The dynamic chemical diversity of elements, ions and molecules that form the basis of life offers both a challenge and an opportunity for study. Small-molecule fluorescent probes can make use of selective, bioorthogonal chemistries to report on specific analytes in cells and in more complex biological specimens. These probes offer powerful reagents to interrogate the physiology and pathology of reactive chemical species in their native environments with minimal perturbation to living systems. This Review presents a survey of tools and tactics for using such probes to detect biologically important chemical analytes. We highlight design criteria for effective chemical tools for use in biological applications as well as gaps for future exploration.

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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...

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metallation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu+ pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wildtype controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.