Recent endeavors on molecular imaging for mapping metals in biology

Abstract

Transition metals such as zinc, copper and iron play vital roles in maintaining physiological functions and homeostasis of living systems. Molecular imaging including two-photon imaging (TPI), bioluminescence imaging (BLI) and photoacoustic imaging (PAI), could act as non-invasive toolkits for capturing dynamic events in living cells, tissues and whole animals. Herein, we review the recent progress in the development of molecular probes for essential transition metals and their biological applications. We emphasize the contributions of metallostasis to health and disease, and discuss the future research directions about how to harness the great potential of metal sensors.

INTRODUCTION

Iron, zinc, and copper, the top three abundant transition metals in the human body, were well-recognized for their essential roles in maintaining homeostasis and sustaining life. There were numerous examples of clinical diseases that were closely associated with metal misregulation, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) (Bleackley and MacGillivray 2011). In this regard, careful maintenance of transition metal homeostasis is required in most living systems.

Labile metal ions, part of the total metal pools, are weakly bound to ligands and can rapidly dissociate (Aron et al. 2015). Redox-active labile metals, such as copper and iron, can be harnessed to create new diagnostics and therapeutics (Chang 2015). Ferrous iron (Fe²⁺) is oxidized into ferric iron (Fe³⁺) through reaction with hydrogen peroxide (H₂O₂), leading to the formation of highly reactive hydroxyl radicals (OH^·), referred to as the Fenton reaction. Fe³⁺ is reduced back to Fe²⁺ via reaction with superoxide radicals (O₂^·–). This redox cycle is called Harber–Weiss reaction (Hassannia et al. 2019). Like iron, copper (Cu⁺ and Cu²⁺) can transform between two oxidation states under biological conditions. These metals can also undergo kinetically appreciable ligand exchange with sensors that respond to metal binding or reaction with an alteration in signal (light or sound) output (Ackerman et al. 2017).

Fluorescence imaging (FLI) has been widely used in identifying diversified living processes. Providing a potentially non-invasive set of tools with spatial and temporal resolution, molecular imaging drives scientists to peer into biological environments, and unravels the mysteries of metal ions in a sophisticated milieu. Small-molecule indicators have been assorted into two basic categories for achieving specificity: (1) chelation-based and (2) reaction-based (Ackerman et al. 2017). Fundamental mechanisms such as fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), and intramolecular charge transfer (ICT) integrate the foundation for most sensor designs (Chang et al. 2020). Traditional FLI normally utilizes excitation light < 600 nm, which can lead to auto-fluorescence as well as cell damage, and can be vigorously scattered in deep tissues. To circumvent the high background and low sensitivity, many efforts have been made to develop probes under near infrared (NIR) light excitation and emission. Accordingly, other imaging modalities such as two-photon imaging (TPI), bioluminescence imaging (BLI) and photoacoustic imaging (PAI) have been developed and attracted much attention. Table 1 points to a contrastive analysis of the three imaging modalities.

Table 1. Strengths and weaknesses of representative imaging modalities (Baker 2010) .

Modality	Labels	Readout	Depth	Resolution	Pros	Cons
TPI	Fluorophores with two-photon absorption (700–1100 nm) cross-section in GM units	Vis–NIR light	~1 mm	1–2 μm	Less photo-damage and photo-bleaching than the corresponding FLI	Relatively poor depth penetration
BLI	Bioluminescent enzymes (typically luciferases)	Light (500– 630 nm emission)	~3 cm	3–5 μm	No external light, zero background emission	Inherently weak emission, expensive (~3 × 105 USD) charge-coupled device (CCD) camera
PAI	Probes (ε > 10 4/(M·cm)) that absorb light (680–950 nm) and create sound signals	Sound	5–7 cm	~50 μm	Better depth than light	Information processing and machines still being optimized

Compared with FLI, BLI does not need an excitation light source, resulting in minimized autofluorescence and much higher signal to noise ratio (Li et al. 2013). Beetle luciferases oxidize D-luciferin with ATP and O₂, generating primarily yellow–green light, whereas marine luciferases release blue photons via the oxidation of imidazopyrazinone analogs (Yao et al. 2018). A growing field of “PAI”, which combines optical and ultrasound imaging, shows advantages of high resolution and high contrast at centimeter imaging depths. Theoretically, the design of molecular PAI sensors is primarily based on extending a large π-conjugated backbone with strong PA signal, for example, porphyrin, boron-dipyrromethene (BODIPY), and cyanine derivatives. Excellent reviews of Chan and coworkers discussing the development of PA agents can be found elsewhere (Knox and Chan 2018; Reinhardt and Chan 2018), and fluorescent sensors for measuring metal ions in living systems are also recommended (Carter et al. 2014; Liu et al. 2013; Park et al. 2020; Trusso Sfrazzetto et al. 2016).

METALLOSTASIS

Zinc

In the cellular environment, zinc is redox inert and is present in the +2 oxidation state. As the second-most abundant metals in the human body following iron, Zn²⁺ is a major cofactor in DNA replication, protein synthesis and cell differentiation (Szewczyk 2013). Some evidence demonstrates that zinc homeostasis in mammals is controlled by the two major families denoted as ZIP (SLC39A) (Eide 2004) and CDF/ZnT (SLC30A) (Palmiter and Huang 2004).

ZIP transporters confer cytoplasmic Zn²⁺ uptake (Huang and Tepaamorndech 2013). Zinc is first absorbed from the diet by ZIP4 (Fig.1), and is delivered to the basolateral membrane or bound to metallothioneins (MTs); the molecular mechanism behind this process has not yet been elucidated (Lazarczyk and Favre 2008; Nishito and Kambe 2018). An excess of unbound cytoplasmic Zn²⁺ is exported outside cells by ZnTs, predominantly ZnT1, and transported to peripheral tissues. Zn²⁺ possibly crosses the outer membrane of mitochondria through porin channels, and then bound to MTs in the intermembrane space or further shipped into the mitochondrial matrix via unknown proteins (Lazarczyk and Favre 2008; Nishito and Kambe 2018). Furthermore, a mutual exchange of Zn²⁺ between the ER and Golgi apparatus through antero- and retrograde vesicular transport has been postulated. Zn²⁺ is delivered in the opposite direction, from organelles to the cytosol by ZIPs, ZIP1 and ZIP7 (ER and Golgi apparatus) are closely related to this process. The ZIP and ZnT families are highly conserved, and their corresponding genes were found in zebrafish as well. The mRNA level of zinc exporter ZnT1 was upregulated in fish subjected to zinc overload and downregulated through zinc deprivation (Zheng et al. 2008).

Figure 1.

A simplified overview of zinc, copper and iron handling pathways in mammalian cells. ZIP: Zrt/Irt-like protein; ZnT: zinc transport protein; CTR1: copper transporter 1; ATOX1: anti-oxidant 1; ATP7: adenosine triphosphatase 7; CCS: copper chaperones for superoxide dismutase; SOD1: superoxide dismutase 1; MTF1: metal regulatory transcription factor 1; COX: cytochrome c oxidase; MT: metallothionein; ISC: iron-sulfur cluster; TF: transferrin; TFR: transferrin receptor; FPN: ferroportin; FTN: ferritin; NCOA4: nuclear receptor coactivator 4; STEAP3: six-transmembrane epithelial antigen of prostate 3; DMT1: divalent metal transporter 1; NRF2: nuclear factor erythroid 2-related factor 2; HMOX1: heme oxygenase 1

Copper

As the third-most abundant trace metal in the human body, copper not only plays a critical role in physiological processes (Valko et al. 2016), but also associates with cell proliferation and major diseases (Brady et al. 2014). It is revealed that biological functions of copper are essentially dependent on Cu²⁺/Cu⁺ transition, also associated with the production of reactive oxidative species (ROS). Abnormal Cu⁺/Cu²⁺ equilibrium can lead to dyshomeostasis, causing many genetic disorders such as Menkes (MNK) disease (Andersson et al. 2014), Wilson disease (WD) (Bandmann et al. 2015), neurodegenerative diseases (Müller et al. 2017), and cancers (Wang et al. 2015).

Cu²⁺ is reduced to Cu⁺ by an elusive mechanism and can enter cells through copper transporter 1 (CTR1), the principal high-affinity copper importer (Cotruvo et al. 2015). Upon entry, copper interacts with cellular ligands and chaperones that adjust its shipping to specific proteins. Glutathione (GSH) can buffer Cu⁺ pools and mediate Cu⁺ transfer between CTR1 and metallochaperones. Carriage of copper to cytosolic superoxide dismutase 1 (SOD1) proceeds through copper chaperones for superoxide dismutase (CCS). This metallochaperone for Cu/Zn SOD is a known marker for alterations in copper metabolism that inversely correlates with intracellular copper bioavailability (Bertinato et al. 2003; Prohaska et al. 2003). Metal regulatory transcription factor 1 (MTF1) transcriptionally activates MT genes and other targets. Cytosolic copper is delivered to mitochondria for loading onto cytochrome C oxidase (COX) (Festa and Thiele 2011). Atox1, homolog of Atx1, is responsible for copper delivery to two P-type ATPases, who also participate in copper efflux from cells. They are ATP7A and ATP7B in the trans-Golgi network, where most cuproproteins are metallated. MNK is an X-linked recessive disorder caused by mutations in genes coding for the copper-transport protein ATP7A, leading to copper deficiency. Conversely, WD is a rare genetic disorder characterized by excess copper stored in various body tissues (Bansagi et al. 2016).

Iron

As the most abundant transition metal in the human body, iron is essential and required for a variety of physiological and pathological processes (Theil and Goss 2009). Iron deficiency anemia (IDA) is imposed by a lack of iron, whereas hereditary hemochromatosis (HH) is an iron-overload disorder (Andrews 2000). Once iron is put into circulation, it is first bound to transferrin (TF) as Fe³⁺ and can subsequently enter cells, which maintain iron homeostasis through a balance of import proteins including transferrin receptor (TFR), divalent metal transporter 1 (DMT1), storage proteins like ferritin (FTN), and export proteins like ferroportin (FPN) (Aron et al. 2018). It is worth noting that metalloreductase STEAP3 (six-transmembrane epithelial antigen of prostate 3) converts Fe³⁺ to Fe²⁺. In mitochondria, iron-sulfur (Fe/S) cluster proteins (ISCs) are generated from iron and cysteine and are indispensable cofactors for proteins in mitochondrial respiration and other cellular activities (Braymer and Lill 2017).

Aberrant iron levels are implicated in ailments such as pyroptosis and ferroptosis (Zhou et al. 2018). Ferroptosis is a newly recognized cell death modality marked by the oxidative modification of phospholipid membranes via an iron-dependent mechanism and that has drawn constantly increasing attention in chemical biology (Conrad et al. 2016; Friedmann Angeli et al. 2019). Augmented levels of nuclear factor erythroid 2-related factor 2 (NRF2) protein after treatment with erastin and sorafenib (inducers of ferroptosis) render cells resistant to ferroptosis. This protection is traced to upregulation of NRF2 target genes like heme oxygenase 1 (HMOX1). Lysosomes can accumulate a large quantity of iron through degradation of FTN (coined ferritinophagy). Inhibition of lysosomal activity or silencing of nuclear receptor coactivator 4 (NCOA4), a cargo receptor recruiting FTN to autophagosomes for lysosomal degradation and iron release, suppresses ferroptosis (Hassannia et al. 2019). Remarkably, a novel quinolone-derived fluorescent turn-on probe IQ44 has been enabling the visualization of autophagosome-lysosome fusion in the process of autophagy (Lim et al. 2019).

MOLECULAR IMAGING

Zinc

Our laboratory reported a turn-on Zn²⁺ sensor NBD-TPEA, the first example for monitoring zinc ions in neuromasts of zebrafish via fluorescence (Qian et al. 2009). By replacing −NO₂ moiety with −SO₂NH₂, we devised an ICT ratiometric fluorescent probe, SBD-TPEA, for Zn²⁺ imaging in biological systems (Liu et al. 2014). In the presence of Zn²⁺ (50 mmol/L HEPES/DMSO = 99.85/0.15, pH 7.2), SBD-TPEA exhibits an emission band centered at 585 nm, with an excitation maximum at 466 nm. However, addition of Zn²⁺ to the probe leads to a distinct hypsochromic emission shift from 585 to 545 nm with a well-defined isoemission point at 585 nm. This change is attributed to ICT alteration when Zn²⁺ binds to the probe. The dissociation constant (K_d) of the SBD-TPEA/Zn²⁺ complex was estimated to be 2.1 nmol/L and its limit of detection (LOD) for Zn²⁺ was 0.5 nmol/L. Because SBD-TPEA responded selectively to Zn²⁺ without interference from other metal ions, it was utilized to image Zn²⁺ in HepG2 cells and zebrafish larvae (Fig. 2).

Figure 2.

A Emission spectra of 3 mmol/L SBD-TPEA (λ_ex = 460 nm) in buffer (50 mmol/L HEPES/DMSO = 99.85/0.15, pH 7.2) obtained by adding aliquots of Zn²⁺ solution (1.2 mmol/L); Inset in A: chemical structure of SBD-TPEA. B–E Confocal fluorescence ratiometric Zn²⁺ imaging in the head of a 3-day-old zebrafish larva stained with SBD-TPEA. B Overlay of bright field and fluorescence image of the SBD-TPEA-stained zebrafish larva from the green channel. C Ratiometric image based on the fluorescence images of the zebrafish larva in B. D Overlay of bright field and fluorescence image of the SBD-TPEA-stained zebrafish larva from the green channel after feeding the larva with Zn²⁺ solution (100 mmol/L) for 1 h. E Ratiometric image of the larva in D. Green channel images obtained with a bandpath of 510–560 nm, and red channel images obtained with a bandpath of 580–630 nm upon excitation at 488 nm. Adapted from Liu et al. 2014

Through integrating a Zn²⁺ ionophore N,N’-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (BPEA) as the ICT donor of the ANaph fluorophore, we constructed Naph-BPEA with nuclear envelope penetrability (Zhang et al. 2013). Of note, this probe did not merely localize in the cell nucleus, it also fluoresced in the cytoplasm. More importantly, our group summarized photoluminescence Zn²⁺ imaging in living subjects before 2015 (Chen et al. 2015). Apart from biological functions and sensing mechanisms of labile Zn²⁺, Zn²⁺ roles within an array of subcellular compartments such as mitochondria, lysosome, endoplasmic reticulum (ER), and Golgi apparatus were illuminated in detail, with corresponding Zn²⁺ sensors displayed. Localization is usually defined by comparing the colocalization of the sensor with a well-established organelle marker and quantifying the overlap using Pearson’s correlation coefficient (PCC) given by a imaging software (Carter et al. 2014). Organelle-specific Zn²⁺ probes have been developed to study Zn²⁺ distribution in different subcellular compartments (Fig. 3), we will only introduce some representative works due to limited space of this review, readers are encouraged to refer to another review for more detailed information (Zhu et al. 2016).

Figure 3.

Organelle-specific reversible Zn²⁺ probes. Golgi-targeting SZnC, ER-targeting ZR1, mitochondria-targeting DA-ZP1-TPP, and lysosome-targeting LysoDPP-C4. Adapted from Zhu et al. 2016

Golgi-targeting

Golgi apparatus contain various metalloproteases and alkaline phosphatases, whose catalytic activities rely largely on the Zn²⁺ ions. In 2015, the Kim research group reported a Golgi-localized two-photon ratiometric probe for zinc ions (Singh et al. 2015). The dipicolylamine moiety within the compound SZnC selectively makes a complex with Zn²⁺ (30 mmol/L MOP/EtOH = 1:1, pH 7.2), with an emission enhancement peaked at 500 nm. The TP action cross-section values (Φ_δmax) of SZnC in the absence and presence of excess Zn²⁺ were determined to be 16 and 92 GM, while the fluorescence quantum yield Φ were 0.12 and 0.93, respectively. Featuring low cytotoxicity and high photostability, this probe can be leveraged to monitor Zn²⁺ fluctuations in real time and getting a 3D distribution picture in the Golgi apparatus.

ER-targeting

Metal ions, such as Cu⁺ and Zn²⁺, are required for ER functions. Recently, Lippard and coworkers synthesized a new red-emitting fluorescent probe ZR1 to monitor mobile Zn²⁺ in the ER, a benzoresorufin fluorophore functionalized with two 2,2’-dipicolylamine (DPA) arms (Loas et al. 2014). The free probe was non-fluorescent (50 mmol/L PIPES, pH 7.0) due to PET from the Zn²⁺ chelators, while Zn²⁺-binding strongly elevated its emission maximum at 611 nm and an 18-nm Stokes shift. On average, the associated brightness values (εΦ) enhanced 44-fold to 2.43 × 10⁴ L/(mol·cm). In comparison with ZBR4, which anchors to the mitochondria, ZR1 spontaneously localizes to the endoplasmic reticulum of HeLa cells.

Mitochondria-targeting

Using fluorescein fluorophore instead, Lippard and coworkers presented a reaction-based fluorescent sensor DA-ZP1-TPP, which selectively localized at the mitochondria (Chyan et al. 2014). Coordination of Zn²⁺ enhances fluorescence intensity by promoting ester hydrolysis and alleviating PET origination from the DPA motif. Adding nanomolar concentrations of free zinc ions resulted in large increases in both the absorption (λ_abs = 510 nm) and fluorescence (λ_em = 529 nm) spectral bands of DA-ZP1-TPP (50 mmol/L PIPES, pH 7.0). These optical changes combined to yield a >140-fold increase in the fluorescence signal ( Φ_Zn = 0.75). With this probe, it is concluded that tumorigenic cancer cells lose the ability to accumulate Zn²⁺ within their mitochondria in contrast to healthy epithelial prostate cells.

Indeed, close correlations between prostatic Zn²⁺ levels and prostate cancer (PCa) have been reported (Costello et al. 2004). At least three ZIPs and six ZnTs are expressed in a lobe-dependent manner in the prostate (Kelleher et al. 2011). In malignant prostate tissues, there is a dramatic decline in Zn²⁺ concentration (500 nmol/g vs. 3000 nmol/g for cancerous and healthy tissues, respectively), as well as a downregulation of ZIP1 (Kolenko et al. 2013).

Lysosome-targeting

Very recently, LysoDPP-C4 has been developed for the evaluation of low pH and Zn²⁺ in an AND logic fashion to investigate lysosomal Zn²⁺ in prostate cancer cells (Du et al. 2019). A morpholine unit is appended for targeting lysosomes, as confirmed by HeLa cell fluorescence imaging studies. LysoDPP-C4 demonstrated an excellent selectivity toward Zn²⁺ and the resulting LysoDPP-C4/Zn²⁺ complexes proved insensitive to pH 3.5–9. At low pH, Zn²⁺ chelation contributes to a large increase in the fluorescence intensity at 515 nm (λ_ex = 430 nm) ascribed to suppression of PET. The sensor formed a host-guest complex in 1:1 stoichiometry with K_d = 1.91 nmol/L. Besides, histological studies using a human sample revealed that LysoDPP-C4 could distinguish between cancerous prostate tissue and healthy one.

In view of the advances in Zn²⁺-specific chelators (TPEA, BPEA, DPA, etc.) and Zn²⁺-complexation-induced spirolactam ring-opening or hydrolysis (Jin et al. 2018), concurrent with phosphorescence lifetime imaging (PLIM) of labile Zn²⁺ (Zhang et al. 2018), it is not surprising that Zn²⁺ sensors constitute the largest family of fluorescent indicators for transition metals. Some of them are reversible and ratiometric, applied to appealing models. So far, the physiological participation of this ion is not completely clear in spite of the large arsenal of fluorescent Zn²⁺ sensors.

Copper

Molecular sensors are intriguing tools to visualize the distribution and speciation of labile copper. Nevertheless, there are added challenges posed by aiming at labile copper over Zn²⁺ due to the need for discrimination between different oxidation states, the quenching nature of Cu²⁺, and the fact that probes must have enough affinities to compete for copper within its biological window (10⁻²¹−10⁻¹⁷ M) (Wegner et al. 2011). In consequence, only a fraction of copper sensors has been utilized for biologically accessible copper. The Kim group shed light on the pathophysiological part of copper ions and sensors for the measurement thereof (Peter et al. 2015).

Cu⁺

Although Cu⁺ is prone to disproportionation and acquires stabilization by specific ligands in aqueous media (Paredes and Das 2011), it is considered to be the dominant intracellular copper oxidation state of labile copper pools (Festa and Thiele 2011). Most of the probes designed for biological systems target Cu⁺, engaged in two common aspects as follows (Fig. 4).

Figure 4.

Design approaches and selected structures for Cu⁺ detection. A NS₄-based chelation: CNSB. B O₂-dependent C−O bond cleavage: CCL-1

(A) NS₄-based chelation

Thioether (NS₄) receptors can be modified to develop fluorescent Cu⁺ sensors. One notable example is CTAP-1, the first small-molecule sensor for mapping labile Cu⁺ in cells (Yang et al. 2005). Soon after this study, the Chang group presented coppersensor 1 (CS1) with visible excitation and emission profiles (Zeng et al. 2006). Tuning the BODIPY fluorophore of CS1 to a rhodol scaffold produced a more hydrophilic Cu⁺ sensor called copper Fluor-3 (CF3) (Dodani et al. 2014). As described earlier, fluorescent sensors that excited at short wavelengths succumb to limited penetration depths, high autofluorescence, and inevitably induce photobleaching and photodamage. These flaws hinder their use for long-term imaging in tissues and organisms. One way to remove these limitations is by NIR single-photon excitation (Guo et al. 2014). Cao Cu-3 represents a novel NIR fluorescent turn-on probe suitable for imaging endogenous Cu⁺ ions in living systems (Cao et al. 2012).

Another approach is to use two-photon microscopy (TPM), employing NIR photons of lower energy as the excitation source to possess the advantage of deepened penetration (>500 μm) (Kimet al. 2014). Our laboratory presented the first-generation ER-localized TP Cu⁺ sensor CNSB (Guo et al. 2019). There is a distinct overlap between coumarin-A (donor) emission and 4-amino-7-sulfamoyl benzoxadiazole (ASBD, acceptor) absorption, implying these two compounds are qualified candidates as a FRET pair to compose ratiometric sensors. CNSB exhibits ~7.44 × 10⁻¹¹ M K_d value (10 mmol/L Tris-HCl/DMF = 6:4, pH 7.2) and the Cu⁺-enhanced emission ratio F₄₇₀/F₅₆₅ is stable from pH 4.0 to 8.0. The sensor was applied to assessing Cu⁺ fluctuation in MCF-7 cells pre-incubated with tunicamycin, proving the relationship between Cu⁺ augmentation and ER stress. Meanwhile, the spatial distribution of Cu⁺ in the heart slice of a 2-week rat was observed via TPI owing to the two-photon ability of coumarin fluorophore. Negligible enhancement of coumarin emission was observed during the fluorescent titration of CNSB.

(B) O₂-dependent C−O bond cleavage

Inspired by the O₂ activation for copper-dependent enzymes, the Chang group adapted the Cu⁺ chelator tris(2-pyridylmethyl)amine (TPA) to develop copper-caged luciferin-1 (CCL-1) (Heffern et al. 2016), a bioluminescent reporter for monitoring labile Cu⁺ levels in a diet-induced mouse model of non-alcoholic fatty liver disease (NAFLD) that manifests as a hepatic copper deficiency, revealing altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain (Fig. 5). As supported by Western blot, CCS levels were elevated in the high-fat diet (HFD) mice over control diet mice, and the major copper exporter proteins ATP7A and ATP7B were also up-regulated.

Figure 5.

CCL-1 imaging reveals hepatic copper deficiency in a diet-induced murine model of NAFLD. A Representative images of mice from HFD and normal diet groups before the study (day 0) and at the end of the 8-week feeding period (day 56). B Western blot analysis of liver extracts of three mice fed normal diets and three HFD mice. Adapted from Heffern et al. (2016)

Not long ago, linking fluorescein donor and rhodamine acceptor through the TPA bridge, they reported reaction-based ratiometric FRET copper probe FCP-1. Together with its 2-color response (F₅₂₆/F₅₇₆), collective results indicate that oncogene-driven changes in the metabolism of GSH, a major cellular redox buffer, leads to a labile Cu⁺ deficiency with differential expression of CTR1 (Chung et al. 2019). This work connects Cu⁺ dysregulation and GSH stress in cancer, providing a roadmap for studying the crosstalk between metal and redox pathways in physiology and pathology with probes.

Cu²⁺

As synthetic fluorescent probes for monovalent copper have been thoroughly reviewed by others (Cotruvo et al. 2015; Fahrni 2013), we focus our discussion on Cu²⁺ probes that compensate for these areas of copper sensing. A large library of luminescent chemodosimeters for Cu²⁺ bioimaging has been collected by Fuyou Li et al. (Yang et al. 2013). Most of such probes recognize Cu²⁺ by three principal categories: chelation, hydrolysis and oxidation (Fig. 6). Among them, the oxidation pattern draws in the fewest probes while the other two seem more attractive.

Figure 6.

Selected structures of Cu²⁺-responsive probes based on chelation (CSBPA), hydrolysis (NRh) and oxidation (FluHMPP)

(A) Chelation

As mentioned previously, 2,2’-dipicolylamine (DPA) can be manipulated to design probes selective for Cu²⁺ over Zn²⁺ (Ballesteros et al. 2009). Modifying the FRET pair of CNSB with DPA unit forms a reversible ratiometric sensor CSBPA for intracellular Cu²⁺ imaging (Chen et al. 2013). Later we achieved in vivo fluorescence imaging for Cu²⁺ in live mice for the first time by a BODIPY-derived NIR fluorescent sensor BDPA (Xue et al. 2016), profiting from its large Stokes shift (~100 nm), excellent photostability and high quantum yield (Φ = 0.85).

Wang et al. developed a series of activatable PA probes with low molecular weights (<438 Da), and could specifically chelate with Cu²⁺ to form radicals displaying turn-on PA signals in the NIR region (Wang et al. 2019a). Introducing the electron-donating group N,N-dimethylaniline into the probe was found to significantly enhance the radical stability and PA intensity. The best probe in the series, RPS1, produced a fast response (within seconds) to Cu²⁺ with a low LOD of 90.9 nmol/L (0.02 mol/L PBS, pH 7.4). Owing to the low molecular weight and amphiphilic structure, RPS1 could effectively cross the blood−brain barrier (BBB) and thus maps Cu²⁺ in the brains of AD mice via PAI for the first time (Fig. 7).

Figure 7.

A Experimental design of target PA probe RPS1 for Cu²⁺ analysis. B Calibration curve of the PA intensity at 710 nm for measuring Cu²⁺ in the range of 0–35 μmol/L. C Schematic diagram of RPS1 crossing the BBB and detecting Cu²⁺, as observed by PAI. Adapted from Wang et al. 2019a

(B) Hydrolysis

The group headed by Chan reported the synthesis and photoacoustic properties of APC-1 and APC-2, two ratiometric probes for mobile Cu²⁺ (Li et al. 2015). Upon binding Cu²⁺, the sensors display 89- and 101-fold enhancements of normalized ratiometric turn-on responses, respectively. Both APCs are equipped with a 2-picolinic ester sensing module that is readily hydrolyzed in the presence of Cu²⁺ but not by other divalent metal ions. Additionally, ratiometric PAI was realized by using an aza-BODIPY dye scaffold exhibiting two spectrally resolved NIR absorbance bands, which correspond to the 2-picolinic ester capped and uncapped phenoxide forms.

Combining Cu²⁺-promoted hydrolysis with spirolactam ring-opening generates an effective strategy to construct fluorescent Cu²⁺ chemodosimeters. A ratiometric TP probe for Cu²⁺ trafficking based on through-bond energy transfer (TBET), was designed by directly conjugating BODAN (donor, green emission) and rhodamine spirolactam (acceptor, red emission) (Fig. 8) (Zhou et al. 2014). Np-Rh elicits a ratiometric response (EtOH/H₂O = 1:9) upon addition of Cu²⁺, with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. Moreover, the Cu²⁺ distribution in Np-Rh-labeled HeLa cells as well as rat liver frozen slices pretreated with Cu²⁺ was visualized using TPM in the 450–530 and 540–650 nm collection windows.

Figure 8.

Two-photon TBET probe Np-Rh. TPI of a rat liver frozen slice stained with 10 μmol/L Np-Rh at ~95 μm for 60 min (A), followed by treatment with 100 μmol/L Cu²⁺ and incubated for another 60 min (B). Green channel images obtained with a bandpath of 450–530 nm (A), and red channel images obtained with a bandpath of 540–650 nm upon excitation at 780 nm with femtosecond pulses (B). Adapted from Zhou et al. 2014

Similarly, Fuyou Li and coworkers introduced a frequency upconversion luminescence (UCL) Cu²⁺ chemodosimeter NRh (Liu et al. 2016), lighting up efficient single-photon upconversion emission at 730 nm under NIR excitation at 808 nm. This probe can serve as an ideal Cu²⁺ sensor for ex vivo and in vivo assay in a WD mouse model. Impressively, the exploitation of UCL small-molecule probes and their bioimaging applications have been attracting much attention (Dong et al. 2018; Yang et al. 2016; Zhang et al. 2017).

(C) Oxidation

Subsequently, the same group developed an easy-to-use probe CYDA based on UV–Vis–NIR absorption changes with excellent sensitivity and selectivity for Cu²⁺ (Shi et al. 2018). The mechanism of CYDA oxidation by Cu²⁺ was first explored (Fig. 9). In aqueous solution (EtOH/H₂O = 1:1), addition of Cu²⁺ to CYDA induced significant changes in the absorption spectra. When the solution was mildly acidic (pH 6.8), the absorbance at 670 nm went down, with the absorbance at 553 nm (ε = 1.09 × 10⁵ L/(mol·cm)) up, accompanied by the solution color changes from blue to red. In alkaline solutions (pH 8.0), the absorbance at 670 nm (ε = 2.14 × 10⁵ L/(mol·cm)) dropped gradually, whereas the absorbance at 823 nm (ε = 0.22 × 10⁵ L/(mol·cm)) rose with an isosbestic point at λ = 793 nm. In the meantime, the solution color turned into greyish blue. These phenomena confirmed the formation of CY1 and CY2. They further demonstrated that the probe was able to quantify Cu²⁺ in urine from WD patients.

Figure 9.

A Oxidation of CYDA by Cu²⁺ in acidic solution (pH 6.8) and alkaline solution (pH 8.0) respectively. B UV−Vis−NIR absorption spectra (WD patients’ urine/EtOH/HEPES = 10:10:1) of 10 μmol/L CYDA when the pH value was 6.8 (left), 8.0 (right), from top to bottom: CYDA/Urine_start, CYDA/Urine_end and Urine. Adapted from Shi et al. 2018

Even a bit earlier than the above study, oxidative C−O bond cleavage turn-on probe FluHMPP has been prepared (Shi et al. 2013). On addition of 20 equiv of Cu(NO₃)₂ (10 mmol/L Tris-HCl/CH₃CN = 7:3, pH 7.2), there was a strong emission enhancement (30-fold) after 2 h. Confocal microscopy experiments have demonstrated that FluHMPP was membrane permeable and capable of tracing exogenous CuCl₂ in HeLa cells. Nevertheless, such C=N structures are unstable and sensitive to reactive oxygen species.

Iron

Despite great interest in labile iron pool (LIP), its biological function remains insufficiently understood, in part due to a relative lack of tools for directly assessing labile iron in living specimens (Dixon and Stockwell 2014). Molecular imaging using iron selective sensors opens up a broad avenue for better understanding the complex handling of iron in biology. Ideally, probes should be able to track biological iron status, selectively detecting either Fe²⁺ or Fe³⁺ or responding to iron species such as heme-iron or ISCs. Because Fe²⁺ and Fe³⁺ are potent emission quenchers, iron imaging within living cells encountered many challenges. Two early sensors, Calcein and Phen Green SK, exhibit turn-off responses to iron binding (Carter et al. 2014). However, both probes cannot adequately distinguish the two oxidation states of iron but can interact with other metal ions, leading to an interfering signal. In the past few years, a substantial body of efforts has been devoted toward construction of iron probes that may make a stunning breakthrough regarding cellular iron homeostasis.

Fe²⁺

The growing palette of chemodosimeters for Fe²⁺ mapping exploits the potent redox activity of this metal ion, and the last several years have witnessed a tremendous number of novel Fe²⁺ chemoprobes suitable for live-cell imaging experiments. Recent reviews by Hirayama, Aron and coworkers exhaustively profiled the development of molecular probes for Fe²⁺ (Aron et al. 2018; Hirayama 2019). A palette of Fe²⁺-mediated principles have been reported, including N-oxide or nitroxide reduction, cyclization, O₂ activation and endoperoxide cleavage (Fig. 10).

Figure 10.

Reaction-based sensing probes for Fe²⁺ detection. A N-oxide: RhoNox-1. B Nitroxide reduction: Rh-T. C Cyclization: L1. D O₂ activation: LCy7. E Endoperoxide cleavage: Trx-puro

(A) N-oxide reduction

The deoxygenation of tertiary amine N-oxide is preferably mediated by Fe²⁺ as it has a lower redox state than Fe³⁺. Utilizing the reaction, Hirayama and coworkers developed the first turn-on fluorescent Fe²⁺ probe RhoNox-1 (Hirayama et al. 2013), which has been commercially available and applied to detecting Fe²⁺ changes during ferroptosis (Wang et al. 2019b). To date, this Fe²⁺ sensing strategy has emerged as the most widely used one, establishing a series of sensors. For instance, HMRhoNox for visualization of intracellular Fe²⁺ delivered by TF (Niwa et al. 2014), CoNox-1 and FluNox-1 for scrutiny of intracellular redox equilibrium shift toward Fe²⁺ in hypoxic HepG2 cells (Hirayama et al. 2017), and a membrane-anchoring probe Mem-RhoNox to scrutinize Fe²⁺ release during endocytotic iron uptake (Niwa et al. 2018).

Recent research expanded the applicability to establish a series of organelle-specific fluorescent probes selective to Fe²⁺, i.e., MtFluNox, Lyso-RhoNox, ER-SiRhoNox and Gol-SiRhoNox. The former three chemosensors (Fig. 11) demonstrated similar off/on contrast and reaction rates (2.1 × 10⁻³ s⁻¹, 2.2 × 10⁻³ s⁻¹, and 1.7 × 10⁻³ s⁻¹), investigated Fe²⁺ specifically at the targeted organelles (PCC = 0.81, 0.80, 0.80), and depicted fluorescence enhancement at 535 nm (green, 100-fold), 575 nm (orange, 60-fold), and 660 nm (magenta, 60-fold), respectively. Furthermore, these probes indicated the aberrant elevation of labile Fe²⁺ levels in the lysosomes and ER but no Fe²⁺ fluctuation in the mitochondria prior to HT1080 cell death initiated by erastin (Hirayama et al. 2019b).

Figure 11.

Chemical structures of the fluorescent Fe²⁺ probes used MtFluNox, Lyso-RhoNox and ER-SiRhoNox. And fluorescence microscopic analysis for multi-color detection of Fe²⁺ at each targeted organelle. Adapted from Hirayama et al. 2019b

As depicts in Fig. 1, DMT1 serves as a primary transporter of Fe²⁺. Over the course of the endocytotic process, directing DMT1 toward the cellular membrane via the trans-Golgi network is partially regulated by a retromer-mediated protein-sorting machinery comprising vacuolar protein-sorting proteins (VPSs) (Burd and Cullen 2014). Therefore, the Golgi organelle functions as a node in this dynamic system for intracellular Fe²⁺ species along with DMT1. Dysfunction of VPSs, particularly the mutation of VPS35, can activate the aberrant delivery of DMT1 to lysosomes concomitantly with Fe²⁺ ions (Tabuchi et al. 2010). The last one Gol-SiRhoNox revealed an abnormal cellular Fe²⁺ distribution induced by dysfunction of VPS35 with a molecular chaperone R55 (Hirayama et al. 2019a).

(B) Nitroxide reduction

In parallel studies, a related Fe²⁺-promoted nitroxide reduction reaction has been employed for reaction-based sensing of Fe²⁺ (Maiti et al. 2015). A rhodamine-linked nitroxide probe, Rh-T, bears a pendant paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) group that quenches fluorescence. Fe²⁺ reduces the receptor radical to a diamagnetic hydroxylamine, resulting in a significant fluorescence turn-on (~2.5-fold) with a LOD ~0.75 µmol/L. Fe²⁺-dependent reduction of the TEMPO radical of Rh-T triggers both a fluorescence turn-on and a change in electron paramagnetic resonance (EPR) signal. Following this example, Zhu group reported NT-Fe with the fastest response (<50 s), as well as the sensitive LOD of 89 nmol/L (Zhanget al. 2020). In zebrafish, NT-Fe responds to the addition of exogenous Fe²⁺, and its signal decreases when pretreated with Fe²⁺ scavenger 2,2’-bipyridine (Bpy).

(C) Cyclization

By exploiting the Lewis acidity of Fe²⁺, a recent report highlighted the application of an Fe²⁺-specific cyclization of a phenolic unit adjacent to a C=N bond appended to a coumarin derivative (Long et al. 2018). Upon cyclization, generation of a benzoxazole ring handicaps C=N bond isomerization and rotation, turning on fluorescence in aqueous solution and HepG2 cells, which has also been confirmed by TD-DFT calculation. Significantly, the sensing reaction completed in 2 min with high sensitivity (LOD = 45 nmol/L). However, more analogous probes should be rationally constructed to verify the validity of the proposed mechanism.

(D) O₂ activation

Prior work by Chang laboratory on creating Cu⁺ indicators, presaged the general utility of biomimetic oxygen activation for Fe²⁺ surveillance. Inspired by the O₂ chemistry of Fe²⁺, and in particular, the 2-His-1-carboxylate triad motif found in mononuclear non-heme Fe²⁺ enzymes (cytochrome P450), they engineered iron probe 1 (IP1) (Au-Yeung et al. 2013). IP1 was capable of monitoring elevated intracellular labile Fe²⁺ levels in HepG2/C3A cells caused by treatment with hepcidin or ascorbic acid.

Replacement of the fluorescein alcohol substituent with a cyanine dye (Cy7), the Tang group yielded another NIR fluorescent probe (LCy7) (Wu et al. 2020), comprising a tris(pyridine) carboxylate-type ligand cage as an iron recognition and activity site. Upon Fe²⁺ coordination, O₂ activation triggers intramolecular C–O bond cleavage and oxidation to release the fluorophore, resulting in an increase (~3.7-fold) in the fluorescence emission peaked at 690 nm. Utilizing LCy7, dynamic Fe²⁺ enhancement was distinctly monitored in HL-7702 cells under ER stress by acetaminophen (APAP) stimulation. In vivo FLI disclosed the conspicuous Fe²⁺ ascent in the liver of mice during drug-induced liver injury (DILI).

(E) Endoperoxide cleavage

Motivated by Fe²⁺-reactive endoperoxide activation observed in antimalarial drugs (e.g., Artemisinin), Renslo and coworkers designed Trx-puro, a histochemical stain for labile Fe²⁺ comprised of a trioxalane-caged puromycin (Spangler et al. 2016). Upon reaction of the probe with Fe²⁺, free puromycin is released, which can be translated onto nascent peptides and quantified via a conventional immunostaining method by a puromycin antibody after fixation. Application of Trx-puro to a panel of cancer cells (PC-3, U-2 OS, MCF10A and RKO) reveals FTN/FPN overexpression and declined Fe²⁺ stores. Linking fluorescein and Cy3 through this bioinspired endoperoxide trigger, Chang et al. envisioned FRET iron probe 1 (FIP-1) for ratiometric fluorescence Fe²⁺ imaging upon 35MEW28-induced ferroptosis in MDA-MB-231 cells (Aron et al. 2016).

Expanding the scope of oxidative cleavage to BLI, the same group then reported the first bioluminescent indicator for Fe²⁺ (Aron et al. 2017), iron-caged luciferin-1 (ICL-1), to surveil labile Fe²⁺ accumulation in a luciferase-expressing murine (FVB-Luc⁺) model of Acinetobacter (A.) baumannii infection (Fig. 12). The probe also retains high selectivity and sensitivity for Fe²⁺ over a variety of biologically-relevant metal ions, oxidants, and reductants and is feasible for detecting both Fe²⁺ rise and fall in PC3M-luc cells and other Fe²⁺-supplemented/depleted models.

Figure 12.

A Fe²⁺-selective cleavage of ICL-1, an endoperoxide-luciferin conjugate and in vivo probe of Fe²⁺. B Representative images of FVB-Luc⁺ mice mock-infected (PBS) or infected with A. baumannii through retroorbital injection (dorsal images at 30 min postinjection of ICL-1) and imaged with ICL-1 (25 nmol) at 24 h postinfection. Adapted from Aron et al. 2017

Despite the exquisite selectivity toward Fe²⁺ for bioimaging, these reaction-based probes were not shown to have a reversible monitoring mode, hampering the capability to probe Fe²⁺ fluxes. If a probe is able to synchronously measure Fe²⁺ and Fe³⁺ without interference from other metal ions, the privileged part of iron taking in ferroptosis will no longer remain a mystery.

Fe³⁺

In contrast to the growing usage of chemical probes for bioimaging of Fe²⁺, measurement of Fe³⁺ in biological environments is rarer. Sahoo et al. reviewed advances on Fe³⁺-specific fluorescent probes (Sahoo and Crisponi 2019; Sahoo et al. 2012). Due to the paramagnetic nature of ferric iron, its previous recognition by fluorescent probes involves different quenching mechanisms, which is improper for bioimaging applications. A new class of turn-on Fe³⁺ sensors can be classified into two major categories, chelation and spirolactam ring-opening (Fig. 13). Furthermore, some ratiometric sensors recognizing the metal with an appropriate ligand group have been proposed to interrogate intracellular Fe³⁺ ions.

Figure 13.

Selected structures of Fe³⁺ probes. Turn-on sensors. Chelation: L2 and L3; Spirolactam ring-opening: L4 and ratiometric probe TPE-o-Py

(A) Turn-on Fe³⁺ probes

(a) Chelation

The modularity of the PET platform has been applied to developing Fe³⁺-triggered fluorescent turn-on probes with enhanced properties. A novel PET-based reversible fluorescence turn-on probe L2 for selective assay of Fe³⁺ was introduced (Sui et al. 2014). The probe was comprised of BODIPY fluorophore with a 1,10-diaza-18-crown-6-based cryptand that acted as the analyte binding unit. Upon addition of Fe³⁺ in aqueous media (H₂O/CH₃CN = 9:1), the weakly fluorescent L2 (PET process is active) displayed 23-fold fluorescence enhancement at 512 nm (λ_ex = 480 nm). The significant fluorescence enhancement occurred due to inhibition of PET from cryptand to BODIPY fluorophore upon complexation with Fe³⁺. The probe displayed a LOD of 0.13 μmol/L and was applied to sensing intracellular Fe³⁺ ions in HCT-116 cells.

Our group introduced a simple anthracene-based fluorescent turn-on probe L3 containing N²-hydroxyethyldiethylenetriamine (HEDTA) for inferring Fe³⁺ concentrations (Qiu et al. 2014). When excited at 373 nm, the [2+2] macrocycle fluorescent sensor (20 mmol/L Tris-HCl/MeOH = 1:1, pH 7.2) exhibits weak emission at 398, 421, and 447 nm. In the presence of Fe³⁺, the formation of a L3-Fe³⁺ complex in 1:2 ratio restricts the PET process, causing significant fluorescence enhancement. Probe L3 shows a linear response ranging from 1 to 10 µmol/L with the LOD of 0.58 µmol/L Fe³⁺. Confocal imaging disclosed that the probe possesses the ability of tracing cytosolic Fe³⁺ in SKOV-3 cells.

(b) Spirolactam ring-opening

Subsequently, we took advantage of the HEDTA ligand to develop another turn-on Fe³⁺ probe Mito-RhFe by appending the HEDTA receptor onto rhodamine B as a cationic, lipophilic tag to localize the probe to mitochondria (Zhu et al. 2018). In UV−Vis Fe³⁺ titration of this probe (20 mmol/L Tris-HCl/MeOH = 1:1, pH 7.2), the solution color changes from colorless to magenta, thereby offering Mito-RhFe as an Fe³⁺-induced ‘naked-eye’ chemosensor. With this probe, the mitochondrial labile Fe³⁺ fluctuation in adherent HeLa cells (PCC = 0.90) upon ferric ammonium citrate (FAC) incubation was visualized via confocal imaging, and the flow cytometric assay for mitochondrial Fe³⁺ in suspension MEL cells, which is difficult to be monitored in an imaging manner, was also developed. Erythroid differentiation is normally accompanied by hemoglobin biosynthesis and upregulation of the mitochondrial iron importing protein mitoferrin-1 (MFRN1) containing ISC (Shaw et al. 2006). Interestingly, the labile Fe³⁺ drop in mitochondria of human K562 erythroleukemia cells undergoing the DMSO-stimulated erythroid differentiation was observed for the first time (Fig. 14).

Figure 14.

A Chemical structure of probe Mito-RhFe. B Photograph of Mito-RhFe solution before (left) and after (right) Fe³⁺ addition (20 mmol/L Tris-HCl/MeOH = 1:1, pH 7.2). C Colocalization of Mito-RhFe with MitoTracker deep red 633 in MCF-7 cells. The cells were stained firstly by 10 μmol/L probe in PBS for 30 min at 37 °C. D Labile Fe³⁺ flow cytometric assay of K562 cells incubated in RPMI1640 medium containing 2% DMSO. E Western blot analysis of the cells in D. The cells were cultured at 37 °C for 4 days, and stained with Mito-RhFe at 0 °C for 30 min before assay. Cells cultured without DMSO were adopted as the control. Adapted from Zhu et al. 2018

In addition to Zn²⁺ and Cu²⁺, Fe³⁺ can trigger the hydrolysis and spirolactam ring-opening process of rhodamine derivatives, which has provided a valuable method to design Fe³⁺-selective luminescent sensors with a turn-on response. Lee et al. have presented six rhodamine-derived Schiff base probes' selectivity of the Fe³⁺ ion in biological systems (Lee et al. 2016). After exposure to Fe³⁺, the probes appeared a new absorption peak at 526 nm, with a concomitant emission maximum at 551 nm. Confocal microscopic analysis showcased that these probes underwent turn-on changes caused by cellular Fe³⁺ ions, and demonstrated a largely enhanced fluorescence upon iron overloading of the HepG2 cell line. They also found that the Fe³⁺-mediated fluorescence augmentation was mainly localized in the ER. Among them, probe L5 exhibited the highest selectivity for ER localization (PCC = 0.7854) over other organelles.

(B) Ratiometric sensors for Fe³⁺

Ratiometric probes are highly valued because their intrinsic internal standard can correct potential variations in light intensity, dye localization, and other experimental imaging conditions (Ackerman et al. 2017). The aforementioned two turn-on platforms can be adapted to quantify labile Fe³⁺ concentrations in a ratiometric readout, such as a bithiazole derivative (Geng et al. 2016), and a spirolactam analog RQBTE (Das et al. 2016)

It has been reported that ftn-1 (encoding the iron sequestering protein FTN) is upregulated in the presence of exogenous FAC supplementation, and downregulated upon treatment with an iron chelator deferoxamine (DFO) in the nematode Caenorhabditis (C.) elegans (Valentini et al. 2012). Goel et al. created the first dual colorimetric and ratiometric fluorescent Fe³⁺ probe NAP-3 (Goel et al. 2015), consisting of a naphtho[2,1-b]-[1,10]-phenanthroline (NAP). After exposure to increasing concentrations of Fe³⁺ ions (DMSO/H₂O = 1:9, pH 7.4), a new peak with maximum at 605 nm emerged and the peak at 544 nm decreased in the emission spectrum, with a final enhancement factor (I₆₀₅/I₅₄₄) over 8.5-fold. It can approximately measure as low as 9.1 nmol/L Fe³⁺. Possibly due to steric hindrance, Fe³⁺ chelator DFO failed to remove Fe³⁺ from the complex (NAP-3/Fe³⁺). Common ions do not imply any significant interference in the determination of Fe³⁺ ion. The indicator enabled direct visualization of exogenous Fe³⁺ in HepG2 cells, and endogenous Fe³⁺ in a multicellular organism, ftn-1 silenced C. elegans (Fig. 15).

Figure 15.

A Ratiometric fluorescence spectra (DMSO/H₂O = 1:9, pH 7.4) of 25 μmol/L NAP-3 (λ_ex = 365 nm) after 1 h upon addition of increasing concentrations of Fe³⁺ (0–0.68 mmol/L). Inset in A: chemical structure of NAP-3. B Wild-type and ftn-1 silenced C. elegans treated with NAP-3 (3 μmol/L, 24 h). Green channel images obtained with a bandpath of 505–550 nm upon excitation at 405 nm, red channel images obtained with 575 nm long pass upon excitation at 561 nm, and the corresponding ratiometric images (F_red/F_green). Adapted from Goel et al. 2015

In fact, most conventional organic dyes exhibit aggregation-caused quenching (ACQ) when aggregated. Aggregation-induced emission (AIE) phenomena, originating from the restriction of intramolecular motions (RIM), is thus a unique turn-on approach for addressing the ACQ issue (Chen et al. 2019a). AIE luminogens (AIEgens) tend to behave in a non-emissive manner when well dissolved in some solvents, but emit intense fluorescence when poorly dissolved by means of forming aggregates (Mei et al. 2015). AIEgens have emerged as a powerful methodology for the assay of transition metal ions such as Zn²⁺ (Jung et al. 2015), Cu²⁺ (Feng et al. 2014), and Hg²⁺ (Chen et al. 2016).

By making use of the position-dependent substituent effects, an AIE featured Fe³⁺ probe (TPE-o-Py) from ortho-substituted pyridinyl-functionalized tetraphenylethylene is synthesized by Tang’s research group (Feng et al. 2018). From weak blue to strong red emission (peak 472 nm to 573 nm) under 365 nm UV irradiation, TPE-o-Py displays high metal selectivity and oxidation-state specificity (THF/H₂O = 3:7) arising from the position isomer of ortho-substitution. And it has a low acid dissociation constant (pK_a ~3.27) that is close to that of hydrolyzed Fe³⁺. More importantly, the light-up fluorescent probe TPE-o-Py can be applied to sensing Fe³⁺ in HeLa cells with a pronounced red shift in fluorescence emission.

It should be noted that the reduction potential in the cytosol favors Fe²⁺ over Fe³⁺ (Aron et al. 2018), and Fe³⁺ is poorly soluble at neutral pH in aqueous media. On the other hand, the vast majority of Fe³⁺ probes have not been well suited for endogenous investigations due to a lack of high sensitivity. In this context, exploring improved motifs to decode Fe³⁺ roles in biology is of tremendous significance.

SUMMARY AND PERSPECTIVES

In this review, we summarized endeavors made in molecular imaging for transition metal cations. Specifically, we highlighted synthetic sensors that are utilized to image zinc, copper, and iron in biological systems (Table 2). Great challenges still remain in the field to be confronted with:

Table 2. Summary of selected probes for metal detection.

Probe	Modality	Readout	Cells	Biological highlights
“n.d.”: not determined. TPI and UCL belong to FLI.
DA-ZP1-TPP	FLI	F529	HeLa, WPE-1, RWPE-2, PC-3	Mobile Zn2+ in mitochondria of healthy vs cancerous prostate cells
LysoDPP-C4	FLI	F515	HeLa, PC3, DU145	Cancerous prostate nodules via Zn2+-induced fluorescence signaling
CNSB	TPI	F470/F565	MCF-7	Fluctuation and distribution of Cu+ under ER stress in MCF-7 cells
CCL-1	BLI	n.d.	PC3M-luc	Cu+ deficiency in a murine model of nonalcoholic fatty liver disease
RPS1	PAI	PA710	bEnd.3	Cross the blood–brain barrier for Cu2+ visualization in mice with Alzheimer’s disease
NRh	UCL	UCL730	HeLa	In vivo detection of Cu2+ in Wilson disease
LCy7	FLI	F690	HL-7702	Labile Fe2+ in ER stress-mediated drug-induced liver injury
ICL-1	BLI	n.d.	PC3M-luc	Labile Fe2+ accumulation in a murine model of A. baumannii infection
Mito-RhFe	FLI	F580	MCF-7	Labile Fe3+ drop in mitochondria of K562 cells undergoing the DMSO-stimulated erythroid differentiation
NAP-3	FLI	F605/F544	HepG2	Endogenous labile Fe3+ pools in ftn-1 silenced C. elegans

(1) Expanding metal probes bioavailable. It is the quality of probes, including solubility, sensitivity and selectivity (3S), not the quantity that matters. Deliberate design of metal sensors with improved bioavailability is of paramount importance. In addition, other transition metals like manganese (Mn) also take active part in health and disease (Avila et al. 2013). Yet Mn sensors are still on an early stage, and there have been far fewer small-molecule probes prepared for Mn²⁺ detection (Bakthavatsalam et al. 2015; Liang and Canary 2010).

(2) Combining super-resolution techniques. Super-resolution techniques, such as stimulated emission depletion (STED), structured illumination microscopy (SIM), and stochastic optical reconstruction microscopy (STORM), provide a valuable starting point to study analytes in nano-dimension (Kozma and Kele 2018). Application of this technique to molecular probes for mapping metals is an important and arduous task.

(3) Designing dual-responsive probes. Using two selective probes simultaneously to monitor multiple species is troublesome, and it is therefore preferable to pursue dual-responsive single probes, operating as a logic gate in response to two elements (Kolanowski et al. 2018). In this case, interrelations between multiple elements in biology can be deciphered. For instance, if a probe is able to synchronously measure Cu⁺ and GSH without any interference from other analytes, more straightforward data would be offered with less work in NAFLD mentioned above.

(4) Mining multi-mode imaging. Given that diverse imaging modes can be complementary, our laboratory recently reported a FLI/PAI dual-modality probe HS-CyBz (Chen et al. 2019b), realizing ratiometric in/ex vivo tracking for stimulated H₂S fluctuations in mice. Integrating several imaging modes into a single metal probe, should be appropriately achieved in the longer term.

To sum up, roles for labile metals in cells and tissues, have been elucidated to a certain extent using these molecular probes. Continued innovations in exploiting chemical reagents, in cooperation with physicists and biologists, presage further opportunities for uncovering how metal cations work in biology.

Conflict of interest

Jing Gao, Yuncong Chen, Zijian Guo and Weijiang He declare that they have no conflict of interest.