Pugliese, L.; De Lorenzi, V.; Ferri, G.; Vo, H.; Lindquist, A.; Tesi, M.; De Luca, C.; Suleiman, M.; Marselli, L.; Zhao, Y.; Marchetti, P.; Beltram, F.; Cardarelli, F.

Aims/hypothesis Quantitative nanoscale analysis of insulin secretory granules (ISGs) in human pancreatic tissue has been limited by the lack of imaging methods that combine high resolution with large-scale sampling. We aimed to establish expansion microscopy (ExM) as a platform for in situ, quantitative analysis of ISG organisation in human {beta}-cells and to assess whether type 2 diabetes (T2D) is associated with alterations in granule size, abundance or spatial organisation. Methods We applied Magnify ExM to PFA-fixed, paraffin-embedded pancreatic tissue sections from 6 human donors, 3 non-diabetic (ND) and 3 T2D, enabling super-resolution optical imaging of insulin-labelled granules. Insulin-positive structures were segmented and analysed using a morphometric pipeline to quantitatively assess size, shape and spatial features. Granule clustering was quantified based on combined area and roundness criteria. Results The diameter distribution of highly circular granules was similar between ND and T2D samples and estimates of granule number per cell indicated only a modest reduction in T2D (~25%). In contrast, mapping insulin-positive structures in a roundness-area space revealed a marked enrichment of large, irregular objects consistent with granule clustering in T2D. The fraction of clustered granules was significantly increased in T2D and strongly inversely correlated with insulin stimulation index (r = -0.85). Conclusions/interpretation These results establish expansion microscopy as a powerful platform for quantitative nanoscale analysis of human pancreatic tissue and identify altered spatial organisation of insulin granules, rather than marked granule depletion, as a prominent feature associated with {beta}-cell dysfunction in T2D.