CD649.2 is a responsive fluorescent probe specifically designed for Cu(II) detection, exhibiting selectivity for metals and oxidation states. It allows for the monitoring of intracellular active Cu(II) levels via live-cell confocal microscopy. This probe provides fundamental insights into the existence and regulation of active Cu(II) levels, such as identifying divalent metal transporter 1 (DMT1) as an importer of Cu(II), observing the increase of active Cu(II) under oxidative stress induced by glutathione depletion, and examining the interaction between increased active Cu(II) and decreased active Cu(I) caused by cancerous mutations that promote oxidative stress.