X-ray Spectromicroscopy Research Articles

Impact of Autophagy and Aging on Iron Load and Ferritin in Drosophila Brain.

Biometals such as iron, copper, potassium, and zinc are essential regulatory elements of several biological processes. The homeostasis of biometals is often affected in age-related pathologies. Notably, impaired iron metabolism has been linked to several neurodegenerative disorders. Autophagy, an intracellular degradative process dependent on the lysosomes, is involved in the regulation of ferritin and iron levels. Impaired autophagy has been associated with normal pathological aging, and neurodegeneration. Non-mammalian model organisms such as Drosophila have proven to be appropriate for the investigation of age-related pathologies. Here, we show that ferritin is expressed in adult Drosophila brain and that iron and holoferritin accumulate with aging. At whole-brain level we found no direct relationship between the accumulation of holoferritin and a deficit in autophagy in aged Drosophila brain. However, synchrotron X-ray spectromicroscopy revealed an additional spectral feature in the iron-richest region of autophagy-deficient fly brains, consistent with iron–sulfur. This potentially arises from iron–sulfur clusters associated with altered mitochondrial iron homeostasis.

Root-driven weathering impacts on mineral-organic associations in deep soils over pedogenic time scales

Abstract Plant roots are critical weathering agents in deep soils, yet the impact of resulting mineral transformations on the vast deep soil carbon (C) reservoir are largely unknown. Root-driven weathering of primary minerals may cause the formation of reactive secondary minerals, which protect mineral-organic associations (MOAs) for centuries or millennia. Conversely, root-driven weathering may also transform secondary minerals, potentially enhancing the bioavailability of C previously protected in MOAs. Here we examined the impact of root-driven weathering on MOAs and their capacity to store C over pedogenic time scales. To accomplish this, we examined deep horizons (100–160 cm) that experienced root-driven weathering in four soils of increasing ages (65–226 kyr) of the Santa Cruz marine terrace chronosequence. Specifically, we compared discrete rhizosphere zones subject to root-driven weathering, with adjacent zones that experienced no root growth. Using a combination of radiocarbon, mass spectrometry, 57Fe Mossbauer spectroscopy, high-resolution mass spectrometry, and X-ray spectromicroscopy approaches, we characterized transformations of MOAs in relation to changes in C content, Δ14C values, and chemistry across the chronosequence. We found that the onset of root-driven weathering (65–90 kyr) increased the amount of C associated with poorly crystalline iron (Fe) and aluminum (Al) phases, particularly highly disordered nano-particulate goethite (np-goethite). This increase coincided with greater C concentrations, lower Δ14C values, and greater abundance of what is likely microbially-derived C. Continued root-driven weathering (137–226 kyr) did not significantly change the amount of C associated with crystalline Fe and Al phases, but resulted in a decline in the amount of C associated with poorly crystalline Fe and Al phases. This decline coincided with a decrease in C concentrations, an increase in Δ14C values, and a shift toward plant-derived C. In contrast, soil not affected by root-driven weathering showed comparatively low amounts of C bound to poorly crystalline Fe and Al phases regardless of soil age and, correspondingly, lower C concentrations. Our results demonstrate that root-driven formation and disruption of MOAs are direct controls on both C accrual and loss in deep soil. This finding suggests that root impacts on soil C storage are dependent on soil weathering stage, a consideration that is critical for future predictions of the vulnerability of deep soil C to global change.

Lysine functionalized nanodiamonds as gene carriers - Investigation of internalization pathways and intracellular trafficking

Two major determinants for effective gene delivery are the capability of the carrier to protect genetic material during extra and intracellular trafficking, and the ability to release it at the site of action in an intact form. The cellular uptake pathway is a controlling parameter regarding the fate of nanoparticle-genetic material complexes. This study elucidates the pathways involved in the cellular uptake of pristine nanodiamonds (pND), functionalized nanodiamonds (lys-NDs) and diamoplexes (ND/genetic material complexes, namely lysND-siRNA). Changes in membrane morphologies corresponding to a specific route of internalization were studied using transmission electron microscopy. Variations in cellular association of diamoplexes after general and targeted inhibition of endocytic pathways were quantified using flow cytometry. Electron micrographs reveal formation of unilateral membrane protrusions suggestive of macropinocytosis as one of the major uptake process for lys-NDs and lysND-siRNA diamoplexes. Formation of pits was also largely detected for lys-NDs but scarcely present for their diamoplexes, attributed to either clathrin-mediated or caveoli-mediated endocytosis. However, inhibition of caveoli-mediated endocytosis did not prevent internalization of the diamoplexes, suggesting the absence of this pathway. Due to the larger aggregate size, pNDs were mainly internalized through macropinocytosis, as indicated by unilateral pseudopods on their micrographs. The cells also showed bilateral pseudopods during internalization suggesting phagocytic uptake, possibly due to random functionalities on the surface of pNDs. Clathrin or caveoli mediated endocytosis was absent. Soft X-ray spectromicroscopy reveals strong sp3-carbon signals confirming that the internalized entities identified by electron microscopy are indeed NDs and their diamoplexes. These findings are the basis of further optimization of amino acid functionalized NDs towards increase of gene delivery efficacy through targeted internalization pathways and overcoming intracellular challenges.

Diffuse Hydrothermal Venting: A Hidden Source of Iron to the Oceans

Iron (Fe) limits or co-limits primary productivity and nitrogen fixation in large regions of the world’s oceans. Hydrothermal supply of Fe to the deep ocean is extensive; however most of this work has focused on examining high temperature, focused flow on ridge axes that create “black smoker” plumes. The contribution of other types of venting to the global ocean Fe cycle has received little attention. To thoroughly understand hydrothermal Fe sources to the ocean, different types of vent site must be compared. To examine the role of more diffuse, higher pH sources of venting, a hydrothermal plume above the Von Damm vent field (VDVF) was sampled for Total dissolvable Fe (unfiltered, TDFe) dissolved Fe (<0.2 µm, dFe) and soluble Fe (<0.02 µm, sFe). Plume particles sampled in-situ were characterised using scanning electron microscopy and soft x-ray spectromicroscopy. The VDVF vents emit visibly clear fluids with particulate Fe (TDFe-dFe) concentrations up to 196 nmol kg-1 comparable to concentrations measured in black smoker plumes on the Mid-Atlantic Ridge. Colloidal Fe (cFe) and sFe increased as a fraction of TDFe with decreasing TDFe concentration. This increase in the percentage of sFe and cFe within the plume cannot be explained by settling of particulates or mixing with background seawater. The creation of new cFe and sFe within the plume from the breakdown of pFe is required to close the Fe budget. We suggest that the proportional increase in cFe and sFe reflects the entrainment, breakdown and recycling of Fe bearing organic particulates near the vents. Fe plume profiles from the VDVF differ significantly from previous studies of “black smoker” vents where formation of new pFe in the plume decreases the amount of cFe. Formation and removal of Fe-rich colloids and particles will control the amount and physico-chemical composition of dFe supplied to the deep ocean from hydrothermal systems. This study highlights the differences in the stabilization of hydrothermal Fe from an off-axis diffuse source compared to black smokers. Off-axis diffuse venting represent a potentially significant and previously overlooked Fe source to the ocean due to the difficulties in detecting and locating such sites.

Insights into past ocean proxies from micron-scale mapping of sulfur species in carbonates

Abstract Geological reconstructions of global ocean chemistry and atmospheric oxygen concentrations over Earth history commonly rely on the abundance and stable isotopic composition (δ34S) of sulfur-bearing compounds. Carbonate-associated sulfate (CAS), sulfate bound within a calcium carbonate mineral matrix, is among the most commonly interrogated sulfur mineral phases. However, recent work has revealed variability in δ34SCAS values that cannot be explained by evolution of the marine sulfate reservoir, challenging the common interpretation that CAS is inherently a high-fidelity record of seawater sulfate. To investigate the source of this inconsistency, we used X-ray spectromicroscopy to map the micron-scale distribution of S-bearing sedimentary phases in Ordovician-aged (ca. 444 Ma) shallow marine carbonates from Anticosti Island, Québec, Canada. Clear differences in the abundance of CAS are observed between cements and fossils, suggesting that variance in bulk-rock data could be a consequence of component mixing and that coupled synchrotron-petrographic screening can identify the carbonate components that are most likely to retain primary CAS. Furthermore, we observe multiple, distinct forms of sulfate (both inorganic and organic). Differences in these forms among fossil clades could provide new insights into biomineralization mechanisms in extinct organisms.

Organic Matter Preservation and Incipient Mineralization of Microtubules in 120 Ma Basaltic Glass

Hollow tubular structures in subaqueously-emplaced basaltic glass may represent trace fossils caused by microbially-mediated glass dissolution. Mineralized structures of similar morphology and spatial distribution in ancient, metamorphosed basaltic rocks have widely been interpreted as ichnofossils, possibly dating to similar to 3.5 Ga or greater. Doubts have been raised, however, regarding the biogenicity of the original hollow tubules and granules in basaltic glass. In particular, although elevated levels of biologically-important elements such as C, S, N, and P as well as organic compounds have been detected in association with these structures, a direct detection of unambiguously biogenic organic molecules has not been accomplished. In this study, we describe the direct detection of proteins associated with tubular textures in basaltic glass using synchrotron X-ray spectromicroscopy. Protein-rich organic matter is shown to be associated with the margins of hollow and partly-mineralized tubules. Furthermore, a variety of tubule-infilling secondary minerals, including Ti-rich oxide phases, were observed filling and preserving the microtextures, demonstrating a mechanism whereby cellular materials may be preserved through geologic time.

Qualifying X-ray and Stimulated Raman Spectromicroscopy for Mapping Cutaneous Drug Penetration.

Research on topical drug delivery relies on reconstructed human skin (RHS) in addition to ex vivo human and animal skin, each with specific physiological features. Here, we compared the penetration of dexamethasone from an ethanolic hydroxyethyl cellulose gel into ex vivo human skin, murine skin, and RHS. For comprehensive insights into skin morphology and penetration enhancing mechanisms, scanning transmission X-ray microscopy (STXM), liquid chromatography tandem-mass spectrometry (LC-MS/MS), and stimulated Raman spectromicroscopy (SRS) were combined. STXM offers high spatial resolution with label-free drug detection and is therefore sensitive to tissue damage. Despite differences in sample preparation and data analysis, the amounts of dexamethasone in RHS, detected and quantified by STXM and LC-MS/MS, were very similar and increased during the first 100 min of exposure. SRS revealed interactions between the gel and the stratum corneum or, more specifically, its protein and lipid structures. Similar to both types of ex vivo skin, higher protein-to-lipid ratios within the stratum corneum of RHS indicated reduced lipid amounts after 30 min of ethanol exposure. Extended ethanol exposure led to a continued reduction of lipids in the ex vivo matrixes, while protein integrity appeared to be compromised in RHS, which led to declining protein signals. In conclusion, LC-MS/MS proved the predictive capability of STXM for label-free drug detection. Combining STXM with SRS precisely dissected the penetration enhancing effects of ethanol. Further studies on topical drug delivery should consider the potential of these complementary techniques.

Electron beam damage of perfluorosulfonic acid studied by soft X-ray spectromicroscopy

Scanning transmission X-ray microscopy (STXM) was used to study chemical changes to perfluorosulfonic acid (PFSA) spun cast thin films as a function of dose imparted by exposure of a 200 kV electron beam in a Transmission Electron Microscope (TEM). The relationship between electron beam fluence and absorbed dose was calibrated using a modified version of a protocol based on the positive to negative lithography transition in PMMA [Leontowich et al, J. Synchrotron Rad. 19 (2012) 976]. STXM was used to characterize and quantify the chemical changes caused by electron irradiation of PFSA under several different conditions. The critical dose for CF2-CF2 amorphization was used to explore the effects of the sample environment on electron beam damage. Use of a silicon nitride substrate was found to increase the CF2-CF2 amorphization critical dose by ∼x2 from that for free-standing PFSA films. Freestanding PFSA and PMMA films were damaged by 200 kV electrons at ∼100 K and then the damage was measured by STXM at 300 K (RT). The lithography cross-over dose for PMMA was found to be ∼2x higher when the PMMA thin film was electron irradiated at 120 K rather than at 300 K. The critical dose for CF2-CF2 amorphization in PFSA irradiated at 120 K followed by warming and delayed measurement by STXM at 300 K was found to be ∼2x larger than at 300 K. To place these results in the context of the use of electron microscopy to study PFSA ionomer in fuel cell systems, an exposure of 300 e−/nm2 at 300 K (which corresponds to an absorbed dose of ∼20 MGy) amorphizes ∼10% of the CF2-CF2 bonds in PFSA. At this dose level, the spatial resolution for TEM imaging of PFSA is limited to 3.5 nm by radiation damage, if one is using a direct electron detector with DQE = 1. This work recommends caution about 2D and 3D morphological information of PFSA materials based on TEM studies which use fluences higher than 300 e−/nm2.

Cr(VI) reduction by an extracellular polymeric substance (EPS) produced from a strain of Pseudochrobactrum saccharolyticum.

A better understanding of the Cr(VI) reduction position and mechanisms by a Cr(VI)-reducing strain is important for the bioremediation of Cr pollution in the environment. In the present study, we were interested in figuring out the role of extracellular polymeric substances (EPS) as the main area for Cr(VI) reduction in the newly reported strain of Pseudochrobactrum saccharolyticum LY10. We investigated the subcellular distribution and reduction capability of each cellular component as the main area of Cr(VI) reduction by scanning electron microscopy and soft X-ray spectromicroscopy. The results suggested that most of Cr was presented in the supernatants as Cr(III) after reduction. In the cells, Cr was mostly distributed in the EPS and cell wall, while the EPS had the maximum Cr(VI) reduction rate (81.5%) as compared with the cell wall (30.1%). Soft X-ray spectromicroscopy analysis indicated that Cr accumulated more in the EPS. Therefore, the results suggested that the EPS were the main area for Cr(VI) reduction in the bacteria of P. saccharolyticum LY10.

Electron beam damage of epoxy resin films studied by scanning transmission X-ray spectromicroscopy

Focused ion beam coupled with scanning electron microscopy (FIB-SEM) is a popular technique for advanced electron microscopy with applications such as, high-precision site-specific lamella sample preparation for transmission electron microscopy (TEM) and slice-and-view FIB 3-dimensional tomography. Damage caused by the electron imaging component of FIB-SEM may be compounded with damage from the ions during the ion milling process. There are known strategies for mitigating damage from ions and electrons (cryo-SEM, dose-control, voltage control), but the electron damage on common embedding resins for EM has not been explored in detail beyond their resistance to shape-change. The relationship between beam parameters and damage mechanisms remains unclear. Since we are relying on the physical, chemical and thermal stability of embedded samples during ion-beam milling, it is important to distinguish electron beam damage from ion beam damage. Scanning transmission X-ray microscopy (STXM) has been used for analyzing the electron beam radiation damage on polymer films by characterizing the chemical bonding changes. In this paper, we focus on the effect of beam voltage and electron dose on electron beam damage to epoxy resin thin films. Irradiated areas on polymer thin films were characterized by near edge X-ray absorption fine structure (NEXAFS) in STXM. We found that, even when using low current and voltage, the electron beam can still cause noticeable chemical changes within the polymer film. The degree of electron beam damage depends not only on the beam energy, but also on the amount of inelastic scattering occurring within the material, as determined by the sample thickness.

Discrimination of DNA and RNA distribution in a mammalian cell by scanning transmission soft X-ray microscopy.

Soft X-ray spectromicroscopy was applied to study the distribution of DNA and RNA in a mammalian cell at the spatial resolution of 400 nm. The relative distribution of DNA and RNA was examined by the SVD (singular value decomposition) method in aXis2000 program using combined full spectra of DNA and RNA at the absorption edge regions of carbon, nitrogen and oxygen. The absorption of nucleic acid was evaluated using 1s-π* transitions in the NEXAFS spectra at the nitrogen K absorption edge and distributed to DNA and RNA according to the relative level obtained above. The present results revealed the usefulness of the SVD method to discriminate closely related molecules such as DNA and RNA.

Nanoscale Examination of Biological Tissues Using X-ray Spectromicroscopy

Nanoscale Examination of Biological Tissues Using X-ray Spectromicroscopy

Using X-ray Spectromicroscopy for Operando Characterization of Li-S Batteries

Using X-ray Spectromicroscopy for Operando Characterization of Li-S Batteries

Understanding Chemomechanical Li-ion Cathode Degradation through Multi-Scale, Multi-Modal X-ray Spectromicroscopy

Understanding Chemomechanical Li-ion Cathode Degradation through Multi-Scale, Multi-Modal X-ray Spectromicroscopy

Optimal Design of Experiment for X-Ray Spectromicroscopy by Machine Learning

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Optimizing Soft X-ray Spectromicroscopy for Fuel Cell Studies: X-ray Damage of Ionomer.

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Extraction of Physical Parameters from X-ray Spectromicroscopy Data Using Machine Learning

Extraction of Physical Parameters from X-ray Spectromicroscopy Data Using Machine Learning

Soft X-ray Spectromicroscopy at the Canadian Light Source

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PEMFC Catalyst Layers: Recent Advances in Process-Structure-Property Correlations

Proton exchange membrane (PEM) fuel cells are being developed as alternative energy sources for both residential and automotive applications. In order for this technology to become fully commercial, reduction of cost and improvements in performance and durability of PEM fuel cells’ membrane electrode assemblies (MEAs) are still required. To address the requirements for further cost reduction, structure and composition of the cathode catalyst layer (CCL) has to be optimized in order to achieve optimum performance and durability while minimizing the platinum group metals (PGM) loading. In order to rationally design CCL structures that meet the performance and durability requirements, a better understanding of structure versus performance relationships is needed. This requires the capability to fabricate different CCL structures, to characterize the spatial distribution of all the phases within the catalyst layer1-3 (carbon, Pt, ionomer, and void), to measure the physico-chemical properties (both ex-situ and in-situ), and, finally, to use these experimental data as inputs for the development of a model-based understanding of the relationship between the CCL structure and CCL performance and durability. Recently, significant progress has been made in reliable measurement and modeling of key CCL properties such as thermal conductivity, electronic conductivity, and gas diffusivity4-7. Dependence of these properties on the CCL structure and manufacturing processes provides support for rational CCL design. References J. Wu, A.P. Hitchcock, M. Lerotic, D. Shapiro, V. Berejnov, D. Susac, J. Stumper, “4D imaging of polymer electrolyte membrane fuel cell cathodes by scanning X-ray microscopy”, Microsc. Microanal., 23, 1784 (2017).V. Berejnov, M. Saha, D. Susac, J. Stumper, M. West, A.P. Hitchcock, Advances in structural characterization using soft x-ray scanning transmission microscopy (STXM): Mapping and measuring porosity in PEM-FC catalyst layers, ECS Transactions, 80, 241 (2017).L.G.A. Melo, A.P. Hitchcock, J. Jankovic, J. Stumper, D. Susac, V. Berejnov, Quantitative mapping of ionomer in catalyst layers by electron and x-ray spectromicroscopy, ECS Transactions, 80, 275 (2017).M. Ahadi, M. Tam, M.S. Saha, J. Stumper, M. Bahrami, Thermal conductivity of catalyst layer of polymer electrolyte membrane fuel cells: Part 1 – Experimental study, J. Power Sources, 354, 207 (2017).M. Ahadi, A. Putz, J. Stumper, M. Bahrami, Thermal conductivity of catalyst layer of polymer electrolyte membrane fuel cells: Part 2 – Analytical modeling, J. Power Sources, 354, 215 (2017).M. Ahadi, M. Tam, A. Putz, J. Stumper, C. McCague, M. Bahrami, Electrical conductivity of PEM fuel cell catalyst layers: Through-plane vs. in-plane, in: European Hydrogen Energy Conference 2018, Costa del Sol, Spain, 14-16th March, paper no. (124), 206 (2018).S. Salari, C. McCague, M. Tam, M.S. Saha, J. Stumper, M. Bahrami, Accurate ex-situ measurements of PEM fuel cells catalyst layer dry diffusivity, ECS Transactions, 69, 419 (2015).

Quantitative analysis of mammalian chromosome by scanning transmission soft X-ray microscopy

Soft X-ray spectromicroscopy was applied to study the quantitative distribution of DNA and protein in a mammalian chromosome at the spatial resolution of 100 nm. The quantities of DNA and protein were evaluated using 1s-π* transition in the NEXAFS spectra at the nitrogen K absorption edge. DNA was not uniformly distributed in the chromosome and DNA/protein ratio was less than 0.497. The present analysis revealed the clues to identify other molecules that contribute to the absorption spectrum of the sample. The results suggested that accumulation of the absorption spectra of relevant molecules would support the refinement of the analysis.