Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.fluorescent sensor | molecular imaging | mobile metals | transition metal signaling M etals are essential components of all living cells, and in many cases cells trigger and utilize dynamic metal movements for signaling purposes. Such processes are well established for alkali and alkaline earth metals like potassium, sodium, and calcium (1-3) but not for transition metals like copper and iron, which are traditionally studied for their roles as static cofactors in enzymes (4-6). We have initiated a program aimed at exploring the concept of mobile transition metals and their contributions to cell physiology and pathology, and in this context, brain neurons offer an attractive model for this purpose owing to their widespread use of potassium and sodium ion channels and calcium release for signaling events (7), as well as a high requirement for copper and iron to meet their steep oxidative demand (8-12). Indeed, the brain needs much higher levels of copper compared to other parts of the body under normal physiological conditions (9, 12), but at the same time mishandling of neuronal copper stores and subsequent oxidative stress and damage events are connected to a variety of neurodegenerative ailments, including Menkes and Wilson's diseases (13, 14), Alzheimer's disease (15-17), familial amyotrophic lateral sclerosis (18,19), and prionmediated encephalopathies (20,21). Previous work hints at the importance of exchangeable copper in neurophysiology, including observations of 64 Cu efflux from stimulated neurons (22, 23), export of Cu from isolated synaptosomes (24), and elevated susceptibility of neurons to excitotoxic insult with copper chelation (25), but none of these reports show direct, live-cell mon...