Calcium imaging is a method that was first developed in the mid-1970s yet kept developing until current days to allow accurate measurement of free calcium ions in tissues. This widely used method has provided significant advances to our understanding of cellular signal transduction, including the discovery of subcellular compartmentalization of neurons and astrocytes, the identification of multiple signaling pathways, and mapping the functional connectivity between astrocytes and neuronal networks. Here we describe a method for the loading and imaging of cell-permeable AM ester calcium-sensitive dyes for the in vitro measurement of free intracellular Ca2+ ions in acute brain slices.