X-ray fluorescence imaging of metals and metalloids in biological systems

Abstract

Metals and metalloids play fundamental roles in many physiological processes in biological systems, but imbalance of these elements in the body may cause many diseases, such as Parkinson’s disease, Alzheimer’s disease, and even cancers. Thus, to better understand the metallome in health and disease, quantitative determination of their localization, concentration, speciation, and related metabolism at cellular or subcellular levels is of great importance. X-ray fluorescence (XRF) imaging, as a new generation of analytical technique, has been reported as an ideal tool to quantitatively map multiple metals and metalloids in tissues with reasonable sensitivity, specificity, and resolution. In the current review, we have introduced the general concept of XRF imaging technique, reviewed the recent advances using XRF imaging to investigate toxicology of metals and metalloids in life science, and discussed the roles of metals and metalloids in various diseases, including cancers and neurodegenerative diseases. We believe that future research on revealing the roles of metals and metalloids in biological systems will directly benefit from the important breakthroughs in developing XRF imaging techniques.

Introduction

In this review paper, metals and metalloids may mean the metal ions and any available forms, such as metal-oxygen anions and metal ion-ligand coordinates, and sometimes not strictly, mean some non-metals, such as As, P, Cl etc. that XRF is able to detect, for simplicity in description.

Metals and metalloids in biological systems

Metals and metalloids perform critical roles in fundamental processes required for all biological species, including osmotic regulation, catalysis, metabolism, biomineralization, and signalling [1,2]. Most biological organisms do not only contain abundant alkali and alkaline earth metals but also store and transport transition metals and metalloids to provide appropriate levels for metalloproteins or cofactors, and protect themselves against the toxic effects [3-5]. In general, the concentration of transition metals and metalloids, such as zinc (Zn), copper (Cu), manganese (Mn), iron (Fe), nickel (Ni), chromium (Cr), arsenate (As) and cadmium (Cd), is much lower than that of alkali and alkaline earth metals [6]. Nevertheless, metalloproteins and metal cofactors are found almost in all plants, animals, and microorganisms [1,7,8].

In living systems, the normal concentration range for each metal or metalloid is narrow (Table 1) [9], while both deficiency and excess of these elements can cause pathological changes [8,10]. For example, the World Health Organization (WHO) and the Food and Agricultural Administration (FAA) suggest that the population mean intake of Cu should not exceed 10-12 mg per day for adults [11]. The deficiency of Cu in the human body would increase the risk associated with developing coronary heart disease [12-14], while the Menke’s disease, Alzheimer’s disease, Parkinson’s disease, Wilson’s disease, and Occipital Horn Syndrome are implicated with the excessive amount of Cu uptake and excretion [13,15-17]. Since metals and metalloids in living systems are essential, researchers in chemistry, biology, and medicine have been attracted to relate their excess or deficiency in living systems to specific diseases [9,16]. Therefore, understanding of the localization, speciation, and metabolism of these metals and metalloids at molecular level is of great importance.

Table 1.

Average relative abundance of selected elements in earth’s crust and mammalian blood plasma [6,9]

Conc. of Metals	Ni	Cd	Zn	As	Cu	Mn	Fe	Cr	Na	K	Ca
Crust (ppm)	75	0.2	70	1.5	55	950	5 × 104	100	3 × 104	3 × 104	4 × 104
Blood (µM)	0.04	-	17	-	8-24	0.1	22	0.5	1 × 105	4 × 103	2 × 103

It is well known that the metals and metalloids in biological samples exist in different forms, such as free ions, metalloproteins or cofactors formed through complexation [7-9], and are not homogenously distributed in biological cells and tissues [18]. As a consequence, in-situ spatiotemporal analysis of their formation, concentration and distribution in living systems remains a great challenge [4,5]. Up to now, a variety of methods have been developed and used to quantitatively determine these metals and metalloids in biological samples. Among these approaches, in-situ imaging technique has been reported as one of the most promising methods for the study of metals in living systems due to its capability of high spatial and temporal resolution [1,16,19]. Recently, several imaging techniques have been proposed to visualize the location and determine the concentration level of specific metals in biological samples [16,20-22]. Table 2 illustrates an overview of the most important imaging-based microanalytical techniques. Imaging mass spectrometry (IMS) technology is used to record spatial and temporal resolution of metals and metalloids in biological samples at the cellular and/or subcellular levels [5,23,24]. Another imaging technology that permits the mapping of a specific metal in living specimens with high sensitivity and spatiotemporal resolution involves utilization of fluorescent biosensors [4,16,25,26]. When the intracellular biosensor interaction with specific metals or metalloids, the location and level of these elements will be immediately recorded as the changes in fluorescence output, either through an intensity increase or an energy shift of the emitted light [25,26]. Other imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), have also been developed for physiological imaging. Although the resolution of these techniques is only at the organ level, their high sensitivity endows them to be widely used in clinical medicine [16,27-31]. These imaging techniques for mapping the metals and metalloids in living systems have recently been comprehensively reviewed elsewhere [4,5,23-26,32,33]. This review will particularly highlight the recent progresses of X-ray fluorescence imaging technology, a new and very promising one developed very recently.

Table 2.

Comparison of the microanalytical techniques [23-31,33-35]

Analytical technique	Detection limit	Spatial resolution	Analytical depth (mm)	Quantification	Ref
MS	0.01 mg/g	15-50 mm	200	Semiquantitative	[23,24]
Fluorescence	pM to nM	2-3 mm (in vivo), 0.2-0.5 mm (in vitro)	< 1 cm	Quantitative/semiquantitative	[25,26]
PET	High pM	1-2 mm	No limit	Semiquantitative	[27-29]
SPECT	Low nM	15-20 mm	No limit	Semiquantitative	[28,29]
MRI	mM to low µM	25-100 mm	No limit	Semiquantitative	[30,31]
XRF	0.1-1 mg/g	0.03-0.2 mm	> 100	Quantitative	[33-35]

X-Ray fluorescence imaging

XRF is an elemental analysis technique, which relies on recording the characteristic secondary X-rays emitted from specific atoms when the materials are irradiated by a focused X-ray beam [34]. The history of XRF dates back to 1895 when German physicist Wilhelm Conrad Röntgen accidentally discovered X-rays during his study of cathode rays in high-voltage, gaseous discharge tube. Based on Röntgen’s discovery, Henry Moseley in 1913 discovered a mathematical relationship between the atom number (Z) and emitted X-ray wavelength. The technique was quickly realized to quantitative analysis of materials using XRF in 1914-1924. While the X-rays have been employed as a standard elemental analysis since 1950s when the first commercial XRF spectrometer was developed [35]. Since then, XRF has attracted increasing consideration and the relevant technique has been widely used for non-invasive imaging of thick and deep biological specimens with high spatial and temporal resolution [36].

As one of the advanced imaging approaches, X-ray fluorescence (XRF) imaging is a powerful technique for the quantitative mapping of distributions and dynamics of elements and chemical species at the spatial submicrometer resolution within biological samples [16,18,34,37]. The physical principle of the XRF is illustrated in Figure 1. Upon excitation by an X-ray photon, a core-shell electron from the specific atom is ejected as a photoelectron. The formed core-hole is then filled by a neighbouring higher energy orbital electron, which results in emission of an X-ray fluorescence photon. The energy of the emitted photon is equal to the difference in binding energies of the two shells involved in the transition [27,37]. Since the binding energy is varied with the nuclear charge, each element has a unique photon energy, i.e., characteristic fingerprint X-ray fluorescence, which enables the multi-element analysis.

Figure 1.

Basic principle of X-ray fluorescence.

Apart from the conventional XRF microprobe, different variations of XRF imaging techniques, such as synchrotron X-ray fluorescence imaging (SXRFI) [16,27,34,37], X-ray fluorescence computed tomography (XRFCT) [16,27,38], confocal XRF (CXRF) [39,40], and total-reflection X-ray fluorescence imaging (TXRFI) [41] have been reported in recent years. Among these techniques, SXRFI has the highest element sensitivity due to absence of the bremsstrahlung background, while XRFCT can provide three-dimensional elemental composition in a sample [27,34]. Therefore, these two techniques are most widely used for the imaging of metals in biological samples.

In particular, SXRFI is a microanalytical technique for mapping the spatial distribution of elements [18,34,42-45]. Due to the unique fluorescence spectrum of each element, simultaneous multi-element analysis can be achieved using SXRFI with qualitative and quantitative modalities. Compared with the conventional micro-XRF (m-XRF), synchrotron X-ray source-equipped SXRFI shows higher sensitivity with the detection limit estimated between 5.0 × 10^-20 and 3.9 × 10^-19 mol/mm² and the spatial resolution improved to 150 nm for the imaging of trace elements [18,46,47]. By virtue of deep penetration of synchrotron X-rays, this technique is highly applicable for mapping the metals in biological samples, such as in whole cells or tissue at single cell and subcellular resolution. A variety of cellular studies utilizing SXRF have been conducted, especially the investigation of distribution and abundance of metals in biological samples.

The apparatus of SXRFI typically consists of X-ray source, undulator, crystal monochromator, focusing optics, motorized sample stage, X-ray fluorescence, and transmission detector (Figure 2) [18,27,34,38,48]. Undulator is used to boast a smaller source size of X-ray and produce a beam with enhanced brightness. To select the band of the incident X-ray beam, a crystal monochromator is equipped. The selected X-ray is then focused on the specimen through a Fresnel zone plate [49]. An energy dispersive detector is applied to record the information of multi-element simultaneously. The orientation of biological samples on the specimen scanning stage is corrected by the help of transmission detector.

Figure 2.

Schematic of an X-ray fluorescence microscope construction.

Due to its capability of 3-D elemental mapping within the sample, XRFCT has attracted increasing attention for imaging metals in biological samples in recently years [50,51]. This technique integrates the CT imaging of X-ray attenuation with typical secondary XRF, and thus the 3-D elemental mapping can be obtained by stacking and combining 2-D XRF imaging. Using XRFCT, the specimen is irradiated by a micro- or nano-focused X-ray beam, and the secondary X-ray is collected with an X-ray dispersive detector to record the energy of each XRF photon [27,34]. The XRF detector is designed to be 90° position to the incident X-ray beam to minimize the elastic and Compton scattered photons and improve the signal-to-background ratio [27,52,53]. The conventional XRFI apparatus (Figure 2) with an additional rotation stage is used as the microscope for the XRFCT [54].

The rotation of the XRFCT microscope is illustrated in Figure 3. For the sample with low elemental abundance, the scanning and rotation of the sample along a series of angles is often used [50,55,56]. The focused X-ray beam is raster scanned through the sample, and the XRF intensity is recorded by the XRF detector at each orientation [27]. Typically, the measurement of a single slice of the sample requires several hours, which limits its application in live biological samples. Fortunately, thanks for the improvement of XRF detectors and fast detector electronics, “on-the-fly” sample scanning modality can reduce the X-ray irradiation time, and the live sample 3-D XRFCT is possible [55,57]. In the case of high elemental abundance (circa > 1 wt%), full-field mode of XRFCT with a wide-fan X-ray beam can be used for the specific elemental mapping. With the wide-fan X-ray beam, only several minutes is required to map the metals in biological samples [34,58]. Thus, a large dimension of the sample can be imaged using such a technique with low risk of radiation damage.

Figure 3.

Schematic of a typical XRFCT rotation stage. The XRF is collected by a XRF detector, and X-ray attenuation is recorded by a transmission detector.

XRFCT is a non-invasive and highly sensitive technique for imaging metals with the 3-D model in living samples. In the XRFCT measurement, the pre-treatment of the biological samples, such as staining, fixation, and washing, is not required, which allows the accurate detection of metals in its native condition. The mapping of elemental abundance in living samples can be in the range of submicrogram per gram with a high spatial resolution (hundreds of nanometers) [59]. In addition, similar to the SXRFI, simultaneous multi-element analysis with qualitative and quantitative mode is also possible for XRFCT [27,34].

Considering the essential roles of metals and metalloids in living systems and the unique advances of X-ray fluorescence imaging, we will highlight the recent progresses in this review on using XRF imaging technique to examine metals and metalloids in biological systems. We will focus on the application of XRF imaging in the investigation of toxicology and roles of metals and metalloids, in particular in various diseases at the organ and tissue level, and at cellular or even subcellular level. In addition, the current and future challenges in this field in terms of X-ray fluorescence development and applications are also discussed.

XRF imaging of toxic elements

It has been reported that some of the heavy metals and metalloids, especially Cr, Co, Pb, Hg, As, are highly toxic to human health and the environment. In biological systems, these elements have been reported to affect cellular organelles and components, such as cell membrane, mitochondrion, lysosome, endoplasmic reticulum, nuclei, and some enzymes involved in metabolism, detoxification, and repair of damages [60]. The relative toxicities, symptoms, and the corresponding treatment are listed in Table 3 [61,62]. It has been demonstrated that the mechanisms of toxicity and carcinogenicity of metals and metalloids are relevant to the reactive oxygen species (ROS) production and oxidative stress. Here, we will briefly summarize the progresses of XRF imaging for the most commonly encountered toxic metalloids and metals, including As, Cr, Co, Pb, and Hg in living systems.

Table 3.

Typical toxicities of the most encountered metals and metalloids, and their treatment

Metal	Acute	Chronic	Toxic concentration	Treatment
As	Nausea, vomiting, “rice-water” diarrhea, encephalopathy, multi-organ dysfunction syndrome, long QT syndrome, painful neuropathy	Diabetes, hypopigmentation/hyperkeratosis, cancer: lung, bladder, skin, encephalopathy	24 h urine: ≥50 µg/L urine, or 100 µg/g creatinine	BAL (acute, symptomatic)
				Succimer
				DMPS (Europe)
Cr	GI hemorrhage, hemolysis, acute renal failure (Cr6+ ingestion)	Pulmonary fibrosis, lung cancer (inhalation)	No clear reference standard	N-cetylcysteine (experimental)
Co	Beer drinker’s (dilated) cardiomyopathy	Pneumoconiosis (inhaled); goiter	Normal excretion: 0.1-1.2 µg/L (serum) 0.1-2.2 µg/L (urine)	NAC
				CaNa2 EDTA
Hg	Elemental (inhaled): fever, vomiting, diarrhea, acute lung injury; Inorganic salts (ingestion): caustic gastroenteritis	Nausea, metallic taste, gingivostomatitis, tremor, neurasthenia, nephrotic syndrome; hypersensitivity (Pink disease)	Background exposure “normal” limits: 10 µg/L (whole blood); 20 µg/L (24-h urine)	BAL
				Succimer
				2,3-dimercapto-1-propane-sulfonic acid
Pb	Nausea, vomiting, encephalopathy (headache, seizures, ataxia, obtundation)	Encephalopathy, anemia, abdominal pain, nephropathy, foot-drop/wrist-drop	Pediatric: symptoms or [Pb] ≥45 µ/dL (blood); Adult: symptoms or [Pb] ≥70 µg/dL	BAL
				CaNa2 EDTA
				Succimer

As imaging

Arsenic (As) is widely distributed in the earth via both natural and anthropogenic pathways [63]. Despite its reputation as a deadly poison, As may be a necessary ultratrace element for some biological systems, such as red algae, chickens, rats, goats, and pigs. Excessive arsenic exposure has been associated with increased incidence of cancers, including lung cancers, skin cancers, and urinary bladder carcinoma in humans [64]. Compared to the reports on heavy metals, limited XRF imaging has been conducted to determine the quantitative spatial distribution of As in live cells and tissues, insects, and vertebrate samples such as fur and feathers.

XRF imaging has been applied to visualize the As distribution in HepG2 human hepatoma cells after exposure to arsenite (1 mM) or arsenate (20 mM). Munro et al. found that As was accumulated in the euchromatin region of the cell nucleus (following arsenite exposure), in accordance with As targeting DNA or proteins involved in DNA transcription [65]. The distribution, toxicity and biotransformation of arsenate in different life stages (larvae, pupae and adults) of a bertha armyworm moth (Mamestra configurata Walker) (Lepidoptera: Noctuidae) were investigated by Andrahennadi et al., and XRF imaging revealed the localized arsenic species, as well as zinc and copper within the gut [66].

Arsenic accumulation induced by chronic intake of arsenic-contaminated water has been examined in rat brain using the XRF imaging. The data showed that the accumulation of As was not linearly proportional to the treated arsenic dose, suggesting the existence of a protection mechanism that limits the transport of inorganic arsenic to the brain. The uniform spatial distribution of As was found, which is probably caused by the homogenous blood spreading to the brain [67]. The spatial distribution of As (and Se) has been evaluated at the cellular and subcellular levels in the skin of mice (Figure 4) [68]. Interestingly, the supplemental Se was found being effective preventing As accumulation in skin, suggesting the As-blocking effect of Se.

Figure 4.

Micro-XRF element mapping of hyperplastic epidermis taken from the skin of a mouse exposed to ultraviolet radiation (UVR) and As (III) for 182 days. These images pointed out the differing subcellular distributions of As and Se in mice given As (III) only, particularly in the overlay, where As was clearly evident throughout the nucleus (excluding the nucleus), whereas Se was concentrated in the peripheral region of the cells. Scale bar 100 mm. Adapted from Environmental Health Perspectives, reference [68].

XRF imaging has also been applied to study the toxicity of As in vertebrate samples, such as fur and feathers. For example, using XRF elemental mapping of the hair, Kempson revealed that the cause-of-death of Phar Lap, a successful and famous racehorse, could be As poison [69].

Hg imaging

Hg is a unique heavy metal that exists in three types of chemical species: elemental Hg, inorganic Hg, and organic Hg [63]. The Hg compounds are well known as the most toxic species [70]. Despite their extreme toxicity, humans and animals are all unable to avoid exposure to some forms of Hg because some Hg compounds are ubiquitous in the environment. However, it remains difficult to investigate the Hg uptake mechanism and the interactions with biological species [71]. XRF has been recently reported to visualize the uptake and distribution of Hg in living systems, such as larval stage zebrafish, human brain tissue, teeth, and human bones.

Using synchrotron X-ray fluorescence mapping, Korbas et al. examined the uptake and localization of organic Hg in zebrafish larvae [72]. They found that methyl and ethyl Hg compounds were mainly accumulated in the rapidly dividing lens epithelium, with lower levels in the brain, optic nerve and various other organs, implying the direct effects of Hg on the ocular tissue. The detailed mechanism underlying organic Hg transport and accumulation has been then investigated [73]. As shown in Figure 5, redistribution of Hg to the eye lens was observed after removal of fish from treatment solutions (contaminated with methyl-Hg L-cysteineate), indicating that eye lens are the major sink for methyl-Hg in early embryonic and larval stages.

Figure 5.

Histological images (lower) of head and XRF imaging (upper) quantitative Hg distributions in zebrafish larvae after a 12-h exposure to 2 µM methyl-Hg L-cysteineate (t = 0) followed by recovery time t = 24 h or 60 h in fresh system water. Scale bar 100 mm, BR: brain, EL: eye lens. Scale bar 100 mm. Adapted from JBIC Journal of Biological Inorganic Chemistry reference [73] with permission.

Inorganic Hg uptake and distribution were also investigated by Korbas et al. [74]. Unlike the methyl-Hg species [75], inorganic Hg was highly concentrated in olfactory epithelium and kidney in the absence of L-cysteine. However, with L-cysteine present in the treatment solution, mercuric bis-L-cysteineate species dominated the treatment, significantly decreasing uptake. In addition, quantitative XRF imaging was used to analyze Hg uptake in the absence and presence of 1-phenyl-2-thiourea (PTU), a widely used inhibitor to generate essentially transparent organisms for larval zebrafish [76]. The Hg concentration in the fish treated with solution of PTU + HgCl₂ was 60-fold lower than that in fish exposed to HgCl₂ solution. In contrast, both head and trunk sections of the larvae treated with CH₃HgCl and PTU had two fold higher Hg than in those treated with CH₃HgCl alone.

XRF imaging has also been employed to study the co-localization of Hg in biological samples [77]. When the human brain tissues were poisoned by methyl-Hg, Hg and Se were co-localized in the grey matter in the form of HgSe nanoparticle [78]. Exposure of larval zebrafish to inorganic Hg also showed nano-scale structures containing co-localized Hg and Se [79]. Supported by the microscaled X-ray absorption analysis, the co-localized deposits were most likely comprised of highly insoluble mixed chalcogenide HgS_xSe_(1-x) where x was 0.4-0.9, probably with the cubic zincblende structure.

Spatial distribution of Hg in human teeth filled with amalgams for more than 20 years has been determined by XRF imaging analysis [80]. Up to ~10 mg/g Hg was detected in the dentinal tubules several millimetres away from the amalgam location. In addition to Hg in human teeth, XRF imaging has been also employed to determine whether Hg is present in the bone as a result of environmental contamination or due to biogenic uptake [81]. The results revealed that Hg existed in part of the localized subset of the osteons.

Pb imaging

Pb is a toxic heavy metal that is taken up by the human body through food, drinking water, and inhalation, and is excreted via the gastrointestinal tract and the renal system [63]. Exposure to Pb is associated with chronic diseases in the nervous, hematopoietic, skeletal, renal and endocrine systems. It is known that Pb accumulates in the skeleton [82]. Thus, the understanding of Pb toxicity is of great importance due to the lack of treatment options for Pb-induced diseases.

XRF imaging could make a significant contribution, although to date only a few XRF imaging studies have been reported about Pb in animal systems. In 2006, Zoeger et al. used high resolution XRF imaging to determine the spatial distribution of Pb and other trace elements in normal articular cartilage and subchondral bone from adult humans with no history of work-related exposure to Pb [83]. As shown in Figure 6, a highly specific accumulation of Pb occurred in the tidemark, the transition zone between calcified and non-calcified articular cartilage. Quantitative fluorescence analysis revealed that a 13-fold higher Pb concentration was in the tidemark of articular cartilage when compared to subchondral bone [83]. The observation was further confirmed by the same research team through investigation of the osteochondral samples, which were long-term treated to increased lead (Pb) concentrations [84,85].

Figure 6.

A. Backscattered electron (BE) image of analyzed chondral/subchondral region of the patella. Non-calcified cartilage (I), tidemark (II), calcified cartilage (III), subchondral bone (IV), and cement-lines (V) can be clearly identified. Length of scale bar: 100 mm. B. Ca, Zn, Sr, Pb signal intensity mapping in the corresponding region. C. Fluorescence intensity profiles along the marked line. Maximum fluorescence intensities were normalized to 10. Pb and Zn maxima can be exactly allocated to the tidemark of articular cartilage. Scale bar 100 mm. Adapted from Osteoarthritis and Cartilage reference [83] with permission.

The distribution of Pb in the human bone tissue has been also determined by XRF imaging [86-88]. The studies conducted by Zoeger et al. revealed that Pb was mostly located at the outer border of the cortical bone in various samples [87]. A remarkable association between Pb and Zn content can also be observed. Pemmer et al. reported that the levels of Pb and Zn were significantly higher in the cement lines than the adjacent mineralized bone matrix, possibly due to the different uptake mechanism [86]. Arora et al. reported the first application of XRF imaging technique in measuring the distribution of Zn and Pb in the ameloblasts in developing Wistar rat teeth. Results showed that Pb was only visualized reliably in developing enamel but not in ameloblasts [89]. Interestingly, XRF imaging has been reported as a useful technology in archaeological studies, and used to analyze bone and hair samples from Ferrante II of Aragon, King of Naples (1469-1496) and Isabella of Aragon, Duchess of Milan (1470-1524). Results showed that Pb was localized to microanatomical locations, consistent with bone remodelling events and compositionally similar to Pb-substituted hydroxyapatite, i.e. Pb binding in bone [90].

Co imaging

Co is very much widespread in the natural environment [91]. For humans, Co is an essential trace element required for the generation of vitamin B₁₂. However, it becomes toxic at high concentrations, thus leading to adverse health effects [16]. There are a few studies done to investigate Co toxicity, uptake, transport, and distribution using XRF imaging analysis [92,93].

In 2009, XRF imaging analysis in tomography mode was performed for the first time on a single cell (HaCaT human keratinocytes) to determine the 3D intracellular distribution of cobalt [93]. The images showed that Co was distributed in the nucleus and perinuclear region in HaCaT cells, implying the possible direct interactions with genomic DNA and nuclear proteins. The perinuclear accumulation in the cytosol suggests that cobalt could be stored in the endoplasmic reticulum or the Golgi apparatus. In addition, when the cells were exposed to exogenous Co, a decreasing level of Zn and Mg was found, indicating a likely replacement Mg and Zn by Co in protein binding sites.

Cr imaging

Cr plays important roles in biological functions of life, but can become toxic at higher doses. Its toxicity is also dependent on its chemical state [63]. It is well known that Cr compounds are stable in both trivalent [Cr(III)] and hexavalent [Cr(VI)] state. Exposure of human beings and animals to Cr has been a major concern because of the high risk of Cr-induced cancers, such as lung cancer [94]. A generally accepted mechanism for Cr(VI)-induced genotoxicity and cancers includes intracellular internalization (through anion channels for water-soluble chromates or phagocytosis of insoluble chromates), transport, reduction to Cr(III), and then formation of Cr-DNA and Cr-protein complexes.

The mechanism of Cr toxicity in live systems can be directly examined by XRF imaging [95]. The investigation conducted by Fayard et al. showed homogenous distribution of Cr in Chinese Hamster Ovary cells (CHO-AA8) exposed in vitro to both soluble and insoluble Cr compounds. Interestingly, Cr(VI) was not detected, suggesting a mechanism of rapid intracellular reduction [96]. Further studies in the same group confirmed the reduction of Cr(VI) to Cr(III) and revealed the distribution in nucleus of Institut Gustave Roussy ovarian cell line 1 (IGR-OV1) cells [97]. Cr seemed to accumulate P-rich regions, such as nucleus and the area outside the nucleus (acidic vacuole), as reported by Dillon et al. in V79 Chinese hamster lung cells. This accumulation may indicate that Cr is capable of targeting the DNA and causing the genotoxic damage [98].

Using XRF imaging, Harris et al. investigated the dynamic process of intracellular uptake, distribution, and biotransformation of Cr(VI) in human lung cells [99]. A549 human lung adenocarcinoma epithelial cells were treated with Cr(VI) at 100 mM for 20 min and 4 h, respectively. As shown in Figure 7, Cr was found in a small area of cytoplasm after 20 min treatment, while distributed to whole cells after 4 h treatment due to uptake of more Cr. Interestingly, a higher Cr concentration in the nucleus and cytoplasmic membrane was still observed, indicating the accumulation in the nucleus and the cytosol [97].

Figure 7.

Distribution mapping (A) and relative amount (B) taken up by single A549 human lung carcinoma cell treated with 100 µM Cr(VI) in comparison with untreated cells. Adapted from JBIC Journal of Biological Inorganic Chemistry reference [99] with permission.

XRF imaging of metals for various diseases

Imaging of metals in cancer research

The association of metal exposure with cancers is well documented [100]. Currently, metals are thought to promote cancer development by a number of mechanisms, such as inducing reactive oxygen species (ROS) to oxidatively damage DNA, protein, and lipids [101,102]. Thus, XRF imaging technique has been actively used to study the relationship between the metal concentrations and a number of cancers, including breast cancer, prostate cancer, and colorectal cancer recently [56,103-105].

XRF has been applied to image K, Fe, Cu, Zn, and Ca in breast cancer [106-108]. Compared to that in the normal tissues, the concentration of assessed metals increased in the tumour region, especially Zn and Cu [106,109,110]. Further investigation by Farquharson et al. confirmed the increasing concentration of Ca, Cu, and Zn in the tumour lesion, but a lower concentration of Fe in some of the tested samples [109,111]. Zn is being considered as a possible marker of human prostate cancer [112]. Using XRF imaging, Ide-Ektessabi reported that the Zn concentration is significantly lower in prostate cancer tissues compared with that in health specimens [113]. This finding was further confirmed by Podgórczyk et al. [112]. The correlation between Zn and Ca suggested that Ca mediated Zn accumulation in prostate cancer tissues [114].

Metals in other cancer lesions and organs have been also investigated using XRF imaging technology. The localization and the relative concentration of Zn, Cu, Fe and Ca in primary colorectal cancer and secondary colorectal liver metastases were examined by Al-Ebraheem et al. Significant increase in the concentration of Zn, Ca, Cu and Fe was found in necrotic tissues [115]. They also quantified the concentration of Fe, Cu, Zn, and K in normal and malignant liver and kidney tissues. The data indicated that the Zn concentration was reduced by 63% and 26% in liver and kidney tumors, respectively. Fe, Cu, and K concentrations were increased in kidney tumors by 150%, 8% and 90%, but reduced in liver tumors by 76%, 29% and 43%, respectively [116].

Among all metals investigated in cancer research, Cu is special due to its special roles in cancer development [111]. It is well known that angiogenesis is vital for supplying oxygen for the tumor growth. Thus, depletion of Cu has been shown to inhibit angiogenesis in a wide variety of cancer cells and xenograft systems [117]. Using XRF imaging, Finney et al. investigated the localization and roles of Cu in angiogenesis system. They found that Cu was remarkably redistributed from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane [118].

Imaging of metals for neurodegenerative diseases

The brain is rich in metals, such as Fe, Cu, and Zn as essential cofactors in metalloproteins, and Hg and Pb as neurotoxins [119,120]. These metals have been implicated in various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), prion diseases, and Huntington’s disease [121]. There is increasing investigation of precise roles of these metals in neurodegenerative diseases using XRF imaging technology [122].

Utilizing XRF imaging, Pushie et al. investigated the level and distribution of Cu, Fe and Zn in the brain of mice that express different levels of prion protein (PrP^C), a family of fatal neurodegenerate diseases [123]. Results suggested that the amount and distribution of specific metals within the central nervous system is regulated by the PrP^C. Subcellular distribution of metals in a childhood neurodegenerative disorder revealed that Zn nuclear-to-cytoplasmic trafficking was perturbed in diseased cells and the Ca subcellular distribution was drastically altered in CbCln6 ^nclf cells [124].

Mn is well known as the magnetic resonance imaging (MRI) reagent in vivo. However, it is reported that Mn is neurotoxic, by accumulating in the hippocampal formation (HPCf) of brain and causing symptoms similar to those associated with Parkinson’s disease [125,126]. Thus, Robison et al. examined the distribution of Mn in the HPCf for Sprague-Dawley rats with chronic Mn exposure, and quantitatively compared Mn distribution with that of other biologically relevant metals, such as Fe, Cu and Zn [127]. In consistence with MRI results, an increasing Mn concentration in hippocampal, especially in the dentate gyrus (DG) and the cornus ammonis 3 (CA3) layer, was observed. In addition, significant spatial correlation of Mn-Zn was observed across the HPCf substructures (Figure 8).

Figure 8.

Analysis of the hippocampal formation regions. A. Two colored images displaying Fe-Mn, Cu-Mn, and Zn-Mn respectively. Note that Mn and Zn co-localized in the CA3 of the HPCf, while a small portion of Mn also co-localized with Fe within the DG as indicated by white pixels in the images. Scale bar: 1 mm. B. Scatter plots of the mean metal concentrations in regions identified by clustering. Scale bar 1 mm. Reproduced from reference [127] with permission of The Royal Society of Chemistry.

XRF imaging was also used to investigate the roles of metals associated with stroke [128] and epilepsy [129-132]. Images showed that Cu plays important roles in the pathogenesis of epilepsy in the Wistar rats. Lower levels of Cu were found in the latent period compared with the control group and even in the acute period [131]. In quantitative analysis of rat brain undergoing pilocarpine-induced epilepsy, a relatively lower Cu level was found in the dentate gyrus, and a lower Zn level in the hippocampus and dentate gyrus, where a higher Ca level was observed in this area [132].

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly, affecting more than 27 million people worldwide (about 2% population in the developed world [133-135]. It is one of the most prevalent and debilitating neurodegenerative diseases and yet it is typically diagnosed only after cognitive symptoms appear, which is normally too late for effective treatment [134]. Pathologically, AD is characterized by extracellular amyloid plaques composed of insoluble amyloid beta (Aβ) protein, and intra-neuronal neurofibrillary tangles (NFTs) containing hyperphosphorylated tau protein. At the molecular level, metals, including Cu, Zn and Fe, have been confirmed as neurochemical factors to be involved in the metabolism and functional expression of Aβ and amyloid precursor protein (APP) [135].

XRF imaging has been developed for the quantitative analysis of metal metabolism in animal models (Figure 9) [136]. The observations reported by Leskovjan et al. revealed that the level of Zn, Cu, Ca, and Fe was all increased in human amyloid plaques, suggesting that these metals are involved in neurodegeneration [137]. Further studies on the time course of the metal concentration and distribution by the same group revealed that Fe in the cortex was 34% higher than age-matched controls at an early stage, corresponding to the commencement of plaque formation [138]. Using XRF imaging, Wang et al. found that Fe and Ca level increased with brain aging in both AD and control mice, while the level of Cu, Fe, Zn and Ca appeared significantly high in AD mice and showed an obvious age-dependent rise [139]. The increase of Zn in a small regional hippocampal was also confirmed by Adlard et al. via XRF imaging [140].

Figure 9.

The quantitative images of Ca, Fe, Cu and Zn in the brain section of Alzheimer’s disease mouse. a: Substantia nigra region, b: Superior colliculus, c: CA3 area of hippocampus, d: Dentate gyrus region. Scale bar 1 mm. Adapted from reference [136] with permission of The Royal Society of Chemistry.

Parkinson’s disease (PD) is a progressive neurological condition [141]. PD is the most common serious movement disorder in the world, and affects about 1% adults older than 60 years and 4% populations older than 80 years. The most characteristic hallmark of PD is confirmed to be the loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNc). Nevertheless, the triggers for these events are still unclear [142,143], while the changes in biometals in brain have long been suspected to play a key role in the PD development. Recently, XRF imaging has been evaluated by mapping and quantifying metals, such as Fe, Zn ad Cu in brain slices from PD and unaffected brains [144,145].

Employing XRF imaging of trace elements in the pathogenesis of PD, Chwiej et al. demonstrated that elements P, Cl, Fe, Cu and Zn played important roles in the process of differentiation between neurons [145]. Due to the importance of Cu, Davies et al. investigated the changes in Cu and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and non-degenerative brain regions. They found a significant decreasing in levels of Cu and Cu-transporter protein 1 in surviving neurons in the SN and LC in PD patients [146].

Robison et al. introduced XRF imaging as a new quantitative tool to determine the Mn distribution in the brain [147]. As shown in Figure 10, the highest Mn level was observed in the globus pallidus (GP), the thalamus (Th), and the substantia nigra pars compacta (SNc) in the brain. But following studies showed that Mn accumulation in SNc is higher than GP and Th. [126]. Dučić et al. showed that Mn was localized in cytoplasmic/paranuclear in dopaminergic neurons after treated with Mn [148], while Mn was accumulated within Golgi apparatus in dopaminergic cells, PC-12 [149].

Figure 10.

XRF imaging of Mn distribution in brain sections of control and treated rats. Mn distribution of coronal sections from untreated (control) rats (A) and Mn treated rats (B). AB, axonal bundle; CPu, caudate putamen; GP, globus pallidus; HPC, hippocampal formation; IC, internal capsule; lv, lateral ventricle; Th, thalamus; SNc, substantia nigra compacta; SNr, substantia nigra reticular. Scale bar 2 mm. Adapted from PLOS ONE, reference [147].

Conclusions and future perspectives

Over the past decade, XRF imaging technique for determining the level and distribution of metals and metalloids has been rapidly developed. This method has been recognized as an ideal tool to reliably determine the elemental distribution in tissue specimens at the cellular, even subcellular level with high sensitivity and low background. In this review, we have presented the general principle of XRF imaging and summarized the recent applications in determining the spatial distribution of metals and metalloids in biological specimens to examine their toxicology and their possible roles in development of various diseases. After a thorough survey of published papers, we are sure that XRF imaging will become a superior tool to study metals and metalloids in animals and humans in the near future.

However, this new technique has faced a few challenges in the future applications in life science. The first challenge is the imaging scan speed bottleneck. At current, the scan speed is relatively slow, so XRF imaging is still not suitable for high-throughput analysis. The second challenge is the limited imaging resolution. It is true that imaging of metals and metalloids has been reported at the cellular or even subcellular levels, higher resolution is still required to determine the distribution and understand the biomolecule-metal interactions as well as metal metabolism, which may help reveal the detail mechanisms that metals or metalloids are involved in disease development. Thus, faster and more efficient imaging system with higher resolution will be an important research direction. The third challenge is the imaging sensitivity limitation. As summarized in this review, current applications of XRF imaging are mainly focused on the toxicology of metals and metalloids, and their possible physiopathology in cancers and neurodegenerative diseases. As a powerful imaging technology, XRF should be widely used to map the trace amount of elements in other diseases, such as cardiovascular diseases [150,151], atheroscelerosis [152], and dental diseases [153,154], improving our understanding of the roles that metals and metalloids play in the development of these diseases.

Disclosure of conflict of interest

None.