<p>By transplanting pancreatic islets onto the dura mater of the mouse brain, we establish a microscopy platform that enables longitudinal intravital imaging of otherwise optically inaccessible tissue. The system combines a cranial window with an air-cushioned floating arena and stable head fixation, providing high mechanical stability for repeated single-cell Ca²⁺ imaging sessions of up to 90 min in awake mice. We show that dura mater-engrafted islets integrate with host vascular and neural networks, and that human islet grafts secrete C-peptide in response to glucose stimulation, indicating metabolic integration. With this platform, we monitor anesthesia-induced changes in capillary blood flow and islet Ca²⁺ dynamics. In awake mice, following subcutaneous glucose injection, we characterize intracellular Ca²⁺ oscillations in insulin-secreting β-cells, revealing changes in amplitude, period, and plateau fraction while network coordination remains stable. The dura mater thus offers long-term optical access to functional endocrine tissue, facilitating stable intravital imaging under anesthesia-free, physiological conditions.</p>