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Increases in cytoplasmic free Ca²⁺ ions ([Ca²⁺]) are a critical signal in pancreatic islet beta-cells and are usually required for insulin secretion in response to glucose or other secretagogues. Changes in Ca²⁺, monitored using high-speed imaging across individual or multiple planes of the islet, can be used to explore the functional networks of beta-cells required for the precise regulation of insulin secretion. These networks are composed of functionally distinct beta-cell subpopulations: first-responders, highly connected hubs, and leader beta-cells, which initiate, connect, and dictate the pattern of spatially organized Ca²⁺ oscillations, respectively. Alterations in Ca²⁺ coordination among beta-cells contribute to defective insulin secretion, which underlies all forms of diabetes mellitus. Here, we provide a detailed protocol to perform Ca²⁺ imaging in isolated rodent islets, focusing on mouse islets expressing the genetic Ca²⁺ sensor, GCaMP6. We provide a step-by-step guide to evaluate general parameters of islet Ca²⁺dynamics, coordination, connectivity, and identification of specific functional subpopulations. This approach can be applied to investigate the role of Ca²⁺ dynamics and coordination in tissues where coordination is critical for normal function.