Pro-inflammatory cytokines contribute to β-cell failure in both Type-1 and Type-2 Diabetes. Data collected so far allowed to dissect the genomic, transcriptomic, proteomic and biochemical landscape underlying cytokine-induced β-cell progression through dysfunction. Yet, no report thus far complemented such molecular information with the direct optical nanoscopy of the β-cell subcellular environment. Here we tackle this issue in Insulinoma 1E (INS-1E) β-cells by label-free fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based super resolution imaging by expansion microscopy (ExM). It is found that 24-h exposure to IL-1β and IFN-γ is associated with a neat modification of the FLIM signature of cell autofluorescence due to the increase of either enzyme-bound NAD(P)H molecules and of oxidized lipid species. At the same time, ExM-based direct imaging unveils neat alteration of mitochondrial morphology (i.e. ~ 80% increase of mitochondrial circularity), marked degranulation (i.e. ~ 40% loss of insulin granules, with mis-localization of the surviving pool), appearance of F-actin-positive membrane blebs and an hitherto unknown extensive fragmentation of the microtubules network (e.g. ~ 37% reduction in the number of branches). Reported observations provide an optical-microscopy framework to interpret the amount of molecular information collected so far on β-cell dysfunction and pave the way to future ex-vivo and in-vivo investigations.
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