An oblique selective plane illumination microscope (oSPIM) has been built and characterized in the first part of the project. The working principle of this system is very similar to other light-sheet architectures.
The optimization of calcium imaging within intact islets with light-sheet microscopy represented a crucial aspect towards the success of this proposal. In the reported period, I managed to optimize the protocol for the imaging experiments, in order to maintain the same physiological condition of culture. By fine tuning of the property of the microscope, I could obtain three dimensional reconstructions of the entire population of alpha cells within a single islet, for multiple islets coming from different animals, and promising preliminary data were collected during the time of the project.
Moreover, I optimized a novel protocol for single molecule fluorescence in situ hybridization in intact islets. This has been a very challenging task, since islets are a very complex environment, and the relatively low signal-to-noise ratio of the single fluorescent mRNAs is hard to distinguish from the background created by scattering. Nevertheless, by changing the protocol and testing different conditions, I was able to overcome this issue, reporting smFISH experiments in intact murine islets using light-sheet microscopy. I tested different probes targeting several genes known to be involved in the regulation of glucagon secretion, such as PDX1, GPR40, GCK (glucokinase), GCG (glucagon), MAFB. GCG showed the best results, given the abundance of the gene in single alpha cells. Interestingly, the fluorescence signature of each cell within the islet showed heterogeneity in the expression of the gene, suggesting for the first time the evidence of the existence of subpopulations oh alpha cells within the intact islet, which supported the hypothesis of the project.
Unfortunately, the ongoing COVID pandemic and the subsequent lockdowns have prevented much of the correlative work that was supposed to be carried out to verify the relationship between heterogeneities in calcium activity and gene expression underlying regulation of glucagon secretion. This part is still work in progress.
At the same way, exploitation and dissemination have been limited to the first part of the project. Nevertheless, I obtained a travel award to participate in the 2019 Biophysical Society meeting, where I was able to present part of the work done in the construction and characterization of the optical architecture.
Later, in February 2019, I was able to travel to Washington University in St. Louis, where I presented my work at a seminar at the Cell Biology & Physiology department.
In march 2019, I presented my work in progress at the San Raffaele Scientific Institute retreat, where I met new colleagues and established new collaborations, especially with the Diabetes Research Institute groups.
Later, I presented my developing project at internal seminars in the institute and used social media to disseminate part of my work. Two publication stemming from the work done during the project are being prepared for submission. No website has been developed for the project.
