An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin

Oxidative stress and Cu²⁺ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu²⁺, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu²⁺-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu²⁺ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn²⁺, Mg²⁺, and Ca²⁺ does not affect Cu²⁺ binding, indicating selectivity of the Cu²⁺-binding site(s) in α-crystallins. Cu²⁺ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu²⁺ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu²⁺ binding. α-Crystallins rescue amyloid beta peptide, Aβ_1-40, from Cu²⁺-induced aggregation in vitro. α-Crystallins inhibit Cu²⁺-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu²⁺-binding protein, does not inhibit this oxidation process significantly. We find that the Cu²⁺-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu²⁺ binding and redox silencing of Cu²⁺ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu²⁺ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.     

紫花苜蓿对铜胁迫生理响应的傅里叶变换红外光谱法研究

为阐明紫花苜蓿(Medicago sativa L.)对铜胁迫的耐性机理,采用准确度好、分辨率高和简便快捷的傅里叶变换红外光谱法(FTIR)研究在不同铜浓度(0、1、5、20、100 mol/L)处理时紫花苜蓿根、茎、叶化学组分的变化。结果表明:随着铜处理浓度的增加,紫花苜蓿根、茎、叶生物量变化不大。其根组织在2924 cm~(-1)处峰高处呈现出先下降后上升的趋势,反映了在低铜(5 mol/L)处理条件下紫花苜蓿分泌的有机酸不断螯合Cu,造成羧酸0-H的减少,但随着Cu含量的升高,其羧酸螯合力变弱,有机酸含量渐渐升高;根组织在1381 cm~(-1)处峰高先下降后上升,反映了含油脂化合物含量先下降后升高。可能与植物在细胞壁结构上增强抗逆性有一定关系,即低Cu处理下细胞壁可能通过提高阳离子交换能力(CEC),增强了耐Cu性;茎组织在2924,1643,1381,1064 cm~(-1)等处峰高无明显变化;叶组织所有峰值在低浓度(5 mol/L)Cu处理下变化不明显,高浓度(5 moL/L)Cu处理下所有峰值先升后降,随着这可能与可溶性糖及可溶性蛋白质等物质含量都呈现先升后降的趋势有关。这表明紫花苜蓿通过根部有机酸含量的变化和提高细胞壁阳离子交换能力,将吸收的Cu大部分积累在根部,阻止Cu向地上部分运输,有效地保护了植物地上部分组织。     

A rhodamine‐triazole fluorescent chemodosimeter for Cu2+ detection and its application in bioimaging

A rhodamine‐based fluorescent chemodosimeter rhodamine hydrazide‐triazole (RHT) tethered with a triazole moiety was developed for Cu²⁺ detection. In aqueous medium, the RHT probe exhibited high selectivity and sensitivity toward Cu²⁺ among other metal ions. The addition of Cu²⁺ triggered a fluorescence emission of RHT by 384‐fold (Φ = 0.33) based on a ring‐opening process and a subsequent hydrolysis reaction. Moreover, RHT also showed a selective colorimetric response toward Cu²⁺ from colorless solution to pink, readily observed with the naked eye. The limit of detection of RHT for Cu²⁺ was calculated to be 1 nM (0.06 ppb). RHT was successfully demonstrated to detect Cu²⁺ in Chang liver cells by confocal fluorescence microscopy.     

Biosorption of Pb<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> by Non-Living Biomass of <Emphasis Type="Italic">Spirulina</Emphasis> sp.

Removal of heavy metals (Pb²⁺, Zn²⁺) from aqueous solution by dried biomass of Spirulina sp. was investigated. Spirulina rapidly adsorbed appreciable amount of lead and zinc from the aqueous solutions within 15 min of initial contact with the metal solution and exhibited high sequestration of lead and zinc at low equilibrium concentrations. The specific adsorption of both Pb²⁺ and Zn²⁺ increased at low concentration and decreased when biomass concentration exceeded 0.1 g l⁻¹. The binding of lead followed Freundlich model of kinetics where as zinc supported Langmuir isotherm for adsorption with their r ² values of 0.9659 and 0.8723 respectively. The adsorption was strongly pH dependent as the maximum lead biosorption occurred at pH 4 and 10 whereas Zn²⁺ adsorption was at pH 8 and 10.     

A N‐(2‐hydroxyethyl)piperazine dangled 2,5‐diphenyl‐1,3,4‐oxadiazole‐based fluorescent sensor for selective relay recognition of Cu2+ and sulfide in water

A new 2,5‐diphenyl‐1,3,4‐oxadiazole‐based derivative (L) was synthesized and applied as a highly selective and sensitive fluorescent sensor for relay recognition of Cu²⁺ and S^2? in water (Tris–HCl 10 mM, pH = 7.0) solution. L exhibits an excellent selectivity to Cu²⁺ over other examined metal ions with a prominent fluorescence ‘turn‐off’ at 392 nm. L interacts with Cu²⁺ through a 1:2 binding stoichiometry with a detection limit of 4.8 × 10^–7 M. The on‐site formed L–2Cu²⁺ complex exhibits excellent selectivity to S^2? with a fluorescence ‘off–on’ response via a Cu²⁺ displacement approach. Copyright © 2016 John Wiley & Sons, Ltd.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

A zinc (II) complex comprising an aminoethyl-nitropyridine-derived N,N,O-donor Schiff base ligand serves as an efficient ON–OFF probe for Cu(II)

A new fluorescent zinc (II) complex-based probe 1 encompassing a Schiff's base (E)-2-methoxy-6-((2-[5-nitropyridin-2-ylamino]ethylimino)methyl)phenol ( HL ) was designed, synthesized, and used for the highly selective detection of Cu²⁺. Ligand HL and complex 1 were characterized using various spectroscopic techniques such as ¹H, ¹³C-NMR, and FTIR spectroscopy, high-resolution mass spectronomy (HRMS), UV/visible light spectroscopy, and fluorescence studies. Ligand HL did not exhibit any considerable change in fluorescence in the presence of various cations. Notably, its Zn(II) complex 1 exhibited highly selective ‘TURN-OFF’ fluorescence signalling towards Cu²⁺ that remained uninterrupted with competing analytes. Probe 1 interacted with Cu²⁺ in 1:2 (1:Cu²⁺) stoichiometry as estimated through a Job's plot. Moreover, the selectivity of 1 was further confirmed through the interaction of the 1 + Cu²⁺ complex with some possible interfering metal ions inducing an insignificant response. Additionally, the association and quenching constant were determined to be 3.30 × 10⁴ M⁻¹ and 0.21 × 10⁵ M⁻¹ through the Benesi–Hildebrand method and Stern–Volmer plot, respectively.     

Ratiometric fluorescent detection of Cu2+ based on dual‐emission ZIF‐8@rhodamine‐B nanocomposites

Zeolitic imidazolate framework‐8 (ZIF‐8) loading rhodamine‐B (ZIF‐8@rhodamine‐B) nanocomposites was proposed and used as ratiometric fluorescent sensor to detect copper(II) ion (Cu²⁺). Scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray powder diffraction, nitrogen adsorption/desorption isotherms and fluorescence emission spectroscopy were employed to characterize the ZIF‐8@rhodamine‐B nanocomposites. The results showed the rhodamine‐B was successfully assembled on ZIF‐8 based on the π‐π interaction and the hydrogen bond between the nitrogen atom of ZIF‐8 and –COOH of rhodamine‐B. The as‐obtained ZIF‐8@rhodamine‐B nanocomposites were octahedron with size about 150–200 nm, had good water dispersion, and exhibited the characteristic fluorescence emission of ZIF‐8 at 335 nm and rhodamine‐B at 575 nm. The Cu²⁺ could quench fluorescence of ZIF‐8 rather than rhodamine‐B. The ZIF‐8 not only acted as the template to assemble rhodamine‐B, but also was employed as the signal fluorescence together with the fluorescence of rhodamine‐B as the reference to construct a novel ratiometric fluorescent sensor to detect Cu²⁺. The resulted ZIF‐8@rhodamine‐B nanocomposite fluorescence probe showed good linear range (68.4 nM to 125 μM) with a low detection limit (22.8 nM) for Cu²⁺ combined with good sensitivity and selectivity. The work also provides a better way to design ratiometric fluorescent sensors from ZIF‐8 and other fluorescent molecules.     

Selective binding of Ni2+ and Cu2+ metal ions with naphthazarin esters isolated from Arnebia euchroma

Naphthazarin esters (C1–C4) isolated from the roots of Arnebia euchroma are found as skilled dual chemosensors for Ni²⁺ and Cu²⁺ among Pb²⁺, Na²⁺, K²⁺, Hg²⁺, Mg²⁺, and Ca²⁺ metal ions. C1–C4 esters exhibited a red shift of 54 nm with Ni²⁺ and 30 nm with Cu²⁺ metal ions in absorption. There is a formation of red-shifted bands between 517 and 613 nm in the absorption spectrum of C1–C4 sensors on binding with Ni²⁺ and Cu²⁺ ions. The addition of Ni²⁺ and Cu²⁺ ions to sensors C1–C4 stimulates a remarkable color change from reddish pink to purple and light blue, respectively. These color changes can be identified with the naked eye. The significant downfield shifts of CO and OH peaks in nuclear magnetic resonance (NMR) spectrum confirm the chelation as binding mechanism. With ultraviolet–visble and NMR studies, it is found that C1–C4 esters possessed notable selectivity and sensitivity toward Ni²⁺ and Cu²⁺ over other metal ions.     

Calix[4]arene based molecular sensors with pyrene as fluoregenic unit: Effect of solvent in ion selectivity and colorimetric detection of fluoride

Fluorescent molecular sensors having excimer emission property have been designed and synthesised incorporating calix[4]arene derivatives in cone and 1,3-alternate conformation as ionophore and two pyrene moieties at close proximity as fluorophore. They exhibit strong excimer emission around 515 nm, which is used to monitor interaction of metal ions with the ionophores. Ion-binding study of these fluoroionophore has been investigated in acetonitrile-chloroform and THF-H₂O with a wide range of cations and anions and the recognition process is monitored by luminescence, UV-Vis and ¹H NMR (for F⁻) spectral changes. The present study demonstrated profound influence of solvent in ion selectivity, in acetonitrile-chloroform they formed complexes with Hg²⁺, Pb²⁺, Cu²⁺ and Ni²⁺, whereas in THF-H₂O they exhibit selectivity only for Cu²⁺. In the case of anions, selectivity for only F⁻ with color change is observed. Composition of the complexes formed was determined from mass spectrometry and the binding constants were determined from fluorescence titration data. The reasons for formation of excimer emission, quenching of it in presence of certain metal ions, role of solvent in selectivity and energy/electron transfer process involved in the ion-recognition event have been discussed on the basis of experimental data.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Selectivity mechanisms in MscS-like channels: From structure to function

The E. coli mechanosensitive (MS) channel of small conductance (EcMscS) is the prototype of a diverse family of channels present in all domains of life. While EcMscS has been extensively studied, recent developments show that MscS may display some characteristics not widely conserved in this protein subfamily. With numerous members now electrophysiologically characterized, this subfamily of channels displays a breadth of ion selectivity with both anion and cation selective members. The selectivity of these channels may be relatively weak in comparison to voltage-gated channels but their selectivity mechanisms represent great novelty. Recent studies have identified unexpected residues important for selectivity in these homologs revealing different selectivity mechanisms than those employed by voltage gated K⁺, Na⁺, Ca²⁺ and Cl^- channels whose selectivity filters are housed within their transmembrane pores. This commentary looks at what is currently known about MscS subfamily selectivity and begins to unravel the potential physiological relevance of these differences.     

A rhodamine-based "turn-on" fluorescent chemodosimeter for Cu2+ and its application in living cell imaging

A new fluorescent probe 1, N-(Rhodamine-6G)lactam-hydrazinecarbothioamide, was synthesized as a fluorescent and colorimetric chemodosimeter in aqueous solution for Cu²⁺. Following Cu²⁺-promoted ring opening, redox and hydrolysis reactions, comparable amplifications of absorption and fluorescence signals were observed upon addition of Cu²⁺; this suggests that chemodosimeter 1 effectively avoided the fluorescence quenching caused by the paramagnetic nature of Cu²⁺. Importantly, 1 can selectively recognize Cu²⁺ in aqueous media in the presence of other trace metal ions in organisms, abundant cellular cations and the prevalent toxic metal ions in the environment with high sensitivity (detection limit < 3 ppb) and a rapid response time (< 2 min). In addition, the biological imaging study has demonstrated that 1 can detect Cu²⁺ in the living cells.     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

A new benzimidazole‐based selective and sensitive ‘on–off’ fluorescence chemosensor for Cu2+ ions and application in cellular bioimaging

Two new twinborn benzimidazole derivates ( L and A ), which bonded pyridine via the ester space on the opposite and adjacent positions of the benzene ring of benzimidazole respectively, were designed and synthesized. Compound L displayed fluorescence quenching response only towards copper(II) ions (Cu²⁺) in acetonitrile solution with high selectivity and sensitivity. However, compound A presented ‘on–off’ fluorescence response towards a wide range of metal ions to different degrees and did not have selectivity. Furthermore, compound L formed a 1:1 complex with Cu²⁺ and the binding constant between sensor L and Cu²⁺ was high at 6.02 × 10⁴ M^?1. Job's plot, mass spectra, IR spectra, ¹H‐NMR titration and density functional theory (DFT) calculations demonstrated the formation of a 1:1 complex between L and Cu²⁺. Chemosensor L displayed a low limit of detection (3.05 × 10^?6 M) and fast response time (15 s) to Cu²⁺. The Stern–Volmer analysis illustrated that the fluorescence quenching agreed with the static quenching mode. In addition, the obvious difference of L within HepG2 cells in the presence and absence of Cu²⁺ indicated L had the recognition capability for Cu²⁺ in living cells.     

Transient fluctuations of intracellular zinc ions in cell proliferation

Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn²⁺). By employing a fluorescent Zn²⁺ probe, FluoZin-3 acetoxymethyl ester, intracellular Zn²⁺ concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn²⁺ concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn²⁺ concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn²⁺ concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn²⁺ concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn²⁺ concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn²⁺ concentrations, suggest a role for Zn²⁺ in the control of the cell cycle. Interventions targeted at these picomolar Zn²⁺ fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.     

Terbium (III) coordination polymer–copper (II) compound as fluorescent probe for time‐resolved fluorescence ‘turn‐on’ detection of hydrogen sulfide

With recognition of the biological importance of hydrogen sulfide (H₂S), we present a simple and effective fluorescent probe for H₂S using a Tb³⁺ coordination polymer–Cu²⁺ compound (DPA/Tb/G–Cu²⁺). Dipicolinic acid (DPA) and guanosine (G) can coordinate with Tb³⁺ to form a macromolecular coordination polymer (DPA/Tb/G). DPA/Tb/G specifically binds to Cu²⁺ in the presence of coexisting cations, and obvious fluorescence quenching is observed. The quenched fluorescence can be exclusively recovered upon the addition of sulfide, which is measured in the mode of time‐resolved fluorescence. The fluorescence intensities of the DPA/Tb/G–Cu²⁺ compound enhance linearly with increasing sulfide concentrations from 1 to 30 μM. The detection limit for sulfide in aqueous solution is estimated to be 0.3 μM (at 3σ). The DPA/Tb/G–Cu²⁺ compound was successfully applied to sense H₂S in human serum samples and exhibited a satisfactory result. It displays some desirable properties, such as fast detection procedure, high selectivity and excellent sensitivity. This method is very promising to be utilized for practical detection of H₂S in biological and environmental samples.     

Recent developments in the creation of a single molecular sensing tool for ternary iron (III), chromium (III), aluminium (III) ionic species: A review

Rational design of a molecular sensing tool is an important topic in molecular recognition, signalling, and optoelectronics that has piqued the interest of chemists, biologists, and environmental scientists. Approximately 150 years have passed since the beginning of the fluorescent chemosensor sector. Due to the paramagnetic properties of Cr³⁺ and Al³⁺, it is tough to prepare a photoluminescence plug-in detector. Most dye-based Al³⁺ sensors must be utilized in organic or mixed solvents for robust hydration of Al³⁺ in water. The sophisticated molecular design of sensors, conversely, allows for the detection of these metal ions in aqueous medium. The design of chemosensors using various fluorophores and their mechanisms of action have been thoroughly discussed. A literature survey covering the design of chemosensors and their mechanisms of action have been thoroughly discussed covering the period 2010–2022 and that was carried out including innovative and exemplary activities from numerous groups throughout the world that have significantly contributed to this sector. The most important advantages of these probes are their aqueous solubility and quick response with outstanding selectivity and sensitivity for temporal distribution with high fidelity of metals in living cells.