Metabolic imaging of beta-cells exposed to glucose and cytokines through fluorescence lifetime microscopy.

Glucose stimulation is connected to insulin secretion by a series of highly regulated biochemical processes that happen in specialized cells of the mammalian pancreas, beta-cells, and, just in view of the significance of this link for systemic glucose homeostasis, non-invasive and quick strategies able to monitor the response to glucose are continuously being tried to find. In this thesis I use the phasor-based approach to Fluorescence-Lifetime Imaging Microscopy (FLIM) to quantify the ratio of protein-bound Nicotinamide adenine dinucleotide (phosphate) in its reduced form (NAD(P)H) to protein-free NAD(P)H, exploiting the autofluorescence coming from cells of the INS-1E cell line, which is a cellular model for beta-cells. Phasor-FLIM analysis permits, on the one hand, to estimate how the bound/free ratio of the NAD(P)H species increases after pulsed glucose stimulation, and, on the other hand, to notice the appearance of Long-Lifetime Species (LLS) as characteristic products of the oxidative stress induced by cytokines, which are a broad and loose category of small proteins important in cell signalling