Mark J. Hackett

Elemental and Chemically Specific X-ray Fluorescence Imaging of Biological Systems

From the perspective of a chemist, biology confers a rich variety of roles on a number of metal ions. It is widely agreed that a large fraction of the genomic output of living things contains metal or metalloid ions, although estimates of this fraction vary widely and depend upon which metal ions are considered.1−3 Moreover, recent reports suggest that, at least in some cases, there are many uncharacterized metalloproteins.4 With inclusion of the s-block metals such as Na, K, Mg, and Ca, the proportion likely approaches 100%; recent estimates from the protein data bank indicate that the prevalence of heavier metal ions of atomic number above 20 within proteins is around 22%,5 with Zn2+ proteins alone constituting about 11%. Living organisms have an inherent and very rich physical structure, with relevant length scales ranging from the nanometer scale for subcellular structure to hundreds of micrometers and above for tissue, organ, or organism-level organization. The ability to derive the spatial distribution of elements on this diversity of length scales is a key to understanding their function. Metals play essential and central roles in the most important and chemically challenging processes required for life, with active site structures and mechanisms that, at the time of their discovery, have usually not yet been duplicated in the chemical laboratory. Furthermore, diseases of metal dysregulation can cause disruption in the distribution of metals.6 For example, Menke’s disease and Occipital Horn Syndrome,7 and Wilson’s disease,8 involve disruption in copper uptake and excretion, respectively, through mutation in the ATP7A and ATP7B Cu transporters.9 The mechanisms of action of toxic elements such as mercury and arsenic are also of interest, as are essential nonmetal trace elements, such as selenium. Likewise, an increasing number of pharmaceuticals include metals or heavier elements; such chemotherapeutic drugs include the platinum derivatives cisplatin and carboplatin,10 some promising new ruthenium drugs,11 and arsenic trioxide, which has been used to treat promyelocytic leukemia.12 Understanding the localization, speciation, and distribution of these at various length scales is of significant interest. A wide variety of heavier elements can be probed by X-ray spectroscopic methods; these are shown graphically in Figure ​Figure1.1. X-ray fluorescence imaging is a powerful technique that can be used to determine elemental and chemical species distributions at a range of spatial resolutions within samples of biological tissues. Most modern applications require the use of synchrotron radiation as a tunable and high spectral brightness source of X-rays. The method uses a microfocused X-ray beam to excite X-ray fluorescence from specific elements within a sample. Because the method depends upon atomic physics, it is highly specific and enables a wide range of chemical elements to be investigated. A significant advantage over more conventional methods is the ability to measure intact biological samples without significant treatment with exogenous reagents. The technique is capable of determining metal and nonmetal distributions on a variety of length scales, with information on chemical speciation also potentially available. Figure ​Figure22 shows examples of rapid-scan X-ray fluorescence imaging at two contrasting length scales: rapid-scan imaging13 of a section of a human brain taken from an individual suffering from multiple sclerosis and showing elemental profiles for Fe, Cu, and Zn;14 and a high-resolution image showing mercury and other elements in a section of retina from a zebrafish larva treated with methylmercury chloride.15 We will discuss both the state of the art in terms of experimental methods and some recent applications of the methods. This Review considers X-ray fluorescence imaging with incident X-ray energies in the hard X-ray regime, which we define as 2 keV and above. We review technologies for producing microfocused X-ray beams and for detecting X-ray fluorescence, as well as methods that confer chemical selectivity or three-dimensional visualization. We discuss applications in key areas with a view to providing examples of how the technique can provide information on biological systems. We also discuss synergy with other methods, which have overlapping or complementary capabilities. Our goal is to provide useful and pertinent information to encourage and enable further use of this powerful method in chemical and biochemical studies of living organisms. Figure 1 Periodic table of the elements showing elements of biological interest that can be probed using X-ray fluorescence imaging. Elements are divided into three categories, those that are physiologically important, those that are pharmacologically active, ...

X-ray-induced photo-chemistry and X-ray absorption spectroscopy of biological samples

As synchrotron light sources and optics deliver greater photon flux on samples, X-ray-induced photo-chemistry is increasingly encountered in X-ray absorption spectroscopy (XAS) experiments. The resulting problems are particularly pronounced for biological XAS experiments. This is because biological samples are very often quite dilute and therefore require signal averaging to achieve adequate signal-to-noise ratios, with correspondingly greater exposures to the X-ray beam. This paper reviews the origins of photo-reduction and photo-oxidation, the impact that they can have on active site structure, and the methods that can be used to provide relief from X-ray-induced photo-chemical artifacts.

X-ray absorption spectroscopy at the sulfur K-edge: a new tool to investigate the biochemical mechanisms of neurodegeneration.

Sulfur containing molecules such as thiols, disulfides, sulfoxides, sulfonic acids, and sulfates may contribute to neurodegenerative processes. However, previous study in this field has been limited by the lack of in situ analytical techniques. This limitation may now be largely overcome following the development of synchrotron radiation X-ray absorption spectroscopy at the sulfur K-edge, which has been validated as a novel tool to investigate and image the speciation of sulfur in situ. In this investigation, we build the foundation required for future application of this technique to study and image the speciation of sulfur in situ within brain tissue. This study has determined the effect of sample preparation and fixation methods on the speciation of sulfur in thin sections of rat brain tissue, determined the speciation of sulfur within specific brain regions (brain stem and cerebellum), and identified sulfur specific markers of peroxidative stress following metal catalyzed reactive oxygen species production. X-ray absorption spectroscopy at the sulfur K-edge is now poised for an exciting new range of applications to study thiol redox, methionine oxidation, and the role of taurine and sulfatides during neurodegeneration.

Prolonged therapeutic hypothermia does not adversely impact neuroplasticity after global ischemia in rats

Hypothermia improves clinical outcome after cardiac arrest in adults. Animal data show that a day or more of cooling optimally reduces edema and tissue injury after cerebral ischemia, especially after longer intervention delays. Lengthy treatments, however, may inhibit repair processes (e.g., synaptogenesis). Thus, we evaluated whether unilateral brain hypothermia (∼33°C) affects neuroplasticity in the rat 2-vessel occlusion model. In the first experiment, we cooled starting 1 hour after ischemia for 2, 4, or 7 days. Another group was cooled for 2 days starting 48 hours after ischemia. One group remained normothermic throughout. All hypothermia treatments started 1 hour after ischemia equally reduced hippocampal CA1 injury in the cooled hemisphere compared with the normothermic side and the normothermic group. Cooling only on days 3 and 4 was not beneficial. Importantly, no treatment influenced neurogenesis (Ki67/Doublecortin (DCX) staining), synapse formation (synaptophysin), or brain-derived neurotropic factor (BDNF) immunohistochemistry. A second experiment confirmed that BDNF levels (ELISA) were equivalent in normothermic and 7-day cooled rats. Last, we measured zinc (Zn), which is important in plasticity, with X-ray fluorescence imaging in normothermic and 7-day cooled rats. Hypothermia did not alter the postischemic distribution of Zn within the hippocampus. In summary, cooling significantly mitigates injury without compromising neuroplasticity.

Subcellular biochemical investigation of purkinje neurons using synchrotron radiation fourier transform infrared spectroscopic imaging with a focal plane array detector

Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air-dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed, the initial rehydration of the dehydrated unfixed tissue can result in distortion of subcellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where postfixation of unfixed tissue sections is required, was employed in place of immersion fixation post spectroscopic analysis. This method provided more accurate histology with reduced distortions as the dehydrated tissue section is fixed in vapor rather than during rehydration in an aqueous fixation medium. With this approach, accurate correlation between infrared images and histology of the same section revealed that Purkinje neurons in the cerebellum are rich in cytosolic proteins and not depleted as once thought. In addition, we provide the first direct evidence of intracellular lactate within Purkinje neurons. This highlights the significant potential for future applications of SR-FTIR-FPA imaging to investigate cellular lactate under conditions of altered metabolic demand such as increased brain activity and hypoxia or ischemia.

In situ biospectroscopic investigation of rapid ischemic and postmortem induced biochemical alterations in the rat brain.

Rapid advances in imaging technologies have pushed novel spectroscopic modalities such as Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) at the sulfur K-edge to the forefront of direct in situ investigation of brain biochemistry. However, few studies have examined the extent to which sample preparation artifacts confound results. Previous investigations using traditional analyses, such as tissue dissection, homogenization, and biochemical assay, conducted extensive research to identify biochemical alterations that occur ex vivo during sample preparation. In particular, altered metabolism and oxidative stress may be caused by animal death. These processes were a concern for studies using biochemical assays, and protocols were developed to minimize their occurrence. In this investigation, a similar approach was taken to identify the biochemical alterations that are detectable by two in situ spectroscopic methods (FTIR, XAS) that occur as a consequence of ischemic conditions created during humane animal killing. FTIR and XAS are well suited to study markers of altered metabolism such as lactate and creatine (FTIR) and markers of oxidative stress such as aggregated proteins (FTIR) and altered thiol redox (XAS). The results are in accordance with previous investigations using biochemical assays and demonstrate that the time between animal death and tissue dissection results in ischemic conditions that alter brain metabolism and initiate oxidative stress. Therefore, future in situ biospectroscopic investigations utilizing FTIR and XAS must take into consideration that brain tissue dissected from a healthy animal does not truly reflect the in vivo condition, but rather reflects a state of mild ischemia. If studies require the levels of metabolites (lactate, creatine) and markers of oxidative stress (thiol redox) to be preserved as close as possible to the in vivo condition, then rapid freezing of brain tissue via decapitation into liquid nitrogen, followed by chiseling the brain out at dry ice temperatures is required.

Long-Range Chemical Sensitivity in the Sulfur K-Edge X-ray Absorption Spectra of Substituted Thiophenes

Thiophenes are the simplest aromatic sulfur-containing compounds and are stable and widespread in fossil fuels. Regulation of sulfur levels in fuels and emissions has become and continues to be ever more stringent as part of governments’ efforts to address negative environmental impacts of sulfur dioxide. In turn, more effective removal methods are continually being sought. In a chemical sense, thiophenes are somewhat obdurate and hence their removal from fossil fuels poses problems for the industrial chemist. Sulfur K-edge X-ray absorption spectroscopy provides key information on thiophenic components in fuels. Here we present a systematic study of the spectroscopic sensitivity to chemical modifications of the thiophene system. We conclude that while the utility of sulfur K-edge X-ray absorption spectra in understanding the chemical composition of sulfur-containing fossil fuels has already been demonstrated, care must be exercised in interpreting these spectra because the assumption of an invariant spectrum for thiophenic forms may not always be valid.

FTIR Imaging of Brain Tissue Reveals Crystalline Creatine Deposits Are an ex Vivo Marker of Localized Ischemia during Murine Cerebral Malaria: General Implications for Disease Neurochemistry

Phosphocreatine is a major cellular source of high energy phosphates, which is crucial to maintain cell viability under conditions of impaired metabolic states, such as decreased oxygen and energy availability (i.e., ischemia). Many methods exist for the bulk analysis of phosphocreatine and its dephosphorylated product creatine; however, no method exists to image the distribution of creatine or phosphocreatine at the cellular level. In this study, Fourier transform infrared (FTIR) spectroscopic imaging has revealed the ex vivo development of creatine microdeposits in situ in the brain region most affected by the disease, the cerebellum of cerebral malaria (CM) diseased mice; however, such deposits were also observed at significantly lower levels in the brains of control mice and mice with severe malaria. In addition, the number of deposits was observed to increase in a time-dependent manner during dehydration post tissue cutting. This challenges the hypotheses in recent reports of FTIR spectroscopic imaging where creatine microdeposits found in situ within thin sections from epileptic, Alzheimers (AD), and amlyoid lateral sclerosis (ALS) diseased brains were proposed to be disease specific markers and/or postulated to contribute to the brain pathogenesis. As such, a detailed investigation was undertaken, which has established that the creatine microdeposits exist as the highly soluble HCl salt or zwitterion and are an ex-vivo tissue processing artifact and, hence, have no effect on disease pathogenesis. They occur as a result of creatine crystallization during dehydration (i.e., air-drying) of thin sections of brain tissue. As ischemia and decreased aerobic (oxidative metabolism) are common to many brain disorders, regions of elevated creatine-to-phosphocreatine ratio are likely to promote crystal formation during tissue dehydration (due to the lower water solubility of creatine relative to phosphocreatine). The results of this study have demonstrated that although the deposits do not occur in vivo, and do not directly play any role in disease pathogenesis, increased levels of creatine deposits within air-dried tissue sections serve as a highly valuable marker for the identification of tissue regions with an altered metabolic status. In this study, the location of crystalline creatine deposits were used to identify whether an altered metabolic state exists within the molecular and granular layers of the cerebellum during CM, which complements the recent discovery of decreased oxygen availability in the brain during this disease.

Distribution of selenium in zebrafish larvae after exposure to organic and inorganic selenium forms

Selenium is an essential micronutrient for many organisms, and in vertebrates has a variety of roles associated with protection from reactive oxygen species. Over the past two decades there have been conflicting reports upon human health benefits and detriments arising from consumption of selenium dietary supplements. Thus, early studies report a decrease in the incidence of certain types of cancer, whereas subsequent studies did not observe any anti-cancer effect, and adverse effects such as increased risks for type 2 diabetes have been reported. A possible contributing factor may be that different chemical forms of selenium were used in different studies. Using larval stage zebrafish (Danio rerio) as a model organism, we report a comparison of the toxicities and tissue selenium distributions of four different chemical forms of selenium. We find that the organic forms of selenium tested (Se-methyl-l-selenocysteine and l-selenomethionine) show considerably more toxicity than inorganic forms (selenite and selenate), and that this appears to be correlated with the level of bioaccumulation. Despite differences in concentrations, the tissue specific pattern of selenium accumulation was similar for the chemical forms tested; selenium was found to be highly concentrated in pigment (melanin) containing tissues especially for the organic selenium treatments, with lower concentrations in eye lens, yolk sac and heart. These results suggest that pigmented tissues might serve as a storage reservoir for selenium.

A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage

An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 μg cm(-2)), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 μg cm(-2)) within 1 day of ICH, relative to sham animals (0.22 μg cm(-2)). Levels of aggregated protein were significantly increased within both the hematoma (integrated band area 0.10 AU) and peri-hematoma zone (integrated band area 0.10 AU) relative to sham animals (integrated band area 0.056 AU), but no significant difference in aggregated protein content was observed between the hematoma and peri-hematoma zone. This result suggests that the chemical form of Fe and its ability to generate free radicals is likely to be a more critical predictor of tissue damage than the total Fe content of the tissue. Furthermore, this article describes a novel approach to colocalize nonheme Fe and aggregated protein in the peri-hematoma zone following ICH, a significant methodological advancement for the field.