The dynamics of cytoplasmic free Ca concentration ([Ca]) in pancreatic β cells is central to our understanding of β-cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non-invasive, imaging site for measuring [Ca] dynamics longitudinally in three dimensions and at single-cell resolution. By applying a fluorescently labeled glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca] changes. We found that almost all β cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the β cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the β-cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca] measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic β-cell [Ca] response patterns at single-cell resolution in health and disease.