| Summary: | Abstract A cascade of highly regulated biochemical processes connects glucose stimulation to insulin secretion in specialized cells of mammalian pancreas, the β‐cells. Given the importance of this process for systemic glucose homeostasis, noninvasive and fast strategies capable to monitor the response to glucose in living cells are highly desirable. Here, we use the phasor‐based approach to Fluorescence Lifetime IMaging (FLIM) microscopy to quantify the ratio between protein‐bound and free Nicotinamide adenine dinucleotide (phosphate) species in their reduced form (NAD(P)H), and the Insulinoma cell line INS‐1E as a β‐like cellular model. Phasor‐FLIM analysis shows that the bound/free ratio of NAD(P)H species increases upon pulsed glucose stimulation. Such response is impaired by 48‐hours preincubation of cells under hyperglycemic conditions. Phasor‐FLIM concomitantly monitors the appearance of long‐lifetime species (LLS) as characteristic products of hyperglycemia‐induced oxidative stress.
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