Mass spectrometry imaging of metals in tissues and cells: methods and biological applications

Abstract

Background

Metals are pervasive throughout biological processes, where they play essential structural and catalytic roles. Metals can also exhibit deleterious effects on human health. Powerful analytical techniques, such as mass spectrometry imaging (MSI), are required to map metals due to their low concentrations within biological tissue.

Scope of review

This Mini Review focuses on key MSI technology that can image metal distributions in situ, describing considerations for each technique (e.g., resolution, sensitivity, etc.). We highlight recent work using MSI for mapping trace metals in tissues, detecting metal-based drugs, and simultaneously imaging metals and biomolecules.

Major conclusions

MSI has enabled significant advances in locating bioactive metals at high spatial resolution and correlating their distributions with that of biomolecules. The use of metal-based immunochemistry has enabled simultaneous high-throughput protein and biomolecule imaging.

General Significance

The techniques and examples described herein can be applied to many biological questions concerning the important biological roles of metals, metal toxicity, and localization of metal-based drugs.

1. Introduction

Metals play essential roles in processes required for normal cellular function. Although this minireview focuses largely on mammalian systems, the roles of metals are highly evolutionarily conserved across species and even kingdoms, a testament to their critical importance.

Metals provide key structural elements by coordinating amino acid ligands, which are required for the function of numerous proteins. Examples of metals that fall into this category include copper, iron, magnesium, manganese, molybdenum, nickel, and zinc [1]. Well-known proteins that contain coordinating metals serving in this role are zinc finger proteins, which function in transcription and chromatin remodeling, DNA polymerases, and glycolytic enzymes such as hexokinase. Metals can also function as components of more complex cofactors. Examples are heme moieties, which consist of iron coordinated in a porphyrin ring, and iron-sulfur clusters, which contain iron coordinated to sulfur atoms. These metal-containing cofactors are required for mitochondrial respiration, DNA repair, and many other critical functions.

In addition to their structural roles, selected metals contribute to the catalytic function of proteins through their ability to gain and lose electrons (i.e., redox cycle). Metals that actively change redox states during catalytic cycles are primarily iron and copper, which play key roles in electron transfer reactions through their association with cytochromes, iron-sulfur clusters, and cupredoxins [2]. Metals also function directly or indirectly in signaling pathways. Notable among these is calcium, which acts as a secondary messenger in functions as diverse as muscle contraction, neuronal transmission, motility, and cell growth. Another example is iron, which serves as a cofactor in proteins that regulate their own metabolism. Iron also plays a role in ferroptosis, which is an iron-dependent form of cell death [3, 4]. These examples illustrate that cellular processes that depend on metals are extensive and diverse.

In addition to their beneficial functions, metals can also exert cytotoxic effects. Toxic environmental metals such as cadmium are well known for their negative effects on health, but endogenous metals can also be toxic when present in excess. For instance, a well-known cytotoxic effect of excess copper and iron is the production of reactive oxygen species (ROS), which can damage a wide range of cellular targets, including DNA, proteins, and lipids. For this reason, the uptake and distribution of metals is carefully managed through a host of exquisitely sensitive regulatory mechanisms. For example, in the case of iron, intracellular management of iron is accomplished by the iron regulatory proteins, which coordinately control the stability and expression of proteins involved in cellular iron uptake, storage, and efflux [5]. At the level of the whole organism, further regulation is accomplished by the peptide hormone hepcidin, which orchestrates iron transit from the diet to the bloodstream [6]. In addition to careful homeostatic regulation, another layer of protection against metal toxicity is afforded by proteins that bind and detoxify metals, such as metallothionines, as well as antioxidant pathways controlled through broadly acting transcription factors such as Nrf2 [7]. Attesting to the critical need for maintaining appropriate metal homeostasis, genetic or dietary metal imbalance is a direct cause of human disorders. For example, iron excess causes hemochromatosis, and conversely, iron deficiency can lead to anemia (the most prevalent nutritional deficiency in the world).

The plasticity and functional diversity of metals has facilitated their deployment for medicinal purposes. The platinum drugs (cisplatin, carboplatin, etc.) are an example of metal-based anti-cancer drugs with a longstanding history of successful clinical use; more recently, ruthenium-based drugs, as well as metal-containing nanoparticles, are being developed as anti-cancer agents [8, 9]. Although still in its early stages, the growing appreciation of the structural role of metals in metal-containing enzymes is facilitating the tailored design of purpose-built metal-containing enzymes for medicinal purposes [10].

Given the pervasiveness of metals in human biology and their role, direct or indirect, in health and disease, in situ imaging of metals has emerged as a critical need. Imaging provides information on spatial distribution, a useful and necessary complement to metal quantitation. For example in toxicology, metal localization is important in identifying sites of metal accumulation and toxicity following exposure to metals such as aluminum, arsenic, and lead [11], as well as in determining the efficacy of therapeutic manipulations. Monitoring the pattern of iron deposition in patients receiving chronic transfusions (such as patients with thalassemia and sickle cell disease) is an important determinant of liver damage, a common and deleterious side effect of transfusion therapy [12]. In cancer research, a key aspect of understanding the efficacy, off-target toxicity, and development of resistance to platinum, ruthenium, and other metallodrugs lies in understanding how they distribute within organs, tissues, and cells [8].

There are a variety of methods for imaging metals within mammalian tissues. Of these, mass spectrometry imaging (MSI) is an approach that offers high sensitivity with elemental and molecular specificity. Many MSI techniques are able to image biochemical species with high spatial resolution, often down to subcellular resolution [13]. Thus, MSI has enabled the mapping of essential, toxic, and exogenous metals, such as drugs and labels, in cells and tissues. A key benefit of this technology is the ability to utilize it in a multi-technique fashion, where MSI data can be directly registered to optical- [14], spectroscopy- [15], and force-based [16] imaging data to provide deeper biological and mechanistic insights into samples of interest. This review focuses on strategies for imaging trace metals, detection of metal-based drugs, and simultaneous imaging of metals and biomolecules with MSI, primarily focusing on research published over the last half decade. We also provide a brief overview of MSI, including highlighting key sample considerations, for those looking to explore the use of MSI for their research.

2. Technique overview

2.1. MSI instrumentation and methods

There are a variety of different MSI approaches available, each with its own strengths and limitations. This brief review is not meant to provide an exhaustive list of MSI methods, but rather to highlight common techniques used for imaging metals and metal-complexes and notable recent advances in the technology (Figure 1). For more detailed reviews of MSI instrumentation and methods, please see elsewhere [13, 17, 18]. The choice of the MSI method employed depends on the spatial resolution needed, the endogenous element or molecule of interest and its abundance, and the sample state. Also, one should consider whether combined imaging approaches are required, which can dictate experimental preparation and workflow [14, 15]. Within MSI instrumentation, there are two main instrumental considerations: the ionization source and the mass analyzer.

Figure 1.

Overview of mass spectrometry imaging (MSI) approaches used for visualizing metals and metal-complexes within biological tissues. (A-C) General ionization schemes of the three different types of desorption/ionization methods utilized in MSI. (D) General considerations for each technique, where the range of sensitivities in (y-axis) is compared against the spatial resolution range (x-axis) for these MSI methods. The spatial dynamic range is illustrated by the transparent tan-colored boxes. Cell size dimensions and the lateral resolution of other structural imaging techniques are displayed along the x-axis for comparison.

2.1.1. Ionization sources

The basic requirement for all mass spectrometry (MS) analyses is that analytes of interest contain a charge to be detected. For MSI methods, this entails analytes in the condensed phase (e.g., endogenous elements and molecules within tissue) being transferred into the gas phase, ionized, and then detected. Essentially, there are three different types of ionization sources for in situ desorption/ionization of chemicals from samples of interest: primary ions, lasers, and liquid junctions (Figure 1A-C, respectively). Furthermore, inductively coupled plasma (ICP) ionization can be combined with laser-based desorption approaches to atomize molecules prior to mass analysis. Most MSI methods utilize the so-called microprobe mode approach, in which the ionization source is manipulated serially across the surface of the sample, by either mechanical movement of the target stage or beam rastering. This generates MS measurements at each point that can be used to create an ion image.

Secondary ion mass spectrometry (SIMS) uses a primary ion beam that bombards the sample producing secondary ions from surface molecules (Figure 1A). SIMS methods offer the highest lateral resolution (regularly <100 nm) [19]. The primary ion beam can be pulsed (sometimes called ‘static mode’ when the ion flux to the sample is maintained below a defined limit) or can constantly bombard the sample (used in ‘dynamic mode’), and these different modes of operation provide varying levels of chemical and spatial information. Note the majority of SIMS measurements require analysis to be performed under high vacuum, and the use of a high-energy primary ion beam for ionization causes excessive fragmentation of biomolecules, which limits the detection of intact species with SIMS. However, SIMS methods are especially powerful for mapping metals within biological samples.

Laser desorption/ionization (LDI) and laser ablation (LA) methods are popular approaches for mapping analytes within a sample. Here, direct irradiation of surfaces with lasers characteristically causes excessive and often complete fragmentation of the ablated molecules (Figure 1B). This phenomenon has been exploited in some applications for elemental surface analysis (e.g., visualizing metal distribution in tissues), which can provide exceptional quantification capabilities. LA-ICP-MS is an especially common method for visualizing the regional and cellular distribution of metals and related metal-containing biomolecules within tissues [20]. Matrix-assisted laser desorption/ionization (MALDI) is perhaps the most common ionization technique used in MSI of biological tissue [21]. In MALDI, samples are coated with a chemical matrix (e.g., a low molecular weight organic acid) that assists in ‘soft’ desorption and ionization (i.e., causing minimal molecular fragmentation) of surface molecules. Since MALDI-MSI methods typically detect intact molecular species, they are commonly used to map the location of metal-coordinated species within a sample (e.g., heme). For all LDI/LA approaches, the lateral resolution is limited by the ability to focus light, which is diffraction limited and depends on the wavelength of the light source. In practice, this translates to the possibility of submicron resolution, but for most applications anywhere from 1 to 50 μm lateral resolution is common (depending on the method). Given the versatility and number of LDI/LA methods, these types of sources are capable of mapping nearly every class of bioactive molecule (e.g., metals to metabolites to large proteins).

Approaches that use liquid junctions for biochemical extraction/desorption have been increasingly popular due, in part, to their ambient ionization ability and their affordability (Figure 1C). These methods use charged solvent droplets (i.e., desorption electrospray ionization; DESI) or a continually refreshed liquid-surface interface that desorbs and ionizes species from the sample (i.e., nanospray-DESI; nano-DESI). These have limited lateral resolution compared to SIMS and laser-based methods (e.g., lateral resolutions commonly ≥ 20-50 μm). Liquid junction-based ionization methods provide the softest ionization, where minimal to no fragmentation of the endogenous molecules is possible. As such, only metal-coordinated molecules are detected with these approaches. A strength of these approaches is that little sample preparation is required, and many samples can be measured ‘as-is,’ which means live cell cultures could be analyzed, for example [22].

2.1.2. Mass analyzers

Besides ionization, the mass analyzer employed is also important in the type of information attainable in an MSI measurement. Quadrupole-, magnetic sector-, and time-of-flight (TOF)-mass spectrometers offer high duty cycles (10s to 100s of MS measurements per second), can be made within a compact footprint, and are robust, but they provide limited mass accuracy and mass resolution (the ability to separate two mass spectral features). High resolution mass analyzers, like Fourier transform ion cyclotron resonance (FTICR)-MS and Orbitrap MS, overcome these limitations, and can provide mass spectral information with the mass accuracy and resolution needed for greater confidence in molecular identifications. For example, FTICR-MS is commonly used to determine the isotopic fine structure of molecules, providing very high confidence molecular formula annotations [22]. However, these MS instruments operate at slow duty cycles (<1-10 MS measurements per second), which limits their use in many imaging applications because the often require 1,000s to 100,000s MS analysis (i.e., a MS analysis is performed at each location/pixel). Another key factor in imaging metals within biological tissues that must be considered is whether the goal is to measure elemental or molecular composition. FTICR-MS and Orbitrap-MS instruments have lower mass limits that prevent, or make it difficult, to measure elemental metal species. Quadrupole-, magnetic sector-, and TOF-MS are especially well suited to map elemental composition within a sample. However, magnetic sectors are only able to measure a few mass channels at a time. Thus, they are not ideal for untargeted approaches. Quadrupole-MS has high mass selectivity, sensitivity, and a high duty cycle, but provides low mass resolution. Alternatively, multi-quadrupole instruments provide increased sensitivity and allow selection of specific ions for targeted analysis. TOF-MS provides high duty cycle data acquisition across the entire mass spectrum, enabling rapid elemental and molecular analysis of large cell arrays or tissue sections [23, 24]. TOF-MS does have a limited mass resolution of small molecules, making it challenging to differentiate isobaric species and provide high confidence molecular annotations. However, the limited mass resolution of TOF-based instruments does not limit their utilization for measuring elemental metal composition, as in mass cytometry by TOF-MS (CyTOF). Notably, both quadrupole- and TOF-based analyzers are used in ICP-MS approaches.

2.2. Sample preparation

Sample preparation is an extremely important step in all MSI workflows, where one must consider how sample handling and preservation affects the chemical makeup and fidelity of the endogenous analytes. Sample preparation methods will differ depending on the sample type, ionization source, and components of interest. However, the workflow generally includes sample collection, preservation, mounting, and ionization aiding. The reader is referred to other reviews for more comprehensive protocols for biological sample preparation for MALDI-MSI [25], SIMS [19, 26], and LA-ICP-MS [27]. Here, we focus on considerations for imaging metal ions within mammalian samples. Sample collection will depend on the sample’s source, such as donor tissue or cultured cells in vitro. Regardless, protocols must always consider the delocalization or degradation of target compounds. When small, diffusible elements such as potassium or sodium are the analyte of interest, tissues must be immediately frozen or fixed to prevent the rapid diffusion of these ions. Often fresh tissue is snap-frozen, which prevents the diffusion of most molecules and will not affect the chemical composition of the sample. Paraffin embedding is also common, but this is not suitable for imaging metals and small molecules that may wash out during the preservation process [17]. For cell culture, chemical fixatives such as glutaraldehyde or osmium tetroxide are often used for crosslinking proteins and lipids, respectively, and immobilizing larger biomolecules [28]. However, to prevent diffusion of small molecules and soluble analytes, samples must be rapidly frozen by freeze-drying or high-pressure freezing; then dehydrated and exposed to fixatives while frozen.

After sample preservation, frozen or embedded samples are often sectioned and mounted on a suitable substrate for each technique. When mounting frozen samples, the use of warm slides can create small water droplets from melting ice crystals on the sample, resulting in analyte delocalization. Transferring samples onto cold substrates leads to better analyte localization [29]. LA-ICP-MS, MALDI-MS, and (nano-)DESI-MS can be performed on glass slides or conductive glass slides (e.g., indium-tin oxide coated). Whether the substrate needs to be electrically conductive depends on several experimental factors (e.g., lateral resolution, probe size, duty cycle, and more). For MALDI-MSI experiments, samples are coated with a matrix (e.g., dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid are commonly used in metal imaging studies). Note that standard matrices may suppress the ion yields of biomolecules and metals [8], although strategies are available to overcome this limitation: for example, treatment with derivatization agents can help recover metallodrugs ion yields [30]. For SIMS samples, ion yields can be suppressed by the biological samples themselves, which are electrically insulating. SIMS samples must therefore be placed on conductive substrates, such as silicon or gold, and/or samples should be sputter coated with a thin layer of a (non-endogenous) metal to increase the conductivity and ion yields [28].

3. Applications in human tissue analysis

As noted, this review focuses on strategies for imaging trace metals, detecting metal-based drugs, and simultaneous imaging of metals and biomolecules. Many biological questions may benefit from using so called ‘multimodal’ approaches, where one or more mass spectrometry approaches, often in conjunction with other imaging methods, map metals and their associated biomolecules within samples of interest.

3.1. Trace metal imaging with LA-ICP-MS

Often metals are at low concentrations in biological tissue, requiring powerful analytic techniques. Among the methods reviewed herein, LA-ICP-MS provides the highest sensitivity for elemental imaging, and has been applied in the characterization of metals in neurobiology [31], nephrology, and cancer studies [32, 33], amongst others. LA-ICP-MS detects sub μg·g⁻¹ elements, allowing the detection of slight changes in concentrations of metals indicative of disease and treatment [34]. For example, recent work investigated the role of metal regulation in Alzheimer’s disease. The amount of trace metals is typically tightly regulated in the brain. However, an increase in copper, iron, and zinc has been associated with Alzheimer’s disease, but the role of these metals in disease progression remains unclear. In a recent study, LA-ICP-MS was used to obtain precise, spatially resolved measurements of brain iron, zinc, copper in a mouse model of tau protein pathology [33]. This study demonstrated an association between tau protein and elevated iron concentration in the hippocampus [35] . In another example, the use of LA-ICP-MS enabled simultaneous imaging of iron and gold nanocluster-labeled ferroportin (an iron efflux pump) in the hippocampus of brains from Alzheimer’s disease patients [35, 36].

Given trace metals can indicate the existence of various disease states, LA-ICP-MS can be a valuable tool for detecting very small changes in metal composition that might be related to disease progression. For example, blood serum may be analyzed to detect trace iron, magnesium, and other metals in the blood, which could provide a noninvasive diagnostic tool [37]. A similar concept can be applied to tissue biopsies, where the atomic metal signals detected with LA-ICP-MS can be correlated with tumor tissue using machine learning algorithms. Such atomic signatures may prove to be powerful new tools in cancer research and disease management [38].

3.2. Imaging trace metals and biomolecules with SIMS

In most cases, imaging biomolecules and metals within tissues requires complementary approaches. However, SIMS can be used to simultaneously image both biomolecules and trace metals. SIMS also provides high lateral resolution (~100 nm-1 μm) and the ability to profile multiple layers of material by using dynamic sputtering to remove material during image acquisition. Nevertheless, a limitation of SIMS in quantifying metal concentrations is that not all biochemicals can be readily ionized using a primary ion beam [19, 39]. To overcome this limitation, SIMS can be correlated with other techniques, such as ICP-MS or electrochemical methods for quantifying metal concentrations and imaging biomolecules [40]. There are a number of recent developments in SIMS instrumentation to image biomolecules. The use of a water gas cluster primary ion beam has allowed simultaneous imaging of intact biomolecules. This primary ion beam does not fragment molecules as much as other primary ion beams used in SIMS [41]. The orbitrap-SIMS instrument also permits high mass resolution for the imaging of biomolecules with submicron lateral resolution [42]. This technique has been utilized in imaging the 3D biomolecular composition of biofilms including elemental potassium and various small lipids and metabolites [43].

Since metal dysregulation is often associated with changes in biological composition, mapping metal, metabolite, and lipid changes can help elucidate the role that metals play in disease. Recent work on tissue sections acquired from pancreatic and lung cancer samples has revealed changes in metabolism and metal concentrations, such as accumulation of magnesium and heme in stromal cells (Figure 2A) [44]. This work demonstrated the ability to use TOF-SIMS analysis to elucidate biochemical changes during cancer progression and characterize heme, histidine, and iron in the tumor microenvironment. Additionally, histological staining and correlated imaging with sum harmonic generation was able to localize blood vessels and specific cell types [44]. Using 3D TOF-SIMS, multiple tissue layers can be probed in a single imaging run. This has been especially useful for probing multiple cell layers in tissue such as lung, where toxic metals may accumulate in specific cell layers. Using 3D TOF-SIMS analysis ZrO⁺ nanoparticles were found primarily in phagocytic cells with only small quantities in the epithelium (Figure2B) [45]. The high lateral resolution of TOF-SIMS has enabled imaging the intracellular heterogeneities of trace metals such as iron, magnesium, and gold simultaneously with lipids and metabolites in biological tissues and single cells [45-47]. The information acquired by TOF-SIMS analyses can be used to identify organelles that exhibit changes in metal content during disease progression. For example, in chronic obstructive pulmonary disease, iron was shown to accumulate within mitochondria and lysosomes, disrupting normal cell function [47].

Figure 2.

Examples of TOF-SIMS studies showing the distribution of metals in tissues. (A) Pancreatic tissue section containing a tumor and blood vessels. (a & e) Hematoxylin & Eosin-stained histology images of the tissue analyzed. TOF-SIMS ion images show the distribution of (b) iron, (c) histidine, and (d) heme fragment ions. (e-h) Illustrate the blood vessel at a higher magnification. (f) the TOF-SIMS overlay displays phosphocholine in red; the sum of iron, heme, and histidine in green; and magnesium in blue. (g) Sum harmonic generation imaging locates the vasculature and (h) autofluorescence images help locate cells. These orthogonal imaging methods (a, e, g, h) provide more biological context to the TOF-SIMS imaging data. Scale bars are 100 μm. (B) Depth profiled lung tissue section shows zirconium-oxide nanoparticles within alveolar compartments. K₂CN⁺ ions show the location of alveoli and K₄PO₃⁺ localizes at alveolar edges. The overlay shows the position of the nanoparticles in the lung tissue. Figures (A) and (B) are adapted from refs 44 and 45, respectively.

Though TOF-SIMS imaging achieves single-cell resolution (~1 μm) [46], high lateral resolution dynamic SIMS, using the Cameca NanoSIMS 50 (so called ‘NanoSIMS’) instrument, can achieve ~30 nm lateral resolution, improves precision in subcellular imaging of metals [19]. This technique has been particularly useful in neurological research where neurotransmitters within single synaptic vesicles have been imaged [48, 49]. The NanoSIMS produces primarily monoatomic and diatomic secondary ions and can collect up to seven (five on older instruments) different m/z values simultaneously using a magnetic sector MS. However, biomolecules of interest must contain a non-abundant element or rare isotope that can be detected via a unique mono- or diatomic ion, such as nitrogen-15 or oxygen-18. Labeling techniques have been established for various lipids, metabolites, and amino acids [48].

3.3. Imaging metals and biomolecules with MALDI and DESI

LA-ICP-MS and SIMS have the highest sensitivity, and are often the best, for detecting trace metals in biological systems. However, many biological questions may benefit from analytical methods that can readily detect larger biomolecules. MALDI-MS and DESI-MS are have been used extensively for imaging biomolecules without fragmenting lipids and metabolites. The reader is referred to [13, 17, 18] for a more comprehensive review on imaging biomolecules with MALDI and DESI. These techniques are also beneficial when analyzing complex biomaterials with moderate metal concentrations in studying drug interactions or metabolism [50, 51]. For example, many therapeutic agents for cancers contain platinum. MALDI-MS can locate such anti-cancer drugs and biomolecules within tumors and healthy tissues [30]. An additional benefit of using MALDI-MS is that it is non-destructive, so chemical information can be correlated with histological staining after MSI analysis [14, 50]. By coupling MALDI-MS with other imaging modalities, lipids and metabolites can be imaged on the same tissue section as elemental analysis. Since MALDI does not require sample washing and it is non-destructive, analytes that may wash out can be imaged post-MALDI. For example, it is possible to image lipids with MALDI then phosphorus, calcium, sulfur, or platinum with micro-X-ray fluorescence or LA-ICP-MS [52, 53]. DESI-MS, which can be operated under ambient conditions, further decreases the amount of sample preparation required, and can detect alkali metal ions simultaneously with lipids, neurotransmitters, and metabolites, as demonstrated in an ischemic stroke model [54]. Overall, LA-ICP-MS provides the most sensitive technique for detecting metal-based drugs in biological tissue, but MALDI-MS or DESI-MS provide techniques for identifying endogenous molecules [55, 56]. As shown in Figure 3, samples that have been imaged with both MALDI-MS and LA-ICP-MS can be co-registered and regions of interest (ROI) can be identified [52]. Analysis of these ROIs can determine distinct characteristics associated with health and disease.

Figure 3.

Example of a multimodal MSI approach for revealing metal and biomolecule distributions within the same tissue sample. Here, a malignant pleural mesothelioma tissue section from a patient treated with cisplatin was imaged with LA-ICP-MS and MALDI-MS. Lipid distributions, detected with MALDI-MS originate from two distinct regions in the tissue, the malignant tissue and recovered tissue indicated in the overlay image in cyan and orange, respectively. Region of interest analysis allows characterization of biomolecules within different tissue areas. The regions found in the LA-ICP-MS images enriched with platinum and phosphorus were used to locate the distinctive lipid signals in these tissues. Ion distributions in the overlay images have a 50% threshold level. Adapted with permission from ref 52.

Perhaps one of the most significant developments in imaging metals with MSI over the last half-decade has been the use of these elements as labels. Specifically, several methods that introduce antibody protein labels with metal tags that can be imaged with MSI techniques have been developed. A benefit of using metal-based labeled antibodies over fluorescently encoded approaches is the increased multiplexing ability, where dozens of labeled proteins can be imaged simultaneously. For example, imaging cyTOF combines LA-ICP-MS with antibody labeled proteins carrying metal isotope labels [57]. This technique can simultaneously image up to 40 proteins, and has been widely used to reveal protein heterogeneity in cancer tissues and other disease processes [57]. In a recent study, image mass cytometry was applied to study the cell distribution for an implantable cell niche used to encourage vasculature in diabetic pancreas tissue [58]. Similar immunolabeling techniques have been used in SIMS imaging. Gold nanoparticles and alkali metal nanobodies have been conjugated to a few proteins [59, 60]. Also, multiplexed ion beam imaging by time-of-flight (MIBI-TOF) uses a TOF-SIMS instrument configuration to image antibodies containing various metal isotopes [61]. Because many metal-isotope labels can be easily distinguished in the TOF mass spectrum, MIBI-TOF can image numerous proteins with 200 nm resolution simultaneously [61]. Additionally, TOF-SIMS enables simultaneous imaging of labeled proteins, endogenous elements such as iron and sodium, and biomolecules. The MIBI approach can be utilized with a NanoSIMS to achieve higher resolution at the expense of high-throughput analysis because only seven different isotopes can be detected simultaneously with a NanoSIMS. However, consecutive scans can image different isotopes without washing and relabeling, unlike immunofluorescence techniques such as Co-Detection by Indexing (CODEX), where washing is required [62].

4. Conclusions and perspectives

Significant advances have been made in imaging of biologically important metals. For example, iron levels in the brain were correlated with protein expression, magnesium and ZrO₂ were located with distinct subcellular compartments, and novel labeling techniques (CyTOF and MIBI-TOF) have enabled simultaneous imaging of metals and proteins, as discussed above. Looking forward, quantitative, spatially- and temporally-defined localization of metals is expected to make increasingly important contributions both to fundamental understanding of metal-dependent biological processes and to medicinal application of metals. For example, accumulating evidence for a contribution of mis-localization of iron, copper, zinc, and other metals to Alzheimer’s disease suggests that tracking these metals will play an important role in diagnosis and therapy in this and other neurological pathologies [31]. Mapping the spatial distribution of iron following exposure to ferroptosis-inducing agents will be of benefit in effectively leveraging ferroptosis in cancer treatment, as well as in blocking excessive ferroptosis in neurological and other disorders. Application of improved imaging techniques may also lead the way to a more nuanced application of metals used to provide contrast in medical imaging, such as gallium. Mapping changes in metal distribution following use of metals or metal-containing drugs as therapeutics will provide important insight into off-target toxicities; it will also increase understanding of biological consequences of exposure to toxic metals.

Data availability

No data was used for the research described in the article.