Dear Tajmalook Khan!

Here is the extensive answe + article! I hope it is proper.

Sincerely yours!

Dr. Bratko Filipič

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In Situ Imaging of Metals in Cells and Tissues

1. Introduction

Approximately a third of the human proteome contains metal cations, either in form of cofactors with catalytic functions, or as structural support elements.1,2 To guarantee a proper maintenance of this metal ion pool, both at the cellular and whole organism levels, nature has evolved a highly sophisticated machinery comprised of a complex interplay between DNA, proteins, and biomolecules.3 Over the past decades, a steadily growing number of diseases have been identified, which are characterized by metal imbalance in cells and tissues. Among the most prominent examples rank Alzheimer’s disease and Parkinson’s disease, two neurodegenerative disorders that involve abnormal accumulation of transition metals in brain tissue.4 While some progress has been made at understanding the molecular basis of these disorders, many important questions remain unanswered. For example, little is known about the cellular structures that are involved in transiently storing metal ions prior to their incorporation into metalloproteins or the fate of metal ions upon protein degradation. An important first step towards unraveling the regulatory mechanisms involved in trace metal transport, storage, and distribution represents the identification and quantification of the metals, ideally in context of their native physiological environment in tissues, cells, or even at the level of individual organelles and subcellular compartments.

Since the inception of the first histochemical methods for the microscopic demonstration of transition metals in tissues more than 140 years ago,5 many highly sensitive microanalytical techniques and instruments have been developed for the in situ analysis of trace metals. The aim of this review is to provide an overview of the most recent achievements in trace metal imaging while at the same time also offering a historical perspective of this rapidly evolving research field. Although this survey has been structured according to the various analytical techniques, particular emphasis is given to the biological background for a better understanding of the context and importance of each discussed study.

An overview of the most important microanalytical techniques currently available for the in situ detection of trace metals in cells and tissues is compiled in Table 1. Depending on the task, each technique may offer specific advantages and, of course, also disadvantages. Currently, synchrotron- and focused ion-beam microprobes presumably offer the best combination of sensitivity and spatial resolution; however, the ionizing high-energy excitation beam is not compatible with studying live organisms. Conversely, techniques that have been specifically developed for physiological imaging in clinical medicine, notably magnetic resonance imaging and positron emission tomography, inherently offer only a low spatial resolution and are merely suitable for obtaining information at the organ or tissue level. Although fluorescence microscopy based methods provide very high sensitivity down to the single molecule level while being at the same time compatible with live cell and tissue studies, scattering and limited penetration depth renders these techniques unsuitable for imaging opaque specimens. There are also important differences regarding the type of quantitative information that can be gained by each of these analytical techniques. For example, the histochemical detection with chromogenic and fluorogenic dyes relies on a competitive exchange of the metal ion within its native environment, most likely coordinated to endogenous ligands. Depending on the exchange kinetics and thermodynamic affinity of the histochemical indicator, only a fraction of the total metal ion contents in a cell or tissue can be probed. Nevertheless, this kinetically labile pool is particularly of interest in context of understanding the uptake, distribution, and regulation of trace elements at the cellular level, and in this regard, these methods offer unique opportunities to dynamically image metal ion fluxes in live cells with high sensitivity and spatial resolution. At the same time, organelles and proteins of interest can be readily labeled with genetically encoded green fluorescent protein tags,12 thus providing direct insights into dynamic processes within a larger cellular and biochemical context. In contrast, similar correlative information is difficult to gain with the fully quantitative micro beam methods, which require xenobiotic elemental tags for identifying subcellular structures. Autoradiographic tracer experiments offer much improved resolution over PET; however, the technique is only applicable to fixed or frozen tissues and cells. Furthermore, tracer studies cannot provide direct information regarding the endogenous metal composition of cells or tissues, and are therefore primarily limited to metal uptake, distribution, and release studies. Finally, mass spectrometric analyses are surface-based methods that destroy the sample while measuring its elemental composition. Clearly, only the combination of several analytical techniques and specific biochemical studies may lead to a fully comprehensive analysis of a biological system.

📷Table 1Spatially resolved microanalytical techniques for in situ imaging of trace metals in biology.6–11Go to:

2. Histochemical Techniques

Histology is the branch of biology dealing with the study of microscopic anatomy of cells and tissues of plants and animals. Histological studies are typically carried out on thin sections of tissue or with cultured cells. To visualize and identify particular structures, a broad spectrum of histological stains and indicators are available. Among the most widely used dyes are hematoxylin and eosin, which stain nuclei blue and the cytoplasm pink, respectively.13 The history of detecting biological trace metal by histological methods dates back more than 140 years. Although these techniques have been today mostly replaced by the much more sensitive modern analytical methods described in this review article, histochemical approaches for visualizing metals mark the very beginning in the exploration of the inorganic physiology of transition metals. Given this special place in history, we deemed it necessary to briefly review some of the early achievements in this field.

2.1. Chromogenic Detection with Chelators and Ligands

Ever since the inception of Perls Prussian blue method for staining of non-heme iron, numerous indicators have been developed for the in situ visualization of trace metals in biological tissues and cells.13 Due to their limited sensitivity; however, most of these techniques were only suitable for the diagnosis of pathological conditions, typically associated with excess metal accumulations, thus preventing their application for routine staining of normal tissue. Furthermore, because the dyes are engaged in a competitive exchange equilibrium with endogenous ligands, histological stains are not suitable for the analytical determination of the total metal contents in tissues and thus limited to the visualization of the histologically reactive fraction of loosely bound labile metal ions.

2.1.1. Histochemistry of Iron

The histochemical demonstration of labile iron reported by Perls in 1867 is among the earliest accounts describing the in situ visualization of a trace metal in biological tissues.5 The method was originally described by Grohe, who observed the formation of a blue coloration when he treated cadaver tissues with potassium ferrocyanide in acidic solution.14 Due to its low cost and simplicity, the technique is still used today for the histological visualization of non-heme iron. Some variations focused on optimizing the concentrations and proportions of the reagents,15–17 among which Lison’s protocol17 appears to be most popular today. An intensification of Perls’ staining can be obtained by exploiting the use of ferric ferrocyanide in catalyzing the oxidation of diaminobenzidine (DAB) to polymeric benzidine black by hydrogen peroxide.18

An alternative method employs the reaction of ferricyanide with Fe(II) resulting in Turnbull blue.19 Since almost all of the Fe in tissues is in the ferric form, the staining procedure requires the in situ conversion of Fe(III) to Fe(II) with ammonium sulfide.15 Due to often incomplete reduction, the method never gained much attention. More recently, an application of Turnbull blue, named the ‘perfusion Turnbull method’ has been developed, where in vivo perfusion of acidic ferricyanide is followed by DAB intensification.20 The direct in vivo perfusion avoids artifacts associated with tissue fixation, including the loss of loosely bound iron and oxidation of Fe(II) to Fe(III). Similarly, Perls method was modified by employing in vivo perfusion with acidic ferrocyanide. Both methods are capable of identifying organs and tissues containing histochemically reactive iron over a broad pH range, including the low endosomal pH.21,22

The history of iron histochemistry would be incomplete without mentioning Quincke’s method, which employed ammonium sulfide for the precipitation of tissue iron as its sulfide.23 A detailed account on the various techniques, including a comprehensive historical overview of non-heme iron chemistry, has been recently published.24

2.1.2. Histochemistry of Copper

The history of histochemical techniques for the identification of copper in a biological environment traces back to the late 19th century, where haematoxylin (1, Chart 1) was suggested as a stain for identifying the distribution of copper in diseased oysters.25 The dye was later applied by Mendel et al. for exploring the distribution of inorganic constituents in the liver of sycotypus canalicalatus.26 Haematoxylin is a natural product isolated from the logwood tree. In its oxidized form, haematein (2), it combines with Al(III) or Fe(III) to give blue-purple colored pigments, which are still used as histological stains for cell nuclei. In 1939, Mallory and Parker further modified the protocol and emphasized the use of fresh ethanolic haematoxylin solutions to avoid interference from hematein formed upon prolonged storage.27 Besides copper, haematoxylin forms colored pigments with a number of other transition metals. This lack of specificity prompted Okamoto in 1938 to explore alternative staining methods based on rubeanic acid (dithiooxamide, 3),28 which became one of the most widely used indicators for Cu. In alcoholic solution, rubeanic acid forms with Cu(II) a dark green precipitate of polymeric copper rubeanate. While the indicator also forms colored complexes with Ni(II), Ag(I) and Co(II), the corresponding precipitates can be visually distinguished based on their colors, and further differentiated based on their solubility in acetate containing ethanol. With a detection limit of approximately 6 µM for Cu(II),29 rubeanic acid is not sufficiently sensitive for visualizing labile copper levels present in normal tissue; however, it has been successfully applied to demonstrate copper in various tissues of Wilson’s disease patients, including the liver,30–32 central nervous system (CNS),31 and kidney.31,32 Recently, Lecca et al. further optimized the rubeanic acid method by incorporation of a microwave treatment, which resulted in better contrast and fewer artifacts.33 Okamoto et al. explored the utility of two additional stains for the detection of copper, rhodanine (p-dimethylaminobenzylidene-rhodanine, 4) and diphenylcarbazide.34,35 Rhodanine forms a reddish brown precipitate with Cu(I) ions and gives a staining that follows a linear relationship with the metal ion concentration;36 however, divalent copper salts do not react. As for the other indicators, the selectivity of diphenylcarbohydrazide towards copper was poor.30 In 1945 Waterhouse introduced sodium diethyldithiocarbamate (DEDTC), which forms with Cu(II) a yellowish-brown precipitate. The limit of detection lies around 3 µM Cu(II) and is comparable with rubeanic acid.37,38 Although the indicator has been successfully used for visualizing copper in different tissues, including liver and putamen,30,39 the yellow color of the precipitate often resembles naturally occurring pigments inside cells, thus limiting its application in light microscopy. Dithizone (5) also forms a yellowish-brown complex with Cu(II) but offers only low sensitivity and selectivity.40 Shikata et al. introduced the use of Orcein (lacmus, litmus) for the histochemical staining of copper.41 Orcein (6) is a mixture of phenoxazone derivatives extracted from orchella weeds. Later it was observed that it also stains copper-associated proteins.42–44

📷Chart 1

The methods described above often produced conflicting results,45 and were only applicable for detecting abnormally high levels of copper that is loosely bound in tissues. Some efforts focused on liberating bound copper with hydrogen peroxide46 or concentrated hydrochloric acid.31 Given the shortcomings of each method, it was recommended to apply typically a series of indicators to independently confirm the results.

2.1.3. Histochemistry of Zinc

The lack of visible color of zinc ions, both as solvated aqua complex or when coordinated to ligand or proteins, rendered the histological identification of zinc a challenging task. In 1905, Mendel and Bradley demonstrated for the first time the presence of labile zinc in hepatopancreatic tissues of sycotypus canalicalatus using sodium nitroprusside followed by alkaline sulfide development.26Due to its low sensitivity, the protocol did not receive much attention at the time, despite the fact that the reaction was later demonstrated to be specific.47 Important methods later developed for the histochemical detection of labile zinc include the dithizone (5) method (Chart 1), Timm’s staining (section 2.3.1), and fluorescence based approaches.48,49 Okamoto introduced the dithizone method for the histochemical demonstration of zinc, which was employed as intravital staining to visualize zinc in islets of Langerhans found in the pancreas.50–53 Dithizone reacts with Zn(II) to give a deep-red colored complex; however, similar complexes are also formed with a number of other transition metals. The specificity of complexation can be improved by adjusting the pH of the medium and the use of additional complex forming reagents.40 For example, one such modification employs a complex forming buffer at pH 5.5 containing tartrate, thiosulphate and cyanide, in which the dye combines predominantly with zinc, thus improving its selectivity by masking the interference of any other metals. The dithizone staining technique has been extensively used for demonstrating zinc in a broad range of samples and tissues, including brain (hippocampus),54–56 pancreatic islets,57,58 prostate,58 and blood cells59 of dogs, humans, rabbits, and rats. Further modification of the method introduced an adduct formation of zinc-dithizone with pyridine resulting in an enhanced positive staining stable up to one week compared with the usual complex dissociation taking place within hours.60 The presence of labile Zn(II) ions in brain tissues was first demonstrated by Maske et al. by means of intraperitonial injection of dithizone to form colored chelates,61although at that time the results were considered inconclusive as the method was believed to be non-specific towards zinc. The report was later confirmed on basis of in situ absorption spectroscopy of a dithizone-stained hippocampus tissue section and comparison with independently prepared reference samples of the Zn(II)-dithizone complex.56 While dithizone staining is adequate for analyzing tissue with a high concentration of labile zinc, the pale and unstable staining, due to chelate decomposition upon exposure to heat, light or solvents, further limits its application.62

2.2. Fluorescence Probes

Compared to chromogenic histochemical stains, fluorescent dyes offer much greater optical sensitivity and harbor the potential for observing biological processes at the single-molecule level.63 Because of their small molecular size, synthetic indicators may passively diffuse across cell membranes and are thus well suited for the non-invasive imaging of cation fluxes in living cells. Given these attractive properties, it is not surprising that the development of new fluorescent probes and indicators represents a very active and steadily growing research area.64 At present, fluorescent indicators have been developed for most biologically relevant metal cations, including calcium, magnesium, sodium, potassium, zinc, copper, and iron.64,65 Furthermore, an increasing number of probes selective towards xenobiotic, toxic heavy metals have been described. Over the past decade, many excellent review articles summarizing these developments have been published, with topical areas covering both the principles and photophysics of probe design,66–69 as well as comprehensive overviews on fluorescence detection of selected metal ions, most notably zinc70–77 and mercury.78 Rather to duplicate these efforts, this section has been limited to outlining a few corner stones in the evolution of Zn(II)-selective fluorescence probes for biological applications, a particularly vital research area that best illustrates the rapid advances in this field.

For many years, dithizone was the only histochemical stain available for demonstrating Zn(II)-ions in tissues.48 In 1969, Mahanand and Houck described the first fluorescence indicator for the selective detection of Zn(II) in blood plasma and urine using 8-hydroxy-quinoline (7, Chart 2).79 While the indicator formed stable complexes with many divalent metal ions, only binding of Zn(II), Mg(II), and Ca(II) led to a strong fluorescence increase, with Zn(II) displaying the highest binding affinity.80 Similarly, 2-methyl-derivative of 8-hydroxy-quinoline was also described as histochemical Zn(II) indicator.81Structurally related to the 8-hydroxy-quinoline indicators, Toroptsev and Eschenko explored the utility of several quinoline sulfonamide derivatives as fluorescence probes for Zn(II).82–85 The bright-green fluorescence was found to colocalize with the dithizone staining in pancreatic beta cells and hippocampal mossy fibers. In the 1980s, Frederickson et al. explored the utility of another quinoline derivative, 6-methoxy-8-p-toluene sulfonamide quinoline (TSQ, 8) as histological indicator for Zn(II).86,87 A comparison of TSQ with the established neo-Timm method demonstrated that the indicator is well suited as stain for demonstrating histochemically reactive Zn(II) in various tissues. In order to improve the water solubility and cellular retention, TSQ was later modified with a carboxylate group.88–91 The resulting probe, zinquin ester (9), has been instrumental in elucidating the role of labile Zn(II) pools in a wide range of biological systems; however, the origin of the distinct vesicular staining pattern observed with zinquin ester remains controversial. To address the question whether these zinc-rich vesicles, also referred to as zincosomes, might arise from a dye-induced sequestration of Zn(II), Wellenreuther et al. performed in situ micro-XANES (section 5.1.3) measurements with RAW264.7 cells.92 The in situ data matched the x-ray absorption near-edge signature of isolated vesicles and implied that Zn(II) is present in complexed form with a coordination environment composed of sulfur, nitrogen (histidine), and oxygen donor atoms.

📷Chart 2

To avoid potentially damaging UV excitation, several fluorescein-based probes tethered to varying chelating units have been recently developed for excitation in the visible spectral range. For example, the ZnAF family of fluorescent probes developed by Nagano and coworkers combined the fluorescein platform with N,N-bis(2-pyridylmethyl)ethylenediamine as Zn(II)-selective binding unit. With a Zn(II) affinity of 2.7 nM, ZnAF2 (10) was successfully used for detecting synaptically released Zn(II) in hippocampal slices.93,94 Further modifications with various chelating moieties furnished a series of indicators with a wide dynamic range for detecting Zn(II) from nM to mM concentrations.95 The fluorescent dyes revealed intriguing concentration differences of synaptically released Zn(II) in acute hippocampal slices.95 In parallel, Lippard and coworkers developed a large family of Zn(II)-responsive indicators, primarily aimed at unraveling the neurobiology of this metal ion. For example, the difluorofluorescein derivative ZP3 (11) was successfully utilized to image endogenous Zn(II) pools in hippocampal slices,96 whereas the cell-impermeant indicator ZP4 (12) proved to be suitable for imaging extracellular Zn(II) and Zn(II)-damaged neurons.97,98 A detailed review of this extensive body of work has been recently published.77

Several efforts focused on developing ratiometric probes for the detection of Zn(II) in biological systems.76 Originally described by Tsien and coworkers for the dynamic visualization of Ca(II)-fluxes,99ratiometric probes undergo a shift of the excitation or emission maxima upon binding of the analyte. By taking the ratio of the emission intensity at two different wavelengths, fluctuations due to uneven dye distribution, cellular uptake, or instrument dependent factors are cancelled out. For example, the iminocoumarin based Zn(II) sensor, ZnIC (13), undergoes a red shift, from 543– 558 nM, associated with an enhanced intramolecular charge transfer upon zinc binding at physiological pH. With a Kd of 1.3 pM, the sensor was successfully used for the ratiometric detection of Zn(II) in cultured cells and in rat hippocampal slices.100

The development of new imaging technologies typically requires also a tailored optimization of the indicator properties. With this goal in mind, Zn(II)-responsive indicators for application in two-photon excitation microscopy,101,102 near-infrared (NIR) fluorescence imaging103–105 have been developed. Due to the increased penetration depth of the low-energy infrared excitation, these two fluorescence microscopy techniques are particularly attractive for imaging thick tissues or potentially for whole animals studies. As illustrated with Figure 1, staining of rat hippocampal slices with the Zn(II)-responsive two-photon indicator AZn2 (14) revealed a characteristic staining pattern of histochemically labile Zn(II) pools. The fluorescence staining was reversed by addition of the high affinity Zn(II)-chelator TPEN (Figure 1c), and a distinct increase in fluorescence intensity was observed upon stimulation with 50 mM KCl, suggesting the release of presynaptic Zn(II) stores (Figure 1e).

📷Figure 1Two-photon excitation microscopy (TPM) images of a rat hippocampal slice stained with 10 µM AZn2 (14). a) At a depth of ca. 120 µm with magnification 10x. Scale bar: 300 µm. b,c) Magnification at 100x in the stratum lucidum (SL) ...

To this point, only few biologically oriented studies took advantage of the capabilities of these newly fluorescent dyes. While the histochemical methods were limited to demonstrating labile Zn(II) in fixed specimens, the inherently high detection sensitivity of fluorescence microscopy combined with a broad range of thermodynamic affinities, Zn(II)-responsive fluorescent indicators harbor great potential for visualizing dynamic Zn(II) fluxes with subcellular resolution in live cells, and thus for elucidating important questions regarding the complex mechanism of cellular zinc homeostasis.

In contrast to the rich literature on Zn(II)-selective fluorescent probes, there are only few reports on the detection of biological copper and iron, despite the fact that both metals are equally important trace elements within the cellular metallome. The fluorescence-based detection of these redox-active metals is particularly challenging due to competing metal-initiated fluorescence quenching pathways, for example through increased triplet conversion rates or energy transfer processes involving energetically low-lying metal-centered states. The adverse fluorescence quenching can be minimized with a rigid fluorophore-ligand architecture, which electronically decouples the metal cation from the fluorescence emitter.68,106Following this concept, fluorescent probes for the detection of Cu(I) in cultured cells have been described.107,108 A rigid probe architecture was also key in the design of a series of Fe(III)-selective probes, although the Fe(III)-induced fluorescence enhancements were only characterized in organic solvents.109 In an alternative approach, Cu(II)- and Fe(III)-selective fluorescence enhancements were achieved through the metal-promoted ring opening of non-fluorescent spirolactam rhodamine derivatives;110,111 however, the sensing process is irreversible and might potentially also be initiated through an oxidative mechanism, thus complicating the interpretation of cellular imaging data. While at present the fluorescence detection of Fe and Cu in a biological environment poses still significant challenges, these initial successes clearly demonstrate its feasibility. Given that most biological laboratories are equipped with fluorescence microscopes, synthetic fluorescent probes remain particularly attractive for routine imaging of labile metal pools and will remain an active research area for developing materials with further improved selectivity and sensitivity.

2.3. Autometallography

A number of endogenous and toxic heavy metals form sulfide or selenide nanocrystals that can be autocatalytically amplified by reaction with Ag ions. The larger Ag nanocluster can then be readily visualized by electron or light microscopy. This property is the basis of all autometallographic amplification techniques, which evolved into an important tool in histochemistry.112 At present, robust protocols for the silver amplified detection of Au(0), Ag(0), Ag-S/Se, Hg-S/Se, Bi-S/Se, and Zn-S/Se nanocrystals have been established. Upon exposure to Ag(I), Hg(II), and Bi(III), organisms metabolically create in vivo composite sulphide and selenide nanocrystals that can be autometallographically detected. In addition, commercially available quantum dots are also autocatalytically active and may be used as histochemical labels. Of the endogenous metal ions, Zn(II) appears to be the sole cation that is converted to Zn-S or Zn-Se nanocrystals upon in vivo perfusion with sulfide or selenide ions, rendering autometallography (AMG) particularly attractive for visualizing Zn stores in tissues with high specificity. While it has been proposed that Cu, Fe, Al, and Pb can be also traced by AMG in tissues and cell cultures, the required high concentrations of sulphide and high pH mobilizes other metal ions from proteins. Under neutral conditions, none of these metals lead to formation of nanocrystals in the presence of sulphide, and neither are nanocrystals formed through metabolic accumulation.

As illustrated with Figure 2, in the autometallographic amplification process Ag ions adhere to the surface of the nanocrystal, where they are subsequently reduced to metallic silver by electrons released from a nearby reductant such as hydroquinone. The silver atoms continue to be incorporated into the original nanocrystal leading to autometallographic silver enhancement. This process continues as long as there is available an adequate supply of both silver ions and reducing molecules (Figure 2).

📷Figure 2Principle of autometallographic silver enhancement (AMG). Electrons released from the reductant (hexagonal molecules) populate the valence band of the nanocrystal, thus increasing the probability for reducing silver ions that subsequently are integrated ...

The developer, which supplies the silver ions and reductant for the amplification process, is critical to the performance of AMG, and many groups devoted efforts to optimize the composition of developers. For example the colloid Gum Arabic, a natural product, employed in the original method can lead to contaminations in the developer was replaced by an industrial product sodium tungstate at pH 5.5.113–116For the analysis of ultra-thin specimens, the AMG emulsion technique has been developed, which utilize the “Gum Arabic Silver Lactate Developer” or its improved variety “Cellulose Silver Lactate Developer”.117 In this technique, the tissue sections are first immersed in a silver containing emulsion, and are then exposed to a chemical developer containing the reductant. As the fluid will pass through the emulsion, it will be enriched with silver ions and thus the fluid penetrating in the tissue section will function as AMG developer.

As outlined in the following section, AMG has been predominantly used for the histochemical detection of labile zinc in tissues, and to a lesser degree for visualizing other transition metals. A comprehensive review detailing the technique and protocols as well as their applications has been recently published.117

2.3.1. AMG Detection of Zinc

The AMG technique originally described by Timm in 1958 was geared towards the general detection of heavy metals in tissues;118,119 however, the method has been later optimized for the selective detection of labile Zn(II) ions. As described in the previous paragraph, the AMG staining relies on the formation of a metal sulfide precipitate in the tissue during fixation by exposure to sulfides, followed by a silver developing process resulting in the deposition of elemental silver at metal localized sites. Haug et al. utilized this method by substituting the original Timm’s method, involving the immersion of the tissue in hydrogen sulfide purged alcohol, with buffered sodium sulfide, and successfully demonstrated labile Zn(II) in the hippocampal mossy fiber system.120 The autometallographic detection of histochemically reactive Zn(II)-ions played a critical role in elucidating the mechanism of Zn(II) translocation into synaptic vesicles, a process that is mediated through the zinc transporter-3 (ZnT-3).121,122 Timm’s original method has been modified over the years, most notably by Danscher and coworkers, who greatly improved its selectivity and sensitivity towards Zn(II).117

For example, the neo-Timm method is based on perfusion with a 0.1% solution of sodium sulfide, which avoids surplus sulfide ions that typically lead to false staining via the formation of Ag-S nanocrystals, and development with silver lactate, which undergoes fast dissociation to produce high levels of Ag(I) ions in the developer.116 Further modification included in vivo Timm’s method employing intravenous injection of sodium sulphide followed by AMG development permitting the visualization of in vivo formed Zn-S nanocrystals.124 The in vivo selenium method was later developed as a greatly improved Zn(II) specific protocol, where the sulfide treatment was replaced by intravital administration of selenide. Presumably due to the increased resistance of zinc selenide nanocrystals towards decomposition at low pH, this method improved the selectivity and broadened the utility of zinc AMG for use with live animals.125,126 As illustrated with Figure 3, histochemically reactive Zn(II) in zinc-enriched neurons (ZEN) in a rat brain slice were captured with great detail, revealing highly ordered glutaminergic ZEN terminals.123

📷Figure 3Histochemically reactive Zn(II) in zinc-enriched neurons (ZEN) in a rat brain slice: (a) Micrograph of a 30-μm-thick sagittal cryostat section of rat brain from an animal treated with sodium selenite and allowed to survive 1.5 h before being sacrificed ...

2.3.2. AMG Visualization of Metals Other than Zinc

Gold

Although gold compounds are being used for treatment of various diseases, most notably rheumatoid and psoriatic arthritis,127 still little is known about their mechanism of action or biodistribution. AMG can be used to trace gold in tissues from individuals treated with gold-containing drugs,128 where they appear to preferentially accumulate in lysosomes.115,129–131 Other applications of AMG-based gold detection include the tracing of gold accumulation around gold implants used as a remedy for osteoarthritis132 and enhancing colloidal gold particles associated with antibodies or enzymes as part of immunohistochemistry (see also section 5.3.2.2).133

Silver

AMG has been successfully used to trace silver in tissues with exposure to silver from different sources, for example, silver nitrate in throat swabs, amalgam fillings. The silver ions released as a result of the decomposition of silver containing molecules in the lysosome can form silver-sulfur nanocrystals, which can be visualized by AMG.134 The silver ions are also known to react with selenium to form silver-selenium nanocrystals, a suitable target for AMG identification.135

Mercury and bismuth

Because mercury metabolically accumulates in lysosomes, leading to formation of mercury-sulfide and mercury-selenide nanocrystals, this metal cation can be readily detected by AMG.136To assess the toxic effect of mercury in central nervous system, Moller-Madsen and coworkers elucidated the detailed distribution of AMG-detectable mercury in the brain and in spinal cord of rats exposed to inorganic, organic, and vaporous forms of mercury.137,138 Similarly, AMG was also applied to bismuth as demonstrated by experiments successfully conducted in mice exposed to bismuth subnitrate.139 In light of the increasing utility of this metal in various fields of science, this method may serve as a valuable tool for exploring the in vivo distribution and toxicology of bismuth.140

Copper

The utility of Timm’s stain for the demonstration of copper in normal tissues remained not without controversy, in part due to difficulties creating nanocrystals by exposure of copper to sulphide at physiological pH or by in vivo exposure to selenium.117 Nevertheless, among traditional histochemical detection methods, the AMG detection of copper appears to be the most sensitive approach according to a study that focused on the demonstration of hepatic copper for the diagnosis of Wilson’s disease.141Despite this lack of specificity, numerous reports in the literature describe the application of AMG for visualizing copper in tissues.119,142,143 To improve the selectivity towards copper, washing with dilute acid144 or trichloroacetic acid145 were proposed, procedures thought to remove other competing metals. Another modification employed the formation of silverdithizonate from copper-dithizonate formed by the reaction between tissue copper and magnesiumdithizonate.118 Application of this method after washing with trichloroacetic acid permitted the histological demonstration of copper in various tissues of normal rats.146

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3. Radioisotope Imaging Techniques

Radioactive isotopes represent the corner stone of nuclear medicine and have found widespread use in other branches of life sciences. Their application as sensitive biochemical markers can be traced back to the work of George de Hevesy in the early 20th century, who initially attempted to isolate a radioactive isotope of lead, at the time known as radium-D, from hundreds of kilograms of lead chloride.147 Because he was not able to separate the two isotopes, he reasoned that the atomic number and not the atomic weight is responsible for the chemical properties of an element. Taking advantage of this observation, he used radium-D for the first time in 1913 as isotope tracer to determine the solubility of lead sulfide and chromate in water,148 and in 1923 to study the uptake and metabolism of lead salts into plants.149

A broad range of radioisotopes are available today as tracers for radioanalytical experiments, most notably 3H, 14C, 35S, 32P, 125I and 131I are routinely used as isotopic labels in biochemical research. Radioactive isotopes have also secured an important place in studying the uptake and distribution of biologically relevant transition metals. An overview of metal isotopes commonly used in biochemical research and nuclear medicine is provided with Table 2. Although many of these radionuclides have been successfully used as biolabels to trace proteins or other molecules of interest, the following discussion focuses primarily on studies concerned with the inorganic physiology of the metal cations themselves. Because all naturally occurring transition metals of biological relevance are composed of stable isotopes, the radiation-based detection is restricted to tracers studies aimed at quantifying the uptake, distribution, and release of metal ions and their complexes. In this context it is important to note that radionuclide imaging techniques can provide little or no information regarding endogenous transition metal levels and their distribution under normal physiological conditions.

📷Table 2Metal Radionuclides Used in Nuclear Medicine and Life Sciences.a

3.1. Autoradiography

Taking advantage of their decay emission, the distribution of radionuclides in biological specimens can be directly visualized by exposing a photographic film or emulsion in close contact. Because the radionuclide-containing specimen itself is the source of radiation, the technique is referred to as autoradiography.150 In 1867, Niepce de Saint-Victor gave the presumably first account of autoradiography, in which he described the blackening of an emulsion of silver chloride and iodide by uranium nitrate and tartrate.151 Curiously, this discovery is older than the knowledge of radioactivity itself, which was pioneered much later by the work of Henri Becquerel in 1896 and the Curies in 1898. For many decades, autoradiographic imaging as a biological technique evolved only very slowly, mostly due to the limited set of naturally occurring radionuclides, such as radium, thorium, or uranium, all of which were of little biological interest. With the invention of the cyclotron by Lawrence in 1930 and the large scale production of radionuclides in nuclear reactors, a broad spectrum of radioisotopes became available. Table 2 gives an overview of metal isotopes that have been used as tracers in biochemical research.

While early autoradiographic methods were limited to larger specimens, where the photographic film was simply pressed against fixed or freeze-dried sections for exposure, newer techniques involving liquid emulsions have been developed that are compatible with cellular or subcellular studies at the light and electron microscopic levels.150 The resolution of an autoradiograph depends on the thickness of the specimen containing the radionuclide, its distance to the photographic emulsion, the thickness of the emulsion layer, and the radiation emitted by the radionuclide. Short-range radiations such as a or low-energy b radiation offer typically the best contrast. The detection sensitivity depends on the exposure time, which in turn varies as a function of the activity and energy of the radionuclide as well as the sensitivity of the photographic emulsion. In combination with transmission electron microscopy, autoradiography may offer a spatial resolution around 0.1 µm.152

3.1.1. Zinc Transport and Distribution in the Brain

Autoradiography has played a particularly important role in studying the transport, distribution, and function of Zn ions in the brain.153,154 Perfusion experiments with 65ZnCl2 provided first insights into the trafficking of Zn(II) ions across the blood-brain or blood-cerebrospinal fluid barrier in rats.155–157 The data revealed slow Zn uptake of approximately 20 nmol/day across cerebral capillaries, and an even lower rate of 0.2 nmol/day across the choroid plexus.157The autoradiographic distribution of 65Zn showed low levels in white matter but relatively high levels in choroid plexus, cerebral cortex, and particularly the dentate gyrus.157 A similar distribution pattern was reported in an earlier histochemical study with the Zn-responsive fluorescent probe TSQ.158 Takeda et al. also concluded based on autoradiographic experiments with 65ZnCl2, that the metal was gradually taken up by the brain via the cerebrospinal fluid in the choroid plexus.159,160 The study showed that 65Zn was largely concentrated in the choroid plexus of rats 1 hour after intravenous injection of 65ZnCl2 and then slowly distributed in the hippocampus and cerebral cortex region. Because the choroid plexus is the site of cerebrospinal fluid (CSF) production, the experiments suggested that Zn is transported via CSF into the choroid plexus. The half-life of elimination of 65Zn ions from the rat brain is in the range of 16–43 days,161 with the longest being associated with the amygdala region consisting of high-density zinc-containing neuron terminals.162

The major carrier protein for labile zinc in the plasma is serum albumin and the other component of exchangeable zinc are the amino acids, histidine and cysteine.163 In order to assess the role of serum albumin as a transporter of zinc to the brain, autoradiographic images of 65Zn distribution in the brain of Nagase analbuminemic rats (NAR) were compared with normal rats.164 NAR has been found to have a genetic mutation which results in lack of serum albumin.165 The study demonstrated that 65Zn distribution in the NAR brain is similar to that in normal rats and suggested that albumin may not be essential for Zn transport into the brain.

In order to understand possible roles of histidine in Zn transport to the brain, 65Zn-His complex or 65ZnCl2were injected intravenously into rats.166,167 In both cases, autoradiographic imaging of brain tissue sections revealed similar 65Zn distribution patterns, indicating that histidine does not block Zn uptake; however, 65Zn-His injection resulted in overall lower Zn levels compared to 65ZnCl2. Despite the similarities in coordination chemistry, intravenously injected 109Cd(II) was not significantly transported into the brain according to a set of autoradiographic experiments.168,169 Detailed binding studies demonstrated that the affinity of Cd(II) to serum proteins is substantially higher compared to Zn(II), and that Cd(II) is not mobilized from proteins by histidine at concentrations present in the plasma.169 Based on these observations the authors propose that the Cd(II) impermeability is due to the avid binding of Cd(II) to plasma proteins.

Infants and school-age children are particularly susceptible to dietary Zn deficiency and malnourishment, often leading to altered growth and behavior problems.170 Several autoradiographic studies were aimed at elucidating possible roles of Zn in brain development and function. For example, imaging of 65Zn distribution in the brains of neonatal, young, and aging rats indicated that Zn is highly demanded in the neonatal brain by the cerebellum, which develops rapidly after birth.171 While older rats showed an approximately two-fold higher total Zn concentration, a more even distribution between the cerebellum and the cerebral cortex was found. A direct relationship between dietary zinc and zinc homeostasis in the brain evolved from a study where rat brains were examined for endogenous Zn level and uptake of Zn after they were fed zinc-deficient diet for 12 weeks.172 The endogenous zinc concentration in the hippocampus was significantly decreased in the brain of rats fed with zinc-deficient diet compared to controls. After intravenously administering 65ZnCl2, Zn uptake in to the brain was significantly higher in Zn-deprived compared to control rats.

Epilepsy is a common neurological disorder manifested by uncontrolled seizures. Several studies directly indicate that alteration of zinc homeostasis in the brain may be associated with epileptic seizures.173–175To study changes in Zn distribution under induced seizure conditions, Takeda et al. intravenously injected 65ZnCl2 into epilepsy (EL) mice, an animal model of genetically determined epilepsy.176 While uptake of zinc by the brain is normal in EL mice, autoradiographic analysis revealed overall reduced Zn concentrations in the brain of seized EL compared to control mice. The concentration of 65Zn was notably decreased in the piriform cortex and the amygdaloid nuclei complex during convulsion. In a related study, epileptic seizure was induced in normal mice by treating with kainate,177 an experimental model for studying temporal lobe epilepsy.178 In agreement with above results, autoradiographic imaging demonstrated that 65Zn concentrations in the brain of kainate-treated mice were much lower compared with normal mice.

Dietary zinc deficiency also impacts tumor growth and malignant proliferation,179–181 an observation that prompted Takeda et al. to study 65Zn uptake in tumors.182 Following subcutaneous implantation of ascites hepatoma (AH7974F) cells into the dorsum, 65ZnCl2 was intravenously injected, and the 65Zn distribution autoradiographically assessed in the whole animal. While the study revealed significantly higher Zn concentrations in the tumor compared to brain tissue, the highest Zn concentrations were found in the liver. In a similar study, following implantation of C6 glioma cells into the hippocampus, 65Zn uptake in the tumor 6 days after the injection of 65ZnCl2 was more pronounced than in other brain regions (Figure 4).183 Based on these results, the authors propose that 69mZn, a short-half life g-emitter (Table 2), might be utilized for the evaluation and viability of brain tumors.

📷Figure 465Zn imaging of brain tissue sections implanted with C6 glioma cells. 65ZnCl2 was intravenously injected into rats 14 days after injection of vehicle (control) or C6 glioma cells into the hippocampus (n = 4). Autoradiography was performed on selected ...

3.1.2. Manganese Transport and Distribution in Brain

Similar to Zn, Mn is also required for proper brain function and development; however, chronic exposure to Mn is toxic and has been linked to neurodegenerative disorders.184 Approximately 80% of the total Mn in the central nervous system is found in the active site of glutamine synthetase, an enzyme that catalyzes the conversion of glutamic acid to glutamine.185 After intestinal absorption, dietary Mn is transported to the liver prior to delivery to the brain.186 The blood-brain and blood-cerebrospinal fluid barriers are two barrier systems in the brain which are critical to normal functioning and have been implicated in various neurodegenerate diseases.187Similar to other trace metals, the blood-brain barrier constitutes the main supply route for Mn to the brain. Autoradiographic studies revealed that Mn enters the brain from the blood mainly across the cerebral capillaries and the cerebrospinal fluid.159,160 Within 1 hour postinjection, the metal accumulated in the choroids plexus, and after 3 days, it redistributed to the dentate gyrus and CA3 of the hippocampus.159The biological half-life for elimination of 54Mn from the brain was determined to be in the range of 51–74 days.161 A study with rats varying in age between 5 days to 95 weeks also underscored the importance of Mn in the developing brain.188 As already observed for Zn, 54Mn uptake was highest for the neonatal age group. The highest Mn concentrations were found in the hippocampal CA3, the dentate gyrus, and the pons, thus contrasting the aging brain, with 54Mn being located in the inferior colliculi, olivary nuclei, and red nuclei.188 These findings strongly suggest that Mn serves a dual role in both brain development and its normal function.

Despite the similarities of Mn uptake into the brain, the transport mechanisms and involved proteins appear to be different. There is evidence that transferrin (Tf), the principal Fe carrier protein, might be also involved in Mn transport to the brain,189,190 although non-protein bound Mn enters the brain more rapidly than Tf.191,192 Given the presence of Tf receptors on the surface of the cerebral capillary endothelial cells,193 it is conceivable that Tf-bound Mn is released within the cells and subsequently transferred to the abluminal cell surface for extracellular release into the interstitial fluid. To examine the role of transferrin in the Mn distribution in the brain, 54Mn concentrations were autoradiographically monitored after intravenous injection under three different conditions: untreated aqueous 54MnCl2, pH 8.6 buffered 54MnCl2, which has a higher affinity for transferrin, and transferrin-bound 54Mn(III).194 One hour after injection, both 54MnCl2 and buffer-treated 54MnCl2 were found to be concentrated in the choroid plexus region. After 6-days, all three tracers were distributed in inferior colliculi, red nuclei and superior olivary complex; however, the radioactivity from transferrin-bound 54Mn(III) was the lowest of all three substrates. The results suggest that Mn is transported into the brain in a pathway which is not solely dependent on transferrin. Another study with hypotransferrinemic mice, an animal model with low plasma transferrin concentration,195 came to a similar conclusion that Tf is not required for Mn transport across the blood-brain barrier.196

3.1.3. Iron Transport and Distribution

In the brain, iron is mostly concentrated in oligodendrocytes and may be required in myelin synthesis.197 The Fe transport protein Tf is presumed to be directly involved in delivery of Fe across the blood-brain barrier through receptor-mediated endocytosis as described in the previous section.198,199 While transferrin is mainly expressed in the liver, a substantial amount of the protein is also found in the brain.200 Iron saturation of plasma transferrin is one of the hallmarks of hereditary hemochromatosis, an iron overload disorder which leads to abnormal iron deposition in tissues, especially the liver.201 The pathological condition of hemochromatosis can be simulated by iron saturation of transferrin with ferric chloride in citrate buffer. Following this protocol, Takeda et al. tested the effect of 59Fe saturation of transferrin on its delivery into the brain by autoradiographic imaging.202 The study showed that 24 hours after injection of 59FeCl3 into the blood stream, the 59Fe concentration in the brain of iron-loaded mice was lower compared to untreated control mice, except within the choroid plexus region which showed an equal concentration. At the same time, the 59Fe concentration in the liver was found to be four times higher compared to control mice. These results suggest that non-transferrin-bound iron is primarily absorbed by the liver, thus leading to a decrease in Fe delivery to the brain through the transferrin mediated pathway. These findings agree with the observation that hereditary hemochromatosis is rarely accompanied by neurological disorders.203

The hypotransferrinemic (HP) mouse is a naturally occurring mutant with a point mutation or small deletion in the transferrin gene, resulting in