Analytical Methods for Imaging Metals in Biology: From Transition Metal Metabolism to Transition Metal Signaling.

The levels, localizations, and redox- and ligand-based speciations of copper and iron in biological systems change over time, as cells, tissues, and organisms grow, age, and respond to their environments. Techniques for visualizing total and/or labile metal pools can enable these changes to be mapped with spatial and temporal resolution and, thus, can aid in gaining insights into the roles of these redox-active metals over the wide range of time scales spanning signaling to metabolism to nutrition and aging. We emphasize the use of a combination of multiple techniques to address these exciting and open questions, as together they can provide a more holistic picture of transition metal dynamics, ranging from localization to oxidation state to bioavailability. Here, we highlight some recent representative examples of studies that combine multiple metal imaging methods to study and understand how copper and iron are sequestered and mobilized in biological systems in physiological and pathological situations. Accumulation of iron in tissues as a function of age has been known since the early 1900s, 193 , 194 but mechanisms by which iron acquisition contributes to senescence and death remain an active area of research. A combination of fluorescent probes and histochemical stains, along with advanced X-ray fluorescence and absorbance imaging techniques, has produced substantial progress toward addressing these questions. Recently, McColl, Bush, and co-workers examined iron accumulation and aging in C. elegans , specifically focusing on the role of ferritin in iron storage. 195 Using XFM, the investigators observed an accumulation of iron in old worms (12 days) compared to young adult worms (4 days), particularly in the intestinal cells ( Figure 8A ). Control measurements show no change in calcium observed in the same time frame. Perl’s stain showed an age-dependent increase in nonheme iron, revealing iron accumulation specifically in intestinal cell nuclei, dense inclusions in the head, and the germ nuclei of postreproductive adults. The punctate iron distribution was confirmed by high-resolution XFM in a subsequent study. 26 To determine whether the labile iron pool expanded with increased total iron, the investigators stained live worms with Calcein, which fluoresces less in the presence of labile iron. Indeed, Calcein fluorescence was lower in old worms than young worms, confirming an increase in labile iron with age ( Figure 8B ). 195 Having demonstrated the expansion of both total and labile iron pools with age, the same researchers examined the role of ferritin, the main iron storage protein, 111 during aging. Size exclusion chromatography experiments revealed that the fraction of iron bound to ferritin decreases as age increases, despite an overall increase in iron levels. While the labile iron pool consists mainly of Fe 2+ , iron is stored in ferritin as Fe 3+ ; therefore, a decrease in the fraction of ferritin-bound iron suggests that the Fe 2+ /Fe 3+ ratio may increase with age. 195 Relative levels of Fe 2+ and Fe 3+ oxidation states in live, hydrated, anaesthetized C. elegans were mapped using fluorescence XANES ( φ -XANES) microscopy, 195 at a radiation dose demonstrated to preserve sample structure ( Figure 8C ). 76 The iron K-edge position shifts to higher energies with oxidation, so its energy can be used to assess the relative fraction of Fe 2+ and Fe 3+ in a sample. 196 Interestingly, young wildtype worms had K-edge positions that corresponded to a mixture of Fe 2+ and Fe 3+ , whereas old wildtype worms had Kedge positions that were lower energy and thus shifted toward Fe 2+ . Taken together, these data suggest that the role of iron in aging is not simply defined by an overall increase in iron levels but includes a shift from Fe 3+ to Fe 2+ in vivo , linked to changes in ferritin loading. At shorter time scales within the span of minutes to hours, cells actively accumulate, store, mobilize, and efflux copper and iron under specific environmental conditions. Metal imaging experiments with high spatial resolution, using a variety of X-ray, mass spectrometry, and chemical probe methods, have played significant roles in identifying and characterizing intracellular structures for metal storage and mobilization. 26 , 162 , 197 , 198 To provide one representative example, the single-cell eukaryotic model organism Chlamydomonas reinhardtii accumulates copper under conditions of environmental zinc deprivation, as demonstrated by bulk ICPMS measurements. 199 Interestingly, this organism responds to zinc deprivation in a manner associated with intracellular copper deficiency by upregulating copper import machinery and downregulating the synthesis of proteins requiring copper cofactors such as plastocyanin. 70 The mismatch between (1) a measurable accumulation of bulk total copper and (2) a functional response characteristic of low intracellular copper suggested that the copper pools accumulating inside these cells might be sequestered into compartments where they are not accessible to the cell’s copper-sensing machinery. To address this question, pilot imaging studies using the small-molecule probe CS3 revealed an increase in fluorescent puncta in zinc-deprived cells, suggesting that copper accumulates in distinct subcellular structures under conditions of zinc deprivation ( Figure 9A ). 70 This increase in fluorescence was not observed using the control probe Ctrl-CS3. Further control experiments with copper supplementation or chelation, along with genetic manipulations of copper homeostasis machinery or lipid transport, all confirm that CS3 responds in this model in a copper-dependent fashion. These experiments led to the direct observation of copper and calcium accumulation in electron-dense structures, termed cuprosomes, using NanoSIMS ( Figure 9B ). Moreover, sequestration of copper in these intracellular cuprosomes is reversible. Upon zinc resupply, fluorescent puncta from CS3 staining started to decrease after 3 h, and the fluorescence signal became more evenly distributed throughout the cell by 24 h. NanoSIMS imaging also showed a decrease in copper at sites of copper accumulation following zinc resupply. Further NanoSIMS analysis with isotope labeling showed that, upon restoration of zinc, copper from these compartments is incorporated into newly synthesized plastocyanin preferentially over copper from extracellular media. Additionally, cells moved from low-zinc media to low-copper media had a growth advantage over cells moved from zinc-replete media to low-copper media, suggesting that the copper accumulated during zinc deprivation becomes bioavailable and may provide an evolutionary advantage under conditions of fluctuating nutrient availability. This example adds to the growing literature concerning the identification of new protein machineries that influence the storage and mobilization of copper in intracellular compartments, including CTR1, 200 CTR2, 162 , 201 ATP7B, 163 metallothionein, and Atox1. 197 In contrast to the wide acceptance of nonredox alkali and alkaline earth metals as mobile metal signals (e.g., Na + , K + , Ca 2+ ), the localization of redox-active transition metals like copper and iron has been thought to be highly restricted to buried protein active sites and other inaccessible stores in order to protect cells, tissues, and organisms against oxidative stress and damage. However, more recent findings have expanded this traditionally narrow view of metals in biology to a new paradigm of transition metal signaling. As signaling elements, copper and iron can be rapidly mobilized in response to external stimuli in order to convey information. The fast and reversible binding of these elements to proteins and other biological targets outside active sites influences the function of these targets in downstream signaling cascades. Our laboratory reported a foundational discovery in redox transition metal signaling by identifying fast copper relocalization within a cell, following an external stimulus, as shown using a primary neuronal cell model. 160 A combination of XFM and imaging using the small-molecule fluorescent probe CS3 revealed dynamic mobilization of copper within seconds from neuronal cell bodies to dendritic spines following depolarization of dissociated hippocampal neurons with potassium chloride ( Figure 10A ). XFM control experiments showed that zinc did not relocalize under the same conditions. Further experiments using both imaging methods established that copper mobilization following membrane depolarization was calcium-dependent, suggesting crosstalk between copper and canonical cell signaling pathways. This work provided direct imaging evidence that complemented older studies on bulk copper release from synaptosomes 202 , 203 and explants, 204 as well as the movement of copper-trafficking proteins in neurons. 205 – 207 Additionally, membrane-impermeable copper chelators disrupt neural function, 164 , 208 which provides evidence for a functional role for copper in neuronal signaling. 209 Indeed, a more recent study from our laboratory has characterized the functional significance of labile copper in neural circuits within intact tissue using fluorescent sensors for dual two-photon copper and calcium imaging ( Figure 10B ). The data show that copper is an endogenous regulator of spontaneous activity, a fundamental property of all neural circuits, by acting as a type of brake to avoid hyperactivity, via the Ctr1 copper ion channel and NMDA receptor. 164 The concept of copper as a representative example of a transition metal signal has been expanded beyond the brain by the identification of copper as an essential regulator of lipolysis, the breakdown of fat to control body weight and energy metabolism. 167 Dynamic fluxes of copper in stimulated adipocytes, as imaged by the copper-responsive fluorescent probe CSR1, reversibly inhibit the enzyme PDE3B, a phosphodiesterase responsible for shutting down the lipolysis pathway by breakdown of the second messenger cAMP. 167 Further biochemical studies characterized a key cysteine residue at an allosteric site outside the active site of PDE3B that modulates its copper-dependent function, linking transition metal signaling to a molecular target. These studies directly tie copper to cAMP/PDE, lipolysis, and metabolic processes related to obesity, and parallel studies have linked copper to other disease pathways such as cancer proliferation 210 – 212 and heart disease. 213 – 215 Interestingly, many of these disease pathways are interconnected. Indeed, obesity is a risk factor for diseases including diabetes, heart disease, cancer, and liver disease. Thus, these fields are ripe for further imaging studies to directly monitor fluctuations in copper in response to biological stimuli and to elucidate how copper may be used to transmit information at the cellular, tissue, and whole-organism levels.

Metals pervade all aspects of the central dogma of biology, as they are required for the synthesis of all DNA and RNA molecules, as well as the proper function of approximately half of all proteins. Because metals can neither be created nor destroyed in biological systems, metal localization is governed by complex acquisition and excretion systems. The ubiquity, necessity, and unique homeostasis of metals drive a desire to study these biomolecules, and the electronic structure and coordination chemistry of metals provide unique chemical signatures that can be harnessed by physical techniques and small molecule probes to map the contributions of metals to the dynamic blueprint of life. We have reviewed the growing collection of analytical methods to monitor both total and labile metal pools in their native biological contexts. These techniques, when used in concert, can provide complementary information on quantity, location, and oxidation state of a given metal over a wide range of length scales and time regimes, particularly spanning the shorter time scales of signaling to medium time scales of metabolism to longer time scales of nutrition and aging. A key principle is the open collaboration between chemistry and biology, where disparate communities will continue to work together to evaluate the best available set of chemical imaging tools for a given biological system and question. Multiple independent measurements and controls from both chemical and biological perspectives will push the field forward. Finally, an emerging theme of these investigations, applied to redox-active transition metals such as copper and iron, is the concept of transition metal signaling. In this burgeoning paradigm, these potent and reactive elements are not only viewed as static cofactors buried within protein active sites and other biomolecules to perform structural and catalytic chemistry, but they are also recognized as dynamic signals that can interact with targets outside active sites on faster time scales to affect function. The coming years are sure to illuminate more aspects of this bioinorganic chemistry and metallobiochemistry beyond the active site.

Analytical methods for assessing labile metal pools 118 , 119 can complement the suite of techniques for direct imaging of total metal pools. The labile metal pool consists of metal ions that are weakly bound to intracellular ligands, such that these ions can be rapidly removed or sequestered by competing metal chelators in the biological environment. Such metal pools may also undergo ligand exchange with fluorescent probes that respond to metal binding and/or reactivity with a change in fluorescence, enabling metal detection with spatial and temporal resolution. These probes can be reversible sensors or irreversible dosimeters. Desirable properties of an effective fluorescent metal probe include (1) high selectivity for the metal of interest, even in the presence of competing metals, other analytes in the cellular milieu, or local changes in pH, redox, and hydrophilicity/hydrophobicity, (2) a large turn-on increase or ratiometric wavelength shift in fluorescence to provide spatial information, in contrast to probes that turn off (i.e., disappear) in the presence of analyte, (3) compatibility with common microscopy laser lines and/or filter sets in terms of excitation/emission wavelengths, (4) visible, red-shifted spectral profiles to minimize sample photodamage and interference from native cellular autofluorescence in blue wavelengths, and (5) predictable localization in a given biological specimen. For redox-active metals such as iron and copper, an additional challenge is to avoid electron- and energy-transfer quenching pathways from transient odd-electron species that can arise from ground or excited states. We will restrict our discussion to probes for labile iron and copper pools that target the Fe(II) and Cu(I) oxidation states, which are dominant within the cell owing to the reducing intracellular environment. We focus on synthetic small-molecule reagents, which have potential for broad application to many cell, tissue, and organism models as they do not require transfection or other manipulations to be introduced into a specimen. At the same time, however, the complexity of biological systems means that there is no one-size-fits-all probe for all systems, and each chemical reagent has to be tested and validated with proper controls in each biological setting and application. Indeed, potential confounding factors and artifacts can include increases and shifts in fluorescence signals due to accumulation, relocalization, or aggregation of dyes. As such, studies that employ imaging of labile metal pools benefit from biological controls with genetic and/or pharmacological manipulation, as well as complementary direct metal imaging methods. Additionally, the pursuit of ratiometric indicators with an internal standard and/or control probes that enable disentangling of dye- versus receptor-dependent signal changes is highly encouraged. Fluorescent indicators fall into one of two basic categories: (1) recognition-based and (2) reaction-based (see Figure 4 ). Recognition-based probes respond to the reversible coordination of a metal to a receptor, whereas reaction-based probes bind a metal to trigger a chemical event that leads to a fluorescence change. Recognition-based detectors are valued for their reversibility but require careful matching of appropriate K d values in order to avoid stripping and redistributing tightly bound metal pools. Reaction-based indicators can be valuable for detecting small changes in metal levels when the reaction is catalytic with respect to the metal, allowing the amplification of signal and integration of signal over time. Such indicators do not permanently bind the metal and thereby avoid perturbing the labile metal pool or undergoing metal-induced fluorescence quenching; however, after the reaction, diffusion of the probe away from the metal leads to a loss of spatial information. Although a wide variety of fluorescent iron and copper probes have been reported in the literature, only a limited subset of these diverse candidates has been satisfactorily characterized in cells, tissues, or animals with comparative images where pharmacological or biological treatments induce metal excess or deficiency, and our discussion focuses on these reagents. Iron, the body’s most abundant transition metal element, presents unique challenges as an analyte, beyond the common challenges of imaging biological metals. This metal readily cycles between Fe 2+ and Fe 3+ under biological conditions, and although mounting evidence has suggested that the intracellular labile iron pool 120 consists mainly of Fe 2+ , 121 the redox activity of iron and its ability to adopt high- or low-spin configurations makes iron a potent potential fluorescence quencher by electron and energy transfer. Fe 2+ is also a weakly coordinating metal on the Irving-Williams series, so developing effective receptors that can selectively bind this ion over competing Cu 2+ , Ni 2+ , and Zn 2+ in particular, is difficult. During probe characterization, potential complications involving iron solubility 122 or uncontrolled Fenton oxidations must be considered. As such, interest in visualizing Fe 2+ in living systems is high, but developing selective and sensitive Fe 2+ -responsive probes remains a significant challenge. Recognition-based fluorescent sensors for turn-on detection of Fe 2+ remain elusive, but several turn-off sensors have been employed to observe changes in labile Fe 2+ levels in cell lysates, live cells, and even model organisms. The two most commonly used sensors, based on a fluorescein scaffold, are the commercially available Calcein and Phen Green SK dyes ( Figure 5 ). Neither sensor shows high iron selectivity, as Calcein responds strongly to Cu 2+ , Co 2+ , and Ni 2+ (>95% fluorescence decrease for each), 123 and Phen Greek SK responds strongly to Cu + and Cu 2+ (97% and 70% decrease, respectively). 124 Oxidation state specificity for Fe 2+ over Fe 3+ is also modest, as Calcein shows nearly quantitative fluorescence quenching in the presence of Fe 2+ but 40–50% quenching in the presence of an equivalent amount of Fe 3+ . Despite the limited selectivity of Calcein and Phen Greek SK for Fe 2+ , iron-specific chelators can be used with these probes to identify changes in labile iron pools. Additional Fe 2+ sensors include the pyridinone-based indicator CP655 ( Figure 5 ) that exhibits improved selectivity for Fe 2+ over other cations, with only Cu 2+ presenting mild cross-recognition (42% decrease). 125 However, CP655 is not selective for Fe 2+ over Fe 3+ and also shows pH sensitivity. Nevertheless, this reagent has been employed to probe labile iron with uniform cellular staining. 126 Finally, RPA, RDA, and PIRO ( Figure 5 ) are rhodamine-based fluorescent iron sensors that localize to the mitochondria owing to the positive charge delocalized over the fluorophore structure. 127 , 128 This set of probes displays decreasing affinity for Fe 2+ (RPA > RDA > PIRO), enabling visualization of endogenous iron (with RPA and RDA) or exogenous iron (with PIRO) depending on the biological situation. Each of the probes recognizes Cu 2+ to some extent (RPA, 73% decrease; RDA, 87%; PIRO, 27%) but has good selectivity over other metals. The rhodamine-based iron sensors have identified a rise in mitochondrial labile iron when heme synthesis is blocked, with a control rhodamine 123 dye showing that mitochondrial membrane potential is not disrupted under these conditions. The growing palette of chemodosimeters for Fe 2+ detection exploits the potent redox activity of this metal ion. A variety of mechanisms, including N-oxide deoxygenation, TEMPO radical reduction, oxygen-dependent oxidative C–O bond cleavage, and endoperoxide cleavage, have been reported. RhoNox-1 ( Figure 5 ) was the first reaction-based Fe 2+ probe to be used in a cellular system. 129 This rhodamine-based probe relies on the ability of Fe 2+ to selectively deoxygenate an N-oxide, converting it to a tertiary amine with a concomitant 30-fold fluorescence turn-on response. RhoNox-1 derivatives such as HMRhoNox-M ( Figure 5 ), which displays a more stable pH profile and a 60-fold turn-on in fluorescence, has been used to visualize iron uptake via transferrin endocytosis 130 and iron accumulation in ovarian endometriosis. 131 A related rhodamine probe, Rh-T ( Figure 5 ), contains a pendant paramagnetic TEMPO group that quenches fluorescence. 132 , 133 Fe 2+ reduces the TEMPO radical to a diamagnetic hydroxylamine, resulting in a 2.5-fold fluorescence turn-on with good selectivity for Fe 2+ over other metal cations, as well as cellular reductants such as ascorbate and NADH. In human fibroblasts, Rh-T responds to the addition of exogenous iron, but its signal does not decrease in response to iron chelation. Our laboratory published Iron Probe-1 (IP-1, Figure 5 ), a first-generation Fe 2+ fluorescent indicator inspired by the oxidative reactivity of mononuclear nonheme iron enzymes. 134 In the presence of Fe 2+ and O 2 , IP-1 undergoes a C–O bond-cleavage reaction to release a reduced fluorescein alcohol derivative, resulting in a 6-fold fluorescence turn-on with high selectivity over competing metal ions in the cell. Only free Co 2+ elicits a response from IP-1 in vitro , but more importantly, the probe does not respond to cyanocobalamin (vitamin B 12 ), the biologically relevant form of cobalt in cells. In HepG2/C3A liver cells, IP-1 localizes to the lysosome, where it is able to sense both increases and decreases in iron levels from iron supplementation and chelation, respectively. Additionally, IP-1 was capable of detecting increases in labile iron following treatment with hepcidin or vitamin C, two natural compounds known to increase labile iron stores. To improve upon the 3-component reaction of IP-1 and provide a direct reaction-based detector for Fe 2+ , we recently presented FIP-1 ( Figure 5 ), a unique ratiometric fluorescent indicator for this metal ion. FIP-1 is a FRET-based probe that uses an endoperoxide trigger 135 to cleave a linker between 5-aminomethyl coumarin and fluorescein in the presence of Fe 2+. 136 This direct and oxygen-independent 2-component reaction proceeds rapidly in the presence of Fe 2+ and results in a 2-fold FRET change. Moreover, FIP-1 distributes evenly within cells and can clearly distinguish both increases and decreases in endogenous labile iron pools. Using FIP-1, we demonstrated that cancer cells possess higher levels of labile iron stores compared to noncancerous cells. Finally, we provided the first evidence of elevations in labile iron during the induction of ferroptosis, 136 a form of iron-dependent cell death. 137 A related puromycin-based endoperoxide probe (Trx-Puro, Figure 5 ) reveals that overexpression of ferritin or ferroportin, iron storage and iron export proteins, respectively, decreases labile iron stores in a variety of cancer cell models. 138 Like iron, copper can cycle between two oxidation states under biological conditions, Cu + and Cu 2+ , presenting a selectivity challenge for probe design and characterization. Previous reports provide evidence that Cu + is the dominant intracellular copper oxidation state for labile pools, 139 , 140 although Cu + is prone to disproportionation in water, which requires stabilization by appropriate ligands. 141 Additionally, both oxidation states of copper are capable of quenching fluorescence, making the design of recognition-based probes particularly challenging as charge-transfer processes can generate transient Cu + and Cu 2+ species. As the field of fluorescence-based Cu + probes has been thoroughly reviewed, 119 , 142 – 144 we focus our discussion on probes that have been employed for biological application. The first sensor for Cu + was introduced by Fahrni and co-workers in 2005. 139 Using a triarylpyrazoline dye and macrocyclic thioether copper-binding motif, CTAP-1 gave a 4.6-fold fluorescent turn-on in the presence of excess Cu + , with no response or interference from other biologically relevant cations ( Figure 6 ). In NIH 3T3 fibroblasts, CTAP-1 showed a significant fluorescence increase in cells cultured in high-copper media compared to basal media. Additionally, its signal showed good agreement with the subcellular distribution of total copper observed by XFM. Subsequent careful studies elucidated photophysical properties to improve signal-to-noise responses 145 – 147 and provide updated CTAP-2 and CTAP-3 versions with improved hydrophilicity ( Figure 6 ). 148 , 149 In particular, CTAP-2, bearing four pendant hydroxyl groups on the thioether macrocycle, was capable of detecting the metalated Atox1 copper metallochaperone on a gel, and CTAP-3, bearing both hydroxyl and sulfonate groups, dissolves directly in water with no nanoparticle formation. In parallel studies, our laboratory developed the first fluorescent copper sensors with visible excitation and emission profiles, which have enabled the study of copper homeostasis in a broad range of biological models used in our work as well as independent investigations by others. The first-generation BODIPY-based copper sensor, Coppersensor-1 (CS1, Figure 6 ), features a bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine (BETA) receptor 150 , 151 and shows high selectivity for Cu + over all biologically relevant cations, with a 10-fold turn-on in the presence of Cu + in vitro . Initial studies showed that CS1 can identify copper-loaded HEK 293 cells compared to control cells, and this work was validated by another study 152 that also showed that this first-generation probe has different localization patterns in two other cell lines, M17 and U87MG. It is not surprising, with the complexity of biological systems, that there is not a one-size-fits-all chemical tool for all applications; as such, it is critical to implement both chemical and biological controls when using a given chemical probe for a given biological model. Indeed, with proper controls in place, CS1 has been employed as a pilot screening tool for assessing fluctuations in labile copper pools in bacteria, 153 yeast, 154 – 156 plant, 157 and mammalian systems. 158 Inspired by work by Nagano on treating fluorescent sensors as electron-transfer cassettes, 159 we developed a next-generation Coppersensor-3 (CS3, Figure 6 ) probe by replacing the fluoro substituents on the BODIPY core with methoxy substituents to improve its brightness (Φ = 0.40 for CS3 vs Φ = 0.13 for CS1) and signal-to-noise response to Cu + (75-fold turn-on for CS3 vs 10-fold for CS1). 160 Notably, the more electron-rich BODIPY core also manifests itself in a tighter Cu + K d for CS3 (9 × 10 −14 M vs 3 × 10 −12 M for CS1). The combination of higher optical brightness, greater turn-on response to Cu + , and tighter K d for CS3 has enabled its use for assessing basal pools of labile copper in a variety of cell types, including neurons, 160 tumor cells, 161 mouse fibroblasts, 162 liver cells, 163 and algae. 70 These studies are bolstered by genetic and pharmacological controls as well as independent measures of the total copper pool. Biological systems are complex mixtures of proteins, nucleic acids, glycans, lipids, and other organic species. Therefore, a balance between the hydrophobicity and hydrophilicity of any probe must be met for the use of a given probe for a given application. Indeed, in the case of copper sensors, similar to CTAP-1, BODIPY-based CS1 and CS3 do not homogeneously stain cells and thus are best suited for use in some biological models but not in others. In an effort to discriminate between dye-dependent fluorescent changes and metal-dependent fluorescent changes, we have introduced the concept of “synthetic mutagenesis” to create matched control probes. A first example is the development of Control Coppersensor-3 (Ctrl-CS3, Figure 6 ), which utilizes the same methoxy BODIPY core as CS3, but where the thioether sulfurs are replaced by isosteric carbons, which is akin to a methionine-to-alanine switch. 70 Thus, Ctl-CS3 cannot bind to copper and does not turn on in the presence of Cu + . In Chlamydomonas reinhardtii , CS3 and Ctl-CS3 were used in pilot screening studies, in conjunction with direct metal imaging techniques, to identify the accumulation of copper in subcellular vesicular compartments, termed cuprosomes; the fluorescence of CS3 increased in the vesicles of experimentally treated cells compared to control cells, but the fluorescence of Ctl-CS3 did not. 70 Neither probe responded to mutant algae with lipid-trafficking deficiencies, showing that fluorescent changes were not due to changes in the hydrophobicity of subcellular environments. Our hope is that continued development of control probes to use in conjunction with analyte-responsive probes will help with the interpretation of imaging data using such reagents. To expand the palette of fluorescent copper probes to more hydrophilic cores with the goal of improving their use in more hydrophobic environments, such as thicker tissue and animal specimens, our laboratory reported a first-generation Copper Rhodol series: CR1–CR5. 164 The most responsive of these sensors, Copper Rhodol 3 (CR3, Figure 6 ), gave a 13-fold turn-on response to Cu + . Replacement of the methyl substituent on the receptor-bearing aryl ring with a bulkier, more electron-withdrawing fluoromethyl substituent, gave Copper Fluor 3 (CF3, Figure 6 ) with a 40-fold turn-on response to Cu + . Partition coefficient measurements demonstrated that CR3 and CF3 were significantly more hydrophilic (log D = 0.96 and 1.15, respectively) than the BODIPY-based CS3 (log D = 3.46). Additionally, both CR3 and CF3 responded to copper selectively in the presence of model liposomes, proteins, glutathione, and cell lysates, whereas control probes based on these scaffolds (Ctl-CR3 and Ctl-CF3, Figure 6 ) did not respond to copper under similar conditions. These new reagents helped to identify an exchangeable pool of copper in developing hippocampal neurons and retinal slices, which regulates normal spontaneous activity in neural circuits. 164 Replacement of the oxygen atom on the rhodol core with a silicon isostere 165 , 166 led to the development of Copper Silicon Rhodol-1 (CSR1, Figure 6 ), a highly photostable fluorescent copper sensor that enables imaging of changes in copper pools in the same sample over long periods of time ( Figure 6 ). 167 CSR1 retains a selective and sensitive response to Cu + (12-fold turn-on) on a hydrophilic probe (log D = 1.15) and was successfully used to monitor changes in labile copper pools in adipocytes, where it stained the cytosol but not lipid droplets. CSR1 discriminated cells pretreated with copper, chelator or vehicle, and it responded to on-stage addition of the membrane-permeable copper chelator, tris((ethylthio)ethyl)-amine (TEMEA). Finally, CSR1 revealed a decrease in labile copper in adipocytes upon stimulation of the beta-adrenergic receptor, concomitant with an increase in lipolysis. Fluorescence from the control probe Ctl-CSR1 ( Figure 6 ) remained stable during parallel experiments, demonstrating the copper-specificity of CSR1 fluorescence in adipocytes. With these pilot imaging studies in hand, we went on to demonstrate that copper is an endogenous modulator of lipolysis through a cAMP signaling cascade where copper acts at the level of the cysteine 768 residue to reversibly inhibit the activity of phosphodiesterase PDE3B. 167 The toolbox of fluorescent copper sensors continues to expand, including sensors with near-IR optical profiles for use in thicker tissue and whole-animal settings, as well as ratiometric and organelle-targeted sensors. For copper sensing in thicker tissue, ACu1 is a 2-photon probe by Cho and coworkers that excites at 750 nm in 2-photon mode (1-photon mode, 365 nm) ( Figure 6 ). 168 Localizing to both mitochondria and Golgi, ACu1 has been used to visualize copper in live hippocampal slices from rats. Additionally, Wan and co-workers published a Cy7 Cu + sensor using the BETA receptor (structure 3 in Figure 6 ), which was used to visualize copper addition and ascorbate-triggered copper mobilization in MG63 osteosarcoma cells. 169 Our laboratory developed a Cy7 Cu + sensor, Coppersensor 790 acetoxymethyl ester (CS790AM, Figure 6 ), which enabled the first fluorescence imaging of labile copper pools in living mice. 170 CS790AM displays a 17-fold turn-on to copper with a highly red-shifted optical profile ( λ abs = 760 nm, λ em = 790 nm). When injected into mice, CS790AM revealed both increased copper from copper injection and decreased copper from injection of the copper-specific chelator ATN-224, the FDA-approved choline salt of tetrathiomolyb-date. Additionally, CS790AM could discriminate between wildtype mice and Wilson’s disease model mice, which lack the copper exporter ATP7B. Compared to wildtype, both the abdomens and isolated livers of ATP7B −/− mice exhibited higher fluorescence signal from CS790AM, indicating an accumulation of copper, which was confirmed by bulk ICPMS and online LC–MS/AA measurements on digested liver tissue. 170 To image labile copper pools at the subcellular level, our laboratory reported Mitochondrial Coppersensor-1 (Mito-CS1, Figure 6 ), 171 the first organelle-targetable copper sensor, by appending a triphenylphosphonium tag 172 onto an asymmetric BODIPY scaffold 173 as a cationic, lipophilic tag to localize the probe to the mitochondria based on mitochondrial membrane potential. Mito-CS1, in conjunction with other biochemical assays, revealed that cells prioritize mitochondria for copper homeostasis over other cellular compartments. This reagent enabled observation of expansion and depletion of the mitochondrial copper pool by copper supplementation and chelation. Interestingly, comparative studies in fibroblasts lacking the mitochondrial copper metallochaperones SCO1 and SCO2 and the copper export pump ATP7A showing that total and labile mitochondrial copper pools remain constant even in situations where whole-cell copper pools are altered. Important control experiments with the cationic dye rhodamine 123 demonstrated that mitochondrial membrane potential was not altered due to copper treatment or deletion of SCO1 or SCO2. A related water-soluble derivative of CS1, OBEP-CS1 ( Figure 6 ), bears an alkylpyridinium group to drive it to the mitochondria in live cells but turns off in response to Cu +. 174 Ratiometric probes are highly valued for their intrinsic internal standard that can correct for variations in dye localization and other experimental imaging conditions. Attachment of the BETA receptor to a naphthyl fluorophore yielded Naphthyl-CS1 ( Figure 6 ), which localizes to both mitochondria and the Golgi apparatus in SH-SY5Y cells and was able to sense changes in copper status with copper supplementation. 175 InCCu1, a ratiometric mitochondrial copper sensor developed by New and co-workers ( Figure 6 ), 176 can specifically monitor increases in mitochondrial copper upon supplementation. Moreover, InCCu1 was used to suggest that cisplatin interferes with copper transport to the mitochondria upon copper supplementation. Finally, Dns-LLC, a Golgi-targeted peptide-based sensor ( Figure 6 ), 177 shows a 12-fold turn-on in the presence of copper with an exceptionally tight binding affinity (12 fM); it responds to increases and decreases in Golgi copper levels following copper supplementation or chelation, respectively. Reaction-based approaches have proved useful for the development of fluorescent probes that go beyond traditional designs based on lock-and-key recognition. 143 , 178 – 181 An elegant bioinspired approach to reaction-based Cu + detection based on oxidative cleavage of the tetradentate ligand, tris[(2-pyridyl)methyl]amine (TPA), was reported by Taki and Yamamoto in 2010 ( Figure 7 ). 182 Upon Cu + binding to the probe FluTPA1, oxidative C–O bond cleavage separates the TPA fragment from the fluorophore and releases the fluorescent dye with a 100-fold turn-on. FluTPA1 and its membrane-permeable FluTPA2 analogue, based on Tokyo Green, 183 show good selectivity over other metal cations, as well as biological oxidants, including hydrogen peroxide, hypochlorite, and hydroxyl radical. FluTPA2 exhibits a notable fluorescence turn-on in HeLa cells after treatment with copper. 182 The TPA ligand has subsequently been used to cage 2-(2′-hydroxyphenyl) benzothiazole, 184 coumarin, 185 xanthone, 186 resorufin, 187 cyanine-quinone, 188 and imino-coumarin 189 ( Figure 7 ), although only the latter three have been applied to cells. Additionally, a mitochondrially targeted reaction-based Cu + probe, RdlTPA-TPP ( Figure 7 ), has been developed using the TPA ligand, circumventing the localization problem associated with this probe. 190 We have expanded the scope of oxidative cleavage reactions to detect cobalt 191 and iron. 134 In addition, we have recently utilized the TPA trigger to develop the first bioluminescent probe for Cu + , Copper Caged Luciferin-1 (CCL-1), which enables the imaging of labile copper levels in cells and living animals. 192 CCL-1 responds selectively to Cu + in vivo with high signal-to-noise, and the combination of a small-molecule caged substrate and genetically encoded enzymatic reporter affords a platform for longitudinal imaging of the same living animal over time with cell- and tissue-specific resolution. In conjunction with biochemical and physiological assays, CCL-1 revealed a liver-specific copper deficiency that accompanies the onset of metabolic symptoms of glucose intolerance and weight gain in a diet-induced mouse model of nonalcoholic fatty liver disease (NAFLD).