CAPTUR3D activities are ideally divided into three major phases: Preparatory, Production, and Exploitation. After 30 months of activity, as can be viewed in the Gantt diagram, the Preparatory phase has been completed, while the other two phases have been activated and are being executed as planned. In more detail, the Preparatory phase was executed by optimizing sample-maintenance, sample-labelling, and sample-imaging procedures. Concerning these latter, unexpected new results were obtained on the metabolic response of α and β cells to glucose by studying tissue autofluorescence lifetime, which are in turn stimulating the development of new machine-learning-based strategies to discriminate α and β cells based on microscopy data. Approximately 12 months from the start, the Italian Ministry of Health blocked the use of tissue explants from human patients for research purposes. As a consequence, Project objectives were re-focused on cell models and an updated DoA approved in April 2022.
The successful completion of the Preparatory Phase laid the ground to the ‘Production Phase’ which comprises three work packages aimed at addressing, at high spatiotemporal resolution, the molecular details of the insulin-granule surroundings, membrane, and lumen. The body of work performed so far produced results on each of these target environments. A few exemplary outcomes are worth of mention. First of all, we provided demonstration of successful feedback-based 3D orbital tracking of single insulin granules, being able to extract information on the dynamics of selected intra-granule molecules during granule trafficking. Second, we successfully tested a genetically-encoded pH biosensor targeted to the insulin granule lumen: the average pH of the granule and its variation in response to glucose were measured and we now expect to measure pH fluctuations at the unprecedented speed of micro-milliseconds. Third, optical super-resolution in the form of Expansion Microscopy (ExM) was introduced in the Project activities to compensate for the lack of STimulated Emission Depletion (STED): ExM was successfully applied to study the effect of pro-inflammatory cytokines on $ \beta $-cells, uncovering a hitherto neglected reshaping of the intracellular landscape and providing a benchmark to interpret previous data and guide future studies.
In the last few months, the Exploitation phase was not only activated but significantly enhanced, leading to high-impact results. Specifically, the agreement to access a biobank of pancreatic tissues from both healthy and diabetic donors (Prof. Marchetti’s Group) has been concretized. This has enabled us to perform ExM experiments on human samples (3 donors per group), which have revealed clear structural alterations in key organelles, such as granules and mitochondria, in diabetic tissue. These results are the subject of a manuscript in preparation and were presented at the EASD 2025 conference.
Concurrently, the technological platform patented by the PI to test drug function has been successfully exploited. We measured the activity and biodistribution of a promising drug, baricitinib, confirming its beta-protective power. These findings have culminated in a major publication in the journal Science Advances. The potential of analyzing the intrinsic fluorescence of drugs, which is at the core of the patent, has become a strategic pillar of the project. It now constitutes the cornerstone of WP4 and the fundamental asset of the ERC Proof of Concept (PoC) project that stemmed from CAPTUR3D and was funded this year.
