Cells employ various metal and metalloid ions to augment the structure and the function of proteins and to assist with vital biological processes. In the brain they mediate biochemical processes, and disrupted metabolism of metals may be a contributing factor in neurodegenerative disorders. In this tutorial review we will discuss the particular role of X-ray methods for elemental imaging analysis of accumulated metal species and metal-containing compounds in biological materials, in the context of post-mortem brain tissue. X-rays have the advantage that they have a short wavelength and can penetrate through a thick biological sample. Many of the X-ray microscopy techniques that provide the greatest sensitivity and specificity for trace metal concentrations in biological materials are emerging at synchrotron X-ray facilities. Here, the extremely high flux available across a wide range of soft and hard X-rays, combined with state-of-the-art focusing techniques and ultra-sensitive detectors, makes it viable to undertake direct imaging of a number of elements in brain tissue. The different methods for synchrotron imaging of metals in brain tissues at regional, cellular, and sub-cellular spatial resolution are discussed. Methods covered include X-ray fluorescence for elemental imaging, X-ray absorption spectrometry for speciation imaging, X-ray diffraction for structural imaging, phase contrast for enhanced contrast imaging and scanning transmission X-ray microscopy for spectromicroscopy. Two- and three-dimensional (confocal and tomographic) imaging methods are considered as well as the correlation of X-ray microscopy with other imaging tools.
Living systems depend on their ability to accumulate, release and use certain metals. With their rich coordination chemistry and redox properties, cells employ a host of biologically essential metal ions to augment protein structure and function and to carry out vital life processes. The spatial distributions of specific metals and their compounds are as important as their chemical properties, because both their localization and their concentration change in biological systems, and their transport and compartmentalization is critical for effective utilization. In the human body, the central nervous system has an immense biological complexity as a command center for cognitive and motor functions [1]. The role that may be played by the gut in regulating central nervous system behavior is a burgeoning area of enquiry [2]. The brain contains numerous endogenous compounds that are involved in signaling, biosynthesis, and metabolic processes. Metals are particularly important during specific neurological events and in many neurodegenerative diseases.
Metal homeostasis is defined as the metal uptake, trafficking, efflux, and sensing that allows organisms to maintain an appropriate (often narrow) intracellular concentration range of essential metals . Metal homeostasis must be maintained by coordinated uptake, trafficking and efflux pathways that place the required amount of the required metal at the required place and time in the cell . The inventory of metals and their species in cells and tissues (including metalloproteins and/or metalloenzymes) is termed as the metallome and the analysis thereof as metallomics . Imaging and quantifying sub-cellular structures provides essential information about cell function, especially if this is done non-destructively without altering the cellular structure . In general, sample preparation methods for chemical imaging analysis should maintain the localization of the analytes of interest without causing any degradation. Since cells and tissue sections are more or less transparent for high-energy X-rays, this allows the investigation of the interior of thick biological samples, without destructive sample preparation, using three dimensional imaging methods.
It was the need to ‘see inside’ opaque objects, especially biological tissues, and to resolve features too small for optical microscopes, or too thick for electron microscopes, that spurred the development of X-ray microscopes to create images with higher resolution than visible or UV light, their wavelength being less than a tenth of a nanometer for X-rays above an energy of 10 keV. This much shorter wavelength means they are less hindered by the diffraction limit which has historically limited spatial observation to micro dimensions for visible or UV light, a disadvantage that could only recently be addressed with super-resolution microscopy techniques . It is possible to use X-rays to visualize cells without the need for chemical fixation, dehydration, or staining of the specimen. As such, X-ray methods are better suited than routine light and electron-based methods (excepting where stabilization with cryo-techniques is possible for imaging native-state specimens at the functionally important spatial resolution of a few tens of nanometers , minimizing interventions which will alter the metal chemistry in the sample such as changes in metal oxidation states. For intracellular imaging of metal species in delicate biological samples such as brain tissues, it is now possible, using the intense X-ray beams of synchrotron X-ray facilities, to achieve nanometer spatial resolution with sub-ppm detection limits for the wide range of metallic that may be present in the normal or malfunctioning brain.
The rationale for investigating metals in the brain is multi-fold. The excellent sensitivity and specificity achievable with X-ray microscopy (XRM) allows the investigation of metal toxicity, for example from environmental exposure to heavy metals such as cadmium, mercury, and arsenic . It also supports studies of the normally functioning brain and investigation of disease-mediated changes to the storage and metabolism of biologically-essential metal elements which may occur in specific intracellular compartments , or as widespread accumulation in multiple regions of the brain . XRM continues to grow in utility for the evaluation of brain tissues during the development of metal-containing compounds and tracers for treatment, clinical imaging and improved diagnostic techniques as reviewed elsewhere . With the emergence of new technologies, pre-clinical imaging of the distribution of metal species and compounds in animal models of disease is an important tool to evaluate the impact of interventions before they are attempted in clinical trial
According to Maret, 21 elements are presently defined as essential for human life, with a number of additional elements known to be beneficial but not yet confirmed as essential . This list includes a controversial one, chromium that in its trivalent valence state is essential and in the hexavalent state toxic. Other elements are essential in some particular species or in particular ecological niches
The main category of elemental constituents of biological materials are those involved in synthetic organic chemistry; hydrogen, carbon, nitrogen, oxygen, chlorine and sulfur show much cellular ultrastructure and are, up to oxygen, difficult to detect in absorption contrast or fluorescence with multi-keV X-rays. They have low fluorescence yields and little absorption contrast. These components are more easily studied with Soft X-ray imaging that utilizes the energy spectra from these elements can provide contextual information about the local environment that complements imaging of other metals (by permitting detailed imaging of tissue structure, and identification of signatures specific to certain proteins,
Alkali and earth alkaline metals such as sodium, potassium, magnesium, and calcium ions are present in ca 0.1 M concentration in tissues and have been studied over a long time in neurobiology [1]. In kinetically labile form, reversibly binding cellular targets, they are involved in active cell transport or cell signaling processes [4]. These elements are not a focus for this review. The comparatively high concentration of these metals in the brain has long-enabled optical imaging, particularly in conjunction with fluorescent probes and indicators [1]. Although synchrotron radiation techniques offer complementary means to investigate structural and temporal aspects of these metals in the brain, optical microscopy continues to underpin many advancements of the field .
Phosphorus is essential as a structural component of cell membranes and nucleic acids and involved in many biological processes. Bromine was added comparatively recently as an essential element for tissue development and architecture [20]. The main role of iodine is as a constituent of thyroid hormones required for brain development. Sulfur and selenium are present in amino acids and play characteristic functions in cells. Because of the versatility of sulfur with its many oxidation states and its prevalence in the environment, sulfur evolved to fill many structural, catalytic, and regulatory roles in biology. The experimental and methodological challenge of sulfur speciation in tissues has been addressed with microfocus X-ray absorption spectroscopy (XAS) in the context of brain tumors [21]. In particular, sulfane sulfur, which is sulfur in the thiosulfoxide, has been found to have regulatory functions in biological systems. The review of Toohey and Cooper outlines the functions of sulfane sulfur, its unique nature, and its bio-generation . Selenium is an essential micronutrient with a brain-specific physiology. While the brain is rather poor in selenium compared to other tissues, Kuhbacher et al. reported that selenium levels in the rat brain were the highest in hippocampus, cerebellum, brainstem and ventricles . Biological functions of selenium manifest themselves via 25 selenoproteins that have selenocysteine at their active center, and the importance of selenium and selenoprotein for brain function, from antioxidant protection to neuronal signaling, is highlighted by Solovyev ]. Selenium is later included in the context of it being a ‘metalloid’; strictly it is not a metal, but it shares certain properties with the metal elements.
Of most concern here are the late first row transition metals: iron, copper, zinc, and manganese, and while less abundant, chromium, cobalt, molybdenum, and nickel are also essential. With their rich chemistry, all of these were incorporated in living organisms quite early in evolution as essential for life. These elements are understood to be present in protein active sites as metabolic cofactors for structural and catalytic functions, but are increasingly also recognized for a second messenger role in cell signaling . As we will see later, there is a complex interplay between these metals in the life processes described by metallomics.
The essential metals must be obtained from the environment and appropriately bound or compartmentalized within the cell for use in biochemical pathways. They are then incorporated into proteins functioning in dioxygen transport, electron transfer, redox transformations, and regulatory control. They are essential for the growth and function of the brain, and become highly concentrated in grey matter with ageing , and play fundamental roles in white matter, for example in the myelination of axons. Their transport into the brain is strictly regulated by the brain barrier system, i.e., the blood-brain and blood-cerebrospinal fluid barriers . The essential elements present a formidable challenge, in that their concentration range in any given compartment must be precisely regulated. Deficiency impedes biological processes, and excess can be toxic. Copper, for instance, is an essential metal that provides catalytic function to numerous enzymes and regulates neurotransmission and intracellular signaling. Conversely, a deficiency or excess of copper can cause chronic disease in humans . Metallothioneins and related sulfur-rich chelators are understood to play important roles in metal ion homeostasis .
Once appropriated, metals must be directed to metalloenzymes or metal storage proteins within the cell. The precise regional, cellular and subcellular locations of these metals are increasingly objects of study. Transition metals can exist in many different forms within cells, including as free ions, coordinatively incorporated in biomolecules such as proteins, or in a labile association with low molecular weight species such as amino acids or glutathione, from which the metal ion could be released by changes in the cellular environment ,While metals show spatial time-averaged heterogeneity, there are also transient changes in concentration occurring as a result of exchange between metal-ion-binding species and labile metal ion pools within cells . Essential elements can undergo complex interactions with non-essential elements and other molecular components. In this context, it is no surprise that metal homeostasis is impacted in neurodegenerative disorders, but it is not yet fully determined in which diseases it is a causative factor as opposed to a consequence of other pathogenic processes.
There remain a large number of non-essential elements (metals and metalloids) in the periodic system of the elements that are not included in the periodic system of biology. Some of them are present at significantly higher overall concentration than the essential elements, while others became more abundant in life forms since the human influence in the Anthropocene . While the bioactivity of some of these elements has positive effects on health, many non-essential elements nevertheless are biologically reactive , in some cases both cumulative and detrimental to health. New applications and manufacturing processes increasingly expose animals and humans to a number of metals to which they have not been exposed to in the past, in particular those from the bottom part of the periodic system. There are many issues of metal toxicity and environmental effects concerning toxic heavy metals (e.g. mercury, lead, cadmium) and other metals (e.g. aluminum which, in the so-called 'Aluminum Age', is now omnipresent in modern life). Food chains and food webs amplify some exposure, which has largely unexplored effects for more recently employed metal ions . The most hazardous of these build up in the biological food chain. For instance, atmospheric deposition of mercury onto sea ice and circumpolar sea water provides mercury for microbial methylation, and contributes to the bioaccumulation of the potent neurotoxin methylmercury in the marine food web . Different methylmercury species (compounds containing the CH3Hg group) cross the blood-brain barrier and are highly neurotoxic. The element can thus affect the human nervous system and harm the brain. XRM of individuals poisoned with high levels of methylmercury species showed elevated cortical selenium with significant proportions of nanoparticulate mercuric selenide plus some inorganic mercury and methylmercury bound to organic sulfur. HgSe is a particularly stable and insoluble form of mercury with molar solubility product Ksp 10− 59. HgSe thus represents an inorganic non-bioavailable form, effectively removing any mercury bound to selenide from involvement in biological processes .
Many metal ions (essential or non-essential) are understood to play critical roles in disorders of the central nervous system including AD, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), prion diseases, and others . For example there is evidence of brain copper dysregulation in AD , with changes in the distribution of copper linked with various aspects of the disease process: protein aggregation, defective protein degradation, oxidative stress, inflammation and mitochondrial dysfunction. Although AD is a multifactorial disease that is likely caused by a breakdown in multiple cellular pathways, copper and other essential metal ions such as iron and zinc play a central role in many of these cellular processes . The role in neurodegenerative disorders of these essential metals, and of those acquired through environmental exposure, requires better understanding.
Understanding the complexity of metallochemistry in the living brain is critical to designing appropriate therapeutic interventions. For example, combinations of iron, zinc, copper and aluminum have been shown in-vitro to influence the formation of amyloid fibrils found in a pathological hallmark of Alzheimer's disease (AD) in a manner which may have consequences for metal chelation therapy Meanwhile, the potential to use iron chelators as therapy to delay PD and related disorders is being explored in clinical trials
In order to fully appreciate the potential of X-ray methodologies for chemical imaging of biological materials, we need to compare their characteristics with those of the “ideal microscope” . In an ideal world, data from one single microscope would be able to yield sufficient information to build a complete picture of a cell in its native (living) state. In reality this is an impossible dream . Different microscopic techniques have particular unique imaging characteristics. Based on particular methodologies, we may discern infrared, visible, UV or Raman microscopy, XRM, electron microscopy, particle induced X-ray emission, mass spectrometry imaging, fluorescent labelling methods, proximal probe microscopies that are capable of generating data within a well-defined window of spatial resolution and information content. The combination of several modes of observation in a single instrument is advantageous. In recent years, correlative microscopy, combining the power and advantages of different imaging systems, incorporating light, electrons, X-ray, nuclear magnetic resonance (NMR) and so forth, has become important, especially for the study of biological materials . Among all the possible combinations of techniques, light and electron microscopy are historically prominent. This review will highlight, amongst others, the possibilities of X-ray imaging techniques in combination with light and electron microscopy and mass spectrometry imaging for more comprehensive analysis of the material complexities of the brain.
Techniques for in-situ metal imaging analysis depend on three key properties: spatial resolution, sensitivity, and selectivity. Spatial resolution and sensitivity are normally negatively correlated, they are connected since the absolute detection limits are defined by the amount of analyte being sampled in a given two-dimensional (2D) pixel or a three-dimensional (3D) voxel. Selectivity concerns the ability to determine the metal's chemical form, oxidation state, coordination environment or association with specific proteins or other molecular structures .
The most important characteristics of the ideal microscope are summarized . The “spatial resolution” at the left in the figure determines the 2D or 3D spatial discrimination level of the measurements. Recent evolution of synchrotron X-ray imaging methods has achieved chemical imaging with a spatial resolution of 10 nm or better, close to the supramolecular interaction level of molecular assemblies in cells. By contrast, laboratory scale instruments combining absorption computed tomography (CT) and XRF-CT have been developed with spatial resolution reaching 20 μm
The term “analytical characteristics” determines the analytical information that is derived from the measurements. Just like other methods in analytical chemistry, chemical imaging analysis is characterized by a number of quality criteria such as sensitivity, selectivity, and accuracy (exactness). Most analytical imaging techniques provide qualitative information; quantitative imaging is often difficult, mainly as a result of matrix effects . Accuracy and consequently quantitative imaging analysis with X-ray imaging tools depend on issues such as linearity of response, the dynamic range of the response curve and the extent of matrix interferences and other measurement artefacts; they will be covered further in this review. We should also distinguish analytical coverage (elemental, molecular, structural and so forth) and the data-generating ability (multi-spectral, hyperspectral, and so forth). The detection limit is determined by the signal-to-noise ratio of the spectral measurements. A higher spatial resolution yields a reduced sample size and hence, for a given probe flux, a reduced signal. Focused beam techniques that increase X-ray flux can achieve higher sensitivity at higher spatial resolution. With SR-XRF, the absolute detection limit has been demonstrated to be as low as 10− 18 g for transition elements such as Fe, detected in a cellular structure with a diameter of 90 nm . Finally, the selectivity determines the potential of a method for discrimination between molecular form, oxidation state, and coordination environment (speciation). There are a number of other characteristics that need to be considered, such as speed of the analysis, degree of automation, the cost of the infrastructure or accessibility of the instrumentation and so forth, demand exceeds the facility time available for XRM at synchrotron sources.
“Sample preservation” (sample integrity, sample health), a particularly important factor for biomaterials, is connected with the way the sample is able to tolerate the measurement process without deterioration. It is affected by factors including the vacuum conditions, hydration state of the sample, temperature, and dose received from the X-ray beam.
Minimizing the radiation dose for a given image resolution and contrast is a primary challenge for XRM. Radiation damage is dose-dependent and alters and subsequently destroys the sample and drastically limits the applicability of any imaging method. SR beamlines enable high-resolution applications but radiation damage becomes more pronounced as the spatial resolution is pushed to smaller values. For delicate biological samples, minimizing the applied dose for a given image resolution is a primary challenge, and biological samples are heterogeneous from the perspective of radiation damage. Resilience is heavily dependent on the properties of the material under investigation and the sample environment. X-rays are less damaging than most other projectiles used in analytical beam techniques. With hard X-rays of 13.8 keV, 3D tomographic reconstructions with a total dose of 1.6 × 105 Gray (J/kg) were documented . Such doses allow multimodal hard-X-ray imaging of a chromosome with nanoscale spatial resolution without detectable radiation damage between two successive scanned images . For analysis of metal ions in brain tissue, it is critical to understand the chemical (and in some cases mineral) modifications to metal elements as a result of the received dose.
Sample history prior to measurement is as important as the analytical environment; some XRM methods may be used to image live cellular material, but the majority of studies utilize archived brain tissue or cells that have been chemically fixed, frozen, and/or dehydrated prior to measurement. The effect of sample processing on tissue integrity and retention of trace metals in mammalian cells and tissues is an important area of study . At room temperature, wet specimens are damaged by impinging radiation due to primary bond breaking as well as hydrolysis of water, so that they suffer from shrinkage as well as material diffusion. Dehydration conveys increased robustness against radiation damage but a significant breakthrough toward accurate imaging of subcellular structures and elemental distributions was achieved by rapidly cooling the fully hydrated sample to a vitrified state and imaging the samples under frozen-hydrated conditions . Such biological samples have better preserved local structure and elemental composition than dehydrated ones [
Temporal characteristics” are important in two respects. First, scanning for the purpose of 2D and 3D imaging analysis is an inherently slow process. It comprises economic factors such as speed and cost. Apart from this economic factor, the total measurement time to generate an image also dictates the scope for dynamic measurements of time-dependent processes. Sensitive approaches are required to follow fluctuations in normal metal homeostasis that accompany processes of development, differentiation, senescence, stress response and so forth, or to acquire knowledge about the redistribution of metals and trace elements accompanying the development of different diseases . Ahmed Zewail, Nobel laureate for chemistry in 1999, summarizes how space-time applications, particularly 4D electron microscopy but also other imaging methods, can be exploited for such work . D electron microscopy is used for studying picosecond dynamics, but even at orders-of-magnitude longer time scales the study of time-dependent processes by XRM has potential to provide important insights. That metal ions can be mobilized via labile pools in cells, which are tightly regulated by complex systems, indicates that in addition to spatial heterogeneity there is an important temporal component that is influenced by specific cellular events. Exploring the metal content with high spatial and temporal resolution requires advanced analytical tools and techniques . There have been advances in imaging metal ions in living cells with high spatial and temporal resolution using optical fluorescence microscopy, and spectroscopic methods (including Fourier transform infra-red and small angle X-ray scattering to study processes such as conformational changes to proteins when they undergo metal binding) are discussed elsewhere , dynamic 4D XRF imaging methods are not presently established
In recent years, there has been rapid improvement in sensitivity and spatial resolution for multi-element (panoramic) bio-imaging of metals, with different methods now providing micron to nanometer spatial resolution, and with detection limits from 0.1 to 100 μg•g− 1. The existing bio-imaging methods that are used at present are based on: (1) mass spectrometry; (2) “beam” methods employing (laser) light, electrons, X-rays or energetic particles to measure characteristic radiation; or (3) methods employing metal-selective probes . Each method has its own advantages and limitations, such as the ability to deliver reliable quantitative analytical results, and the overall cost of the use and accessibility of the instrumentation.
Of the methodologies that are based on excitation of the lower electronic shells, the most powerful is achieved by the combination of high spatial resolution with high sensitivity XRM. This requires instrumentation that is not readily accessible. Electron excitation in electron microscopy techniques (primarily scanning electron microscopy, electron probe microanalysis, and transmission electron microscopy) is orders-of-magnitude less sensitive for elemental analysis [48]. Proton or other heavy ion beam techniques approach the sensitivity and spatial discrimination levels of XRM, but rely also on complex and not easily accessible instrumentation . Mass spectrometry imaging (MSI) techniques have the unique advantage of being able to measure isotope ratios. For elemental analysis, dynamic secondary ion mass spectrometry (D-SIMS) combines spatial resolution down to 35 nm with attogram (or less) detection limits but is limited to the simultaneous measurement of only 5–7 isotopes in the major instrument used for bio-analysis, the Cameca NanoSIMS . Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has detection limits for most elements approaching ppb levels, but is limited to a spatial resolution of the order of μm. This is within the realms of sub-cellular neuronal imaging . The sensitivity of LA-ICP-MS can be an order-of-magnitude superior to that of many SR XRM techniques , but this is being challenged by advances in XRM beamline development. Compared with LA-ICP-MS, μ-XRF can offer higher resolution (tens of nanometers), although the respective merits of the two techniques depends on the elements being studied, the science question being addressed, and sample handling constraints.
Metals are heterogeneously distributed within biological materials. Understanding the functioning of normal and pathophysiological processes requires imaging techniques at different scales of spatial discrimination. As illustrated schematically for the brain in Fig. 2, the constituent material can be considered at three levels of spatial resolution: that of the organ, the tissue architecture composed of individual cells, and the cell and its intercellular structures (organelles). Tissues are complicated assemblies of multiple interacting cell types that communicate with each other to achieve physiological states. At the highest level, imaging of metal species and compounds in biological materials requires nanometer spatial resolution to match the intracellular complexity and to visualize interactions at the molecular and supramolecular level. Metals are found in high concentrations within structures where they react, particularly in organs with high metabolic activity such as the brain. Within cells, metals are localized according to need. For example: mitochondria contain high levels of iron in Fe-S clusters and products of haem synthesis; the nucleus is rich in zinc finger proteins essential for gene transcription; and the Golgi complex is a major regulator of cellular copper levels
The brain has a unique chemical composition and reactivity at the molecular level. The requirements arising from cognitive and motor functions result in its having the highest concentration of metal ions in the body and the highest per weight consumption of body oxygen [1], a side-effect of which is its heightened vulnerability to oxidative stress damage. Transition metals such as copper, zinc, iron, manganese, and cobalt are key cofactors in a wide range of brain cell functions, including cellular respiration, antioxidant removal of toxic free radicals, and oxygen delivery to brain cells. They are also cofactors for cell signaling at synapses. Even minor disruption to, or errors in, the regulation of biometals can impact cell function and, ultimately, neuronal survival . As opposed to most other trace elements, which are coordinated to protein ligands, selenium is covalently bound. As a component of the amino acid selenocysteine, it is incorporated in several selenoproteins which play important roles in brain development and metabolism
Multiple abnormalities occur in the homeostasis of essential endogenous brain biometals in a wide variety of disorders, including epilepsy and neurodegenerative disorders such as AD, PD, ALS, MSA and Huntington's disease. This includes abundant elements such as calcium, the transition metals, and trace elements such as the metalloid selenium. Metal accumulation is frequently associated with microscopic insoluble protein deposits in the proteinopathies, and can be deficient in cells and within cellular compartments. Therefore, it is essential to study them on the microscopic level
Mineralization of certain metals, including iron and calcium, is inherent to certain physiological and pathophysiological processes (e.g. the formation of ferrihydrite cores in the iron storage protein ferritin, and the deposition of calcium in brain tissue in the rare disorder Fahr's disease, respectively). In recent work from Maher and colleagues it is suggested that magnetite nanoparticles observed in post-mortem human brain samples originate from air pollution, and it is noted that similar nanoparticles have been associated with AD pathology . Indeed, evidence for magnetite deposits associated with amyloid pathology has previously been demonstrated by a range of analytical techniques including μ-XAS in tissues surveyed by μ-XRF . However, it was assumed that the magnetite formation was endogenous; potentially a consequence of an interaction between iron stores and aggregating amyloid . The scope for nanoparticulate iron oxide to stimulate excess free radical production is documented elsewhere , and although mishandled iron in the brain may contribute to the toxicity of the hallmark amyloid plaques in AD, there is not yet enough known to establish whether an external source of magnetite from air pollution may be a factor in the disease.
The interplay and complexity of metal ion metabolism is routinely underestimated, with analytical and conceptual constraints leading to many studies where a metal element is studied in isolation. There are the elements known to be essential to normal brain function, such as calcium, copper, zinc, or iron, where aspects of their metabolism are interdependent, and in addition there are elements such as aluminum which are understood to be non-essential but which can amplify catalytic pro-oxidant reactions involving essential metals . This interdependency is critical in conditions of both deficiency and excess. For example, the loss of the main copper transport protein, ceruloplasmin, appears to be responsible for derailing iron homeostasis in aceruloplasminaemia , and there is evidence for localized brain iron accumulation in Wilson's disease, which is primarily a disorder of copper accumulation (including in the central nervous system) . This presents a significant analytical and computational challenge. Despite advances in the development of models of brain metabolism, there is an inevitable dependency on empirical data to determine the impact of interventions such as chelation treatments. Chang recently created an analogy for metallomics in which the essential metal elements are compared with the different instrumental parts in a symphonic work. Describing or modifying the harmony of the orchestration cannot be achieved by following a single instrument (one element); it instead invites comprehension of the contributions from the full orchestra (i.e. the interplay of all contributing elements, essential or otherwise) [4]. Overall, the power of X-ray microscopy techniques is that they offer an unparalleled combination of sensitivity, specificity and spatial resolution to access simultaneously this broad spectrum of metal elements.
In the second half of the 19th century, experiments with highly energetic electron beams gave rise to RÖNTGEN’S fascinating discovery of the X rays. The first radiographs of human hands were made public 125 years ago . The medical personnel of today’s hospitals and dental offices regularly uses hard X-ray radiography and CT mainly for diagnostic purposes. The X-ray sources integrated into these systems still rely on the physical principles RÖNTGEN introduced more than 125 years ago.
Research activities in the field of high-resolution hard X-ray imaging, however, often employ synchrotron radiation facilities, which have many advantages compared to conventional systems. They offer unique brilliance, polarization, and pulsed time structure [2]. The combination of the synchrotron radiation facility’s photon flux with monochromators has led to tunable X-ray beams with reasonably high intensity, i.e. the photon energy can be selected so that the specimen becomes semi-transparent for optimized absorption-based imaging [3]. Artefacts well known from conventional sources including beam hardening are avoided
Despite the numerous advantages, synchrotron radiation facilities have fundamental drawbacks. The imaging experiments have to be carefully planned well in advance, since the usage requires successful beamtime applications. Therefore, liquid metal and inverse Compton scattering X-ray sources [4, 5] were introduced, for example, to exploit phase-contrast imaging in the local laboratory environment
High-resolution imaging experiments, such as phase-contrast imaging and spatially resolved small-angle X-ray scattering, can only be performed on biopsies and human tissue post mortem, because the dose exceeds the medically acceptable limits. Therefore, these X-ray-based techniques are complementary to the ultrasound and magnetic resonance-based in vivo methods with millimeter resolution. Even magnetic resonance microscopy cannot resolve microstructures below about 10 μm
The gold standard for investigating biological tissues at the cellular level is histology. It involves a series of tissue preparation procedures. First, the brain tissue is fixated, for example using formalin solution. Second, the fixated brain tissue is sliced either by sectioning at cryogenic temperatures or after embedding into paraffin. Before optical microscopy is applied, the slices are stained by means of preselected chemicals. This approach is not only established as a part of clinical practice at pathology departments, but also applied for brain research. Despite the two-dimensional nature of histological sections, serial sectioning allows for the three-dimensional visualization of tissues . For example, in 2013 the BigBrain project created an atlas of the full human brain with 20 μm isotropic voxels based on the combination of over 7,000 full brain histological sections with a data size of more than 1 TB [10]. The researchers aligned the two-dimensional sections by registration with an MRI volume recorded prior to histology. It should be noted, however, that the process of serial sectioning introduces tissue deformations such as rips, folds, and shears. Furthermore, the staining often introduces modulations in the brightness of the optical micrographs, especially for slides of centimeter size.
The spatial resolution in conventional histology is limited by the slice thickness, precluding the visualization of cellular or subcellular details in three-dimensional space without specialized, time-consuming acquisition protocols. One such approach involves simultaneous sectioning and optical microscopy, allowing for the atlas of an entire mouse brain (Golgi-Cox-stained and resin-embedded) at voxel sizes of 0.33 μm × 0.33 μm × 1.00 μm and requiring ten days of continuous data acquisition to yield the roughly 8 TB raw volume
Histological sectioning combined with electron microscopy is another approach, but penetration depths below 1 μm result in similar limitations related to sectioning. Other alternatives include magnetic resonance imaging, which offers a non-destructive, isotropic visualization and can be applied to larger volumes. Unfortunately, even the best available magnetic resonance imaging facilities only reach pixel sizes of a few tens of micrometers. Confocal microscopy has been used for volumetric imaging of brain tissue with volumes on the order of 4 mm3 [12]. Confocal light sheet microscopy has been employed for the visualization of an entire mouse brain at around 1 μm voxel length. This technique, however, relies on tissue clearing and on fluorescent dyes , which are usually incompatible with subsequent conventional histology.
Hard X rays have a penetration depth to overcome the limitation of sectioning. and provide micrometer or even nanometer resolution in tomographic imaging. As brain tissues are composed of low Z elements, conventional X-ray absorption provides hardly any contrast. Therefore, the phase-contrast modalities are preferable, especially for non-stained soft tissues . The contrast advantage for phase, i.e. the relation between the real and the imaginary parts of the refractive index, is several orders of magnitude, as pointed by the pioneer in the field, U. Bonse, decades ago.
Currently, there are several methods for phase retrieval in hard X-ray imaging . A powerful and frequently used method was proposed in 2002 by D. Paganin and coworkers, wherein a FOURIER-space filter is applied to standard radiographic projections to retrieve the phase [18]. This phase retrieval approach is ideal for high-throughput studies, as it allows for the fast and relatively simple data acquisition and processing. The interpretation of the derived quantities must be done with care, as it relies on a reasonable estimate of the ratio between the real to the imaginary part of the refractive index and the assumption that it is nearly constant throughout the specimen.
X-ray-based virtual histology, a synonym for high-resolution hard X-ray computed tomography of fixated and embedded tissues, has extended conventional histology to the third dimension with an isotropic resolution below 1 μm . X-ray-based virtual histology with the conventional absorption contrast was applied to bone, as demonstrated by e.g. P. Schneider et al. . X-ray staining protocols have been employed to improve the absorption contrast in X-ray-based virtual histology of soft tissues.
For the visualization of the cellular structure of soft tissues without staining, X-ray-based virtual histology has been based on phase contrast. Here, the X-ray-based virtual histology of lungs is a pioneering and early example, as the air-tissue interfaces yield an especially strong phase contrast [26]. J. Albers and coworkers recently summarized the wide variety of other X-ray-based virtual histology studies for soft tissues. Synchrotron radiation-based phase-contrast X-ray-based virtual histology of lung, brain, and heart tissues shows that the phase contrast imaging provides a quality superior to the conventional approaches involving staining protocols at substantially lower radiation dose . Several research teams have demonstrated phase-contrast X-ray-based virtual histology using advanced laboratory-based systems. For example, M. Töpperwien and coworkers [28] recently published a profound X-ray-based virtual histology study of paraffin-embedded human cerebellum using a liquid-metal jet source. The combination of the tomography data with automatic segmentation algorithms allowed for the three-dimensional localization of more than one million neurons in a volume of about 1 mm3. Compared to the synchrotron facilities, these advanced systems have been available in the laboratory of the researchers throughout the year but yield only limited photon flux even for a broad spectrum. The imaging of large volumes combined with high spatial resolution is, therefore, currently reserved for synchrotron radiation-based virtual histology.
Thanks to the essentially non-destructive nature of hard X rays, the subsequent validation of the imaging results by the established conventional histology is possible. Thus, X-ray-based virtual histology is also applied to select the cutting planes for histological sectioning [29], which has been traditionally done only by optical inspection, i.e. without the knowledge of the tissue features in the bulk. Therefore, X-ray-based virtual histology has been suggested as a valuable addition to the current workflow of histology in clinical and research settings
The extension of high-resolution X-ray-based virtual histology to centimeter-size objects is prevented either by the limited photon-beam size and by the restrictions of the detector. Laboratory-based X-ray sources with their cone beam allow for the full-field illumination of objects as large as the entire human skull, but limited flux reduces high-resolution acquisition for large specimens. Synchrotron radiation sources provide orders of magnitude higher photon flux, but the beam cross section is often limited to square millimeters [. Additionally, currently available detectors have generally 4000 × 4000 pixel arrays at best, limiting the spatial resolution to about a thousandth of the object’s diameter for Ml-field acquisitions. For example, the field-of-view corresponds only to 4 mm when using 1 μm-wide effective pixels.
Local tomography is a technique, where a tomogram is taken from a small part of the specimen . This approach allows for the reconstruction of a part of a specimen, even if the sample is significantly larger than the X-ray beam’s cross section and the effective detector width. A series of local tomography reconstructions can be combined to obtain the complete image of a large object . Due to the truncated projections taken in local tomography, the resulting reconstructions are prone to artifacts . These artifacts can be reduced by prior knowledge of the specimen geometry, extrapolating data into truncated regions , or with specialized reconstruction techniques.
An alternative approach is to create a mosaic projection (or sinogram) by stitching many limited field-of-view acquisitions before reconstruction. The field-of-view can be almost doubled by off-axis acquisition, wherein projections taken 180 degrees apart are combined prior to reconstruction . Even for phase-contrast imaging, this off-axis acquisition techniques allows for increased field-of-view without reducing contrast or spatial resolution . Additionally, stitching radiographs enabled us to image objects, which are more than 30 mm in diameter such as cochlear implants and the surrounding tissues including bone . This approach requires increased effort, since not only the sinograms are larger, but also the number of projections has to be raised. The limits of stitching techniques are currently a matter of discussion . Some research teams use the term tomosaic for techniques involving stitching many fields-of-view . On the one hand, this approach requires significant computational resources and precise translational stages. On the other hand, it provides a dose-efficient acquisition compared to recording numerous local tomography scans [. Recently, this approach has allowed for the imaging of a complete mouse brain with 0.8 μm-wide pixels. It has required the stitching of a mosaic grid of 12 × 11 images per projection and a dedicated data analysis package for handling the tera-voxel dataset . With a volume of one cubic centimeter, this measurement is around three orders of magnitude larger than previously performed studies. A complete human brain atlas, however, requires a 3,000 times larger volume. Consequently, dedicated protocols for the specimen preparation as well as the data acquisition and processing have to be further developed.
Currently, there are several large-scale research initiatives across the globe dedicated to brain research, including major efforts in Europe, the US, Canada, China, Korea, Taiwan, Japan, and Australia [43]. Brain imaging and brain atlases play a dominant role in these projects, as they provide information on the microanatomy of the brain. For example, the Multi-Level Human Brain Atlas is a co-design project of the Human Brain Project, a Flagship project of the European Union, which looks to build a multimodal, hierarchical brain atlas . Public availability and data sharing including high-quality brain atlases are essential for these initiatives [43]. Therefore, most recently, the Taiwanese synchrotron-radiation facility proposed a dedicated beamline, denoted 02A, which will be exclusively used for X-ray imaging for brain science. This beamline will be realized during Phase-II of that synchrotron radiation facility . At the European Synchrotron Radiation Facility (ESRF), the flagship beamline BM18 is under construction and will be operational during 2021. This beamline will have a huge beam diameter and will allow for an automated hierarchical imaging of entire human organs.
There is mutual consensus that hard X-ray computed tomography (CT) with true micrometer resolution should not be applied to living species especially humans, because serious radiation damage is expected, often resulting in diseases including cancer. Therefore, high-resolution hard X-ray studies of human tissues are performed post mortem. Nevertheless, the organ’s anatomy should be visualized as close as possible to the in vivo conditions. Therefore, the human brain was investigated post mortem by means of clinical magnetic resonance imaging (MRI). First, images with the brain inside the skull of the dead body were acquired. After removal of the brain from the skull, it swelled by about 5% . Subsequent to placing the human brain into formalin solution, a dozen MRI datasets during the formalin penetration were recorded, which led to local compressive and tensile strains larger than ± 20 %. The non-rigid registration of the MRI datasets allowed for the precise quantification of the entire formalin-induced deformation fields . This knowledge enables computer-based correction of high-resolution CT-data by less detailed MRI-data.
A. Lareida et al. discovered how to visualize osmium-stained ganglion cells present in the organ of Corti . The nondestructive neuron cell counting in a selected volume of 125 μm × 800 μm × 600 μm gave rise to the presence of 2,000 ganglion cells along one millimeter of the organ of Corti. Therefore, the study demonstrated for the first time that hard X-ray tomography allows visualizing individual cells within human nerve tissue. Although ganglion cells are encapsulated in highly X-ray absorbing bony tissue, individual ganglion cells were counted and measured in size and shape without any sectioning and related artefacts. Consequently, the paper provided an important basis for the direct comparison of healthy and altered inner ear morphologies such as dysplastic malformations down to the sub-cellular level.
Already a year later, G. Schulz et al. visualized individual Purkinje cells in non-stained human cerebellum [7]. The uniqueness of the grating-based hard X-ray-based phase-contrast tomographic imaging became clear by the detailed comparison with MR microscopy and selected histological sections of the same part of the human brain [6]. For such a comparison, one needs powerful registration algorithms [48]. N. Chicherova et al. performed dedicated research activities for the non-rigid slice-to-volume registration as required for the identification of the counterparts of histology slides within CT-data . This task is particularly challenging due to the many degrees of freedom . The registration of twodimensional histology slides to X-ray-based virtual histology volumes allows for the validated interpretation of tomography results and for the colorization of tomography datasets according to the well-known stains in histology, i.e. to extend the histology into the third dimension . This study was mainly based on advanced laboratory CT and demonstrated the power of paraffin embedding in brain imaging. A. Khimchenko et al. have shown for the first time that non-stained cells in human brain tissue can be made visible even with conventional high-resolution CT-systems . Nevertheless, at synchrotron radiation facilities the resolution of tomography systems can be pushed to a fraction of a micrometer and subcellular structures are clearly detectable thanks to the superior density resolution of phase-contrast mode compared with absorption mode. The phase tomography approaches, i.e. grating-based interferometry, holotomography, and single-distance phase retrieval were experimentally compared [. These studies, performed on soft tissues of mice and rats, showed that single-distance phase retrieval minimizes the necessary time for data acquisition and the size of raw data to be recorded. Therefore, single-distance phase retrieval should be considered for future projects on three-dimensional imaging of human brain with hard X rays
Although X-ray-based virtual histology of paraffin-embedded brain tissue based on synchrotron radiation has allowed for the visualization of subcellular details and automated cell counting [54], the application of X-ray optics enabled tomographic imaging beyond the optical limit, as recently demonstrated . Nanoholotomography has pushed X-ray-based virtual histology beyond the 100-nm limit, with isotropic voxel sizes down to 25 nm and the ability to study the anatomical features of individual cells, e.g. organelles, in brain tissues by hard X rays . This approach is unique because not only is spatial resolution superior to optical microscopy, but also the accessible volumes are substantially larger than in electron-microscopy based techniques. Therefore, the gap between conventional histology and electron-based methods has been bridged.