategy, which is unprecedented for these probes. The preliminary results confirmed that the multiphoton approach is a promising alternative for the continuation of this project.
(3) Skills and competences. The project has challenged the methodological limits of the imaging system and the ER has invested considerable time in developing novel approaches. Throughout this process, she had the possibility to improve on her experimental design, quality of collected data and analysis methods, working across disciplines in biophysics and imaging. She has built experience in communication and through presenting her work, participation in conferences and workshop, built her network. She has submitted an application for a next postdoctoral position where she can use her acquired skills and experience.
