We describe a new method for simultaneously collecting particles in exhaled air for subsequent chemical analysis and measuring their size distribution. After forced exhalation, particles were counted and collected in spots on silicon wafers with a cascade impactor. Several phospholipids were identified by time-of-flight secondary ion mass spectrometric analysis of the collected spots, suggesting that the particles originated from the lower airways. The amount of particles collected in ten exhalations was sufficient for characterizing the phospholipid composition. The feasibility of the technique in respiratory research is demonstrated by analysis of the phospholipid composition of exhaled particles from healthy controls, patients with asthma, and patients with cystic fibrosis. We believe this technology will be useful for monitoring patients with respiratory disease and has a high potential to detect new biomarkers in exhaled air.

Background: Aortic valve stenosis (AVS), which is the most common valvular heart disease, causes a progressive narrowing of the aortic valve as a consequence of thickening and calcification of the aortic valve leaflets. The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) in cardiovascular prevention have been recently demonstrated in a large randomized controlled trial. In addition, n-3 PUFA serve as the substrate for the synthesis of specialized pro-resolving mediators (SPMs), which are known by their potent beneficial anti-inflammatory, pro-resolving and tissue-modifying properties in cardiovascular disease. However, the effects of n-3 PUFA and SPMs on AVS have not yet been determined. The aim of this study was to identify the role of n-3 PUFA-derived SPMs in relation to the development of AVS. Methods: Lipidomic and transcriptomic analyses were performed in human tricuspid aortic valves. Apoe-/- mice and wire injury in C57BL/6J mice were used as models for mechanistic studies. Results: We found that n-3 PUFA incorporation into human stenotic aortic valves was higher in non-calcified regions compared with calcified regions. LC-MS-MS based lipid mediator lipidomics identified that the n-3 PUFA-derived SPM resolvin E1 (RvE1) was dysregulated in calcified regions and acted as a calcification inhibitor. Apoe-/- mice expressing the Caenorhabditis elegans Fat-1 transgene (Fat-1tgxApoe-/-), which enables the endogenous synthesis of n-3 PUFA, increased valvular n-3 PUFA content, exhibited reduced valve calcification, lower aortic valve leaflet area, increased M2 macrophage polarization and improved echocardiographic parameters. Finally, abrogation of the RvE1 receptor ChemR23 enhanced disease progression, and the beneficial effects of Fat-1tg were abolished in the absence of ChemR23. Conclusions: n-3 PUFA-derived RvE1 and its receptor ChemR23 emerge as a key axis in the inhibition of AVS progression, and may represent a novel potential therapeutic opportunity to be evaluated in patients with AVS.

A partial ichthyosaur skeleton from the Toarcian (Lower Jurassic) bituminous shales of the ‘Schistes Carton’ unit of southern Luxembourg is described and illustrated. In addition, associated remnant soft tissues are analyzed using a combination of imaging and molecular techniques. The fossil (MNHNL TV344) comprises scattered appendicular elements, together with a consecutive series of semi-articulated vertebrae surrounded by extensive soft-tissue remains. We conclude that TV344 represents a skeletally immature individual (possibly of the genus Stenopterygius) and that the soft parts primarily consist of fossilized skin, including the epidermis (with embedded melanophore pigment cells and melanosome organelles) and dermis. Ground sections of dorsal ribs display cortical microstructures reminiscent of lines of arrested growth (LAGs), providing an opportunity for a tentative age determination of the animal at the time of death (>3 years). It is further inferred that the exceptional preservation of TV344 was facilitated by seafloor dysoxia/anoxia with periodical intervals of oxygenation, which triggered phosphatization and the subsequent formation of a carbonate concretion. 

Background: Smoking, along with many respiratory diseases, has been shown to induce airway inflammation and alter the composition of the respiratory tract lining fluid (RTLF). We have previously shown that the phospholipid and protein composition of particles in exhaled air (PEx) reflects that of RTLF. In this study, we hypothesized that the composition of PEx differs between smokers and non-smokers, reflecting inflammation in the airways. Objective: It was the aim of this study to identify differences in the phospholipid composition of PEx from smokers and non-smokers. Methods: PEx from 12 smokers and 13 non-smokers was collected using a system developed in-house. PEx was analysed using time-of-flight secondary ion mass spectrometry, and the mass spectral data were evaluated using multivariate analysis. Orthogonal partial least squares (OPLS) was used to relate smoking status, lung function and pack years to the chemical composition of RTLF. The discriminating ions identified by OPLS were then used as explanatory variables in traditional regression analysis. Results: There was a clear discrimination between smokers and non-smokers according to the chemical composition, where phospholipids from smokers were protonated and sodiated to a larger extent. Poor lung function showed a strong association with higher response from all molecular phosphatidylcholine species in the samples. Furthermore, the accumulated amount of tobacco consumed was associated with variations in mass spectra, indicating a dose-response relationship. Conclusion: The chemical composition of PEx differs between smokers and non-smokers, reflecting differences in the RTLF. The results from this study may suggest that the composition of RTLF is affected by smoking and may be of importance for lung function.

The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer's disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (∼10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration.

The pathological mechanisms underlying Alzheimer's disease (AD) are still not understood. The disease pathology is characterized by the accumulation and aggregation of amyloid-β (Aβ) peptides into extracellular plaques, however the factors that promote neurotoxic Aβ aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in biological tissues. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used to study Aβ deposition in transgenic mouse brain tissue and to elucidate the plaque-associated chemical microenvironment. The imaging experiments were performed in brain sections of transgenic Alzheimer's disease mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). Multivariate image analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their chemical identity. This include cortical and hippocampal Aβ deposits, whose amyloid peptide content was further verified using immunohistochemistry and laser microdissection followed by MALDI MS analysis. Subsequent statistical analysis on spectral data of regions of interest revealed brain region-specific differences in Aβ peptide aggregation. Moreover, other plaque-associated protein species were identified including macrophage migration inhibitory factor suggesting neuroinflammatory processes and glial cell reactivity to be involved in AD pathology. The presented data further highlight the potential of IMS as a powerful approach in neuropathology. Hanrieder et al. described an imaging mass spectrometry based study on comprehensive spatial profiling of C-terminally truncated Aβ species within individual plaques in tgArcSwe mice. Here, brain region-dependent differences in Aβ truncation and other plaque-associated proteins, such as macrophage migration inhibitory factor, were observed. The data shed further light on plaque-associated molecular mechanisms implicated in Alzheimer's pathogenesis.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer’s disease (AD), in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

A cell preparation method allowing subcellular localization of cholesterol and phosphocholine with imaging ToF-SIMS

Factors determining the pH-controlled dissolution kinetics of nilotinib formulations with the pH-titrable polymer hydroxypropyl methylcellulose phthalate, obtained by carbon dioxide-mediated precipitation, were mechanistically examined in acid and neutral environment. The matrix effect, modulating the drug dissolution, was characterized with a battery of physicochemical methodologies, including ToF-SIMS for surface composition, SAXS/WAXS and modulated DSC for crystallization characterization, and simultaneous UV-imaging and Raman spectroscopy for monitoring the dissolution process in detail. The hybrid particle formulations investigated consisted of amorphous nilotinib embedded in a polymer matrix in single continuous phase, displaying extended retained amorphicity also under wet conditions. It was demonstrated by Raman and FTIR spectroscopy that the efficient drug dispersion and amorphization in the polymer matrix were mediated by hydrogen bonding between the drug and the phthalate groups on the polymer. Simultaneous Raman and UV-imaging studies of the effect of drug load on the swelling and dissolution of the polymer matrix revealed that high nilotinib load prevented matrix swelling on passage from acid to neutral pH, thereby preventing re-precipitation and re-crystallization of incorporated nilotinib. These findings provide a mechanistic foundation of formulation development of nilotinib and other protein kinase inhibitors, which are now witnessing an intense therapeutic and industrial attention due to the difficulty in formulating these compounds so that efficient oral bioavailability is reached.

The transition from terrestrial to marine environments by secondarily aquatic tetrapods necessitates a suite of adaptive changes associated with life in the sea, e.g., the scaleless skin in adult individuals of the extant leatherback turtle. A partial, yet exceptionally preserved hard-shelled (Pan-Cheloniidae) sea turtle with extensive soft-tissue remains, including epidermal scutes and a virtually complete flipper outline, was recently recovered from the Eocene Fur Formation of Denmark. Examination of the fossilized limb tissue revealed an originally soft, wrinkly skin devoid of scales, together with organic residues that contain remnant eumelanin pigment and inferred epidermal transformation products. Notably, this stem cheloniid—unlike its scaly living descendants—combined scaleless limbs with a bony carapace covered in scutes. Our findings show that the adaptive transition to neritic waters by the ancestral pan-chelonioids was more complex than hitherto appreciated, and included at least one evolutionary lineage with a mosaic of integumental features not seen in any living turtle. © 2022, The Author(s).

Konservat-Lagerstätten, such as the Toarcian (Early Jurassic) Posidonia Shale of southwestern Germany, are renowned for their spectacular fossils. Ichthyosaur skeletons recovered from this formation are frequently associated with soft tissues; however, the preserved material ranges from three-dimensional, predominantly phosphatized structures to dark films of mainly organic matter. We examined soft-tissue residues obtained from two ichthyosaur specimens using an integrated ultrastructural and geochemical approach. Our analyses revealed that the superficially-looking ‘films’ in fact comprise sections of densely aggregated melanosome (pigment) organelles sandwiched between phosphatized layers containing fibrous microstructures. We interpret this distinct layering as representing condensed and incompletely degraded integument from both sides of the animal. When compared against previously documented ichthyosaur fossils, it becomes readily apparent that a range of preservational modes exists between presumed ‘phosphatic’ and ‘carbonized’ soft-tissue remains. Some specimens show high structural fidelity (e.g. distinct integumentary layering), while others, including the fossils examined in this study, retain few original anatomical details. This diversity of soft-tissue preservational modes among Posidonia Shale ichthyosaurs offers a unique opportunity to examine different biostratinomic, taphonomic and diagenetic variables that potentially could affect the process of fossilization. It is likely that soft-tissue preservation in the Posidonia Shale was regulated by a multitude of factors, including decay efficiency and speed of phosphatic mineral nucleation; these in turn were governed by a seafloor with sustained microbial mat activity fuelled by high organic matter input and seasonally fluctuating oxygen levels. © 2023 The Authors. 

Background: Patterning medical devices at the nanoscale level enables the manipulation of cell behavior and tissue regeneration, with topographic features recognized as playing a significant role inthe osseointegration of implantable devices. Methods: In this study, we assessed the ability of titanium-coated hemisphere-like topographic nanostructures of different sizes (approximately 50, 100, and 200 nm) to influence the morphology, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Results: We found that the proliferation and osteogenicdifferentiation of hMSCs was influenced by the size of the underlying structures, suggesting that size variations in topographic features at the nanoscale level, independently of chemistry, can be exploited to control hMSC behavior in a size-dependent fashion. Conclusion: Our studies demonstrate that colloidal lithography, in combination with coating technologies, can be exploited to investigate the cell response to well defined nanoscale topography and to develop next-generation surfaces that guide tissue regeneration and promote implant integration.

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C (δ13C as light as -69% V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13Cdepleted carbonates ever reported, δ13C down to -125% V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane.

An isolated, yet virtually intact contour feather (FUM-1980) from the lower Eocene Fur Formation of Denmark was analysed using multiple imaging and molecular techniques, including field emission gun scanning electron microscopy (FEG-SEM), X-ray absorption spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Additionally, synchrotron radiation X-ray tomographic microscopy (SRXTM) was employed in order to produce a digital reconstruction of the fossil. Under FEG-SEM, the proximal, plumulaceous part of the feather revealed masses of ovoid microstructures, about 1.7 μm long and 0.5 μm wide. Microbodies in the distal, pennaceous portion were substantially smaller (averaging 0.9 × 0.2 μm), highly elongate, and more densely packed. Generally, the microbodies in both the plumulaceous and pennaceous segments were aligned along the barbs and located within shallow depressions on the exposed surfaces. Biomarkers consistent with animal eumelanins were co-localized with the microstructures, to suggest that they represent remnant eumelanosomes (i.e. eumelanin-housing cellular organelles). Additionally, ToF-SIMS analysis revealed the presence of sulfur-containing organics – potentially indicative of pheomelanins – associated with eumelanin-like compounds. However, since there was no correlation between melanosome morphology and sulfur content, we conclude these molecular structures derive from diagenetically incorporated sulfur rather than pheomelanin. Melanosomes corresponding roughly in both size and morphology with those in the proximal part of FUM-1980 are known from contour feathers of extant parrots (Psittaciformes), an avian clade that has previously been reported from the Fur Formation.

The capabilities of time-of-flight secondary ion mass spectrometry (TOF-SIMS) with regards to limits in lateral resolution for biological samples are examined using supported lipid bilayers and individual lipid vesicles, both being among the most commonly used cell membrane mimics. Using supported 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) bilayers confined to a SiO2 substrate by a chemically modified gold surface, the edge of the lipid bilayer was analyzed by imaging TOFSIMS to assess the lateral resolution. The results using 80 keV Bi32+ primary ions show that, under optimized conditions, mass spectrometry imaging of specific unlabeled lipid fragments is possible with sub-100 nm lateral resolution. Comparison of the secondary ion yields for the phosphocholine ion (m/z 184) from a POPC bilayer using C60+ or Bi3+ primary ions showed similar results, indicating an advantage of Bi3+ primary ions for high-resolution imaging of lipid membranes, due to their better demonstrated focusing capability. Moreover, using 300 nm vesicles of different lipid composition, the capability to detect and chemically identify individual submicrometer lipid vesicles at separations down to ~ 1 μm is demonstrated.

The organization of lipid membranes plays an important role in a wide range of biological processes at different length scales. Herein, the authors present a procedure based on time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize the nanometer-scale ordering of lipids in lipid membrane structures on surfaces. While ToF-SIMS is a powerful tool for label-free analysis of lipid-containing samples, its limited spatial resolution prevents in-depth knowledge of how lipid properties affect the molecular assembly of the membrane. The authors overcome this limitation by measuring the formation of lipid dimers, originating in the same nanometer-sized primary ion impact areas. The lipid dimers reflect the local lipid environment and thus allow us to characterize the membrane miscibility on the nanometer level. Using this technique, the authors show that the chemical properties of the constituting lipids are critical for the structure and organization of the membrane on both the nanometer and micrometer length scales. Our results show that even at lipid surface compositions favoring two-phase systems, lipids are still extracted from solid, gel phase, domains into the surrounding fluid supported lipid bilayer surrounding the gel phase domains. The technique offers a means to obtain detailed knowledge of the chemical composition and organization of lipid membranes with potential application in systems where labeling is not possible, such as cell-derived supported lipid bilayers.

In recent years, time-of-flight secondary ion mass spectrometry (ToF-SIMS) with cluster ion sources has opened new perspectives for the analysis of lipid biomarkers in geobiology and organic geochemistry. However, published ToF-SIMS reference spectra of relevant compounds are still sparse, and the influence of the chemical environment (matrix) on the ionisation of molecules and their fragmentation is still not well explored. This study presents ToF-SIMS spectra of eight glycerolipids as common target compounds in biomarker studies, namely ester- and ether-bound phosphatidylethanolamine, ester- and ether-bound phosphatidylcholine, ester-bound phosphatidylcholine, ester- and ether-bound diglycerides and archaeol, obtained with a Bi 3 + cluster ion source. For all of these compounds, the spectra obtained in positive and negative analytical modes showed characteristic fragments that could clearly be assigned to e.g. molecular ions, functional groups and alkyl chains. By comparison with the reference spectra, it was possible to track some of these lipids in a pre-characterised organic extract and in cryosections of microbial mats. The results highlight the potential of ToF-SIMS for the laterally resolved analysis of organic biomarkers in environmental materials. The identification of the target compounds, however, may be hampered by matrix effects (e.g. adduct formation) and often require careful consideration of all spectral features and taking advantage of the molecular imaging capability of ToF-SIMS.

The occurrence of microbialites in post-glacial coral reefs has been interpreted to reflect an ecosystem response to environmental change. The greater thickness of microbialites in reefs with a volcanic hinterland compared to thinner microbial crusts in reefs with a non-volcanic hinterland led to the suggestion that fertilization of the reefal environment by chemical weathering of volcanic rocks stimulated primary productivity and microbialite formation. Using a molecular and isotopic approach on reef-microbialites from Tahiti (Pacific Ocean), it was recently shown that sulfate-reducing bacteria favored the formation of microbial carbonates. To test if similar mechanisms induced microbialite formation in other reefs as well, the Tahitian microbialites are compared with similar microbialites from coral reefs off Vanuatu (Pacific Ocean), Belize (Caribbean Sea, Atlantic Ocean), and the Maldives (Indian Ocean) in this study. The selected study sites cover a wide range of geological settings, reflecting variable input and composition of detritus. The new lipid biomarker data and stable sulfur isotope results confirm that sulfate-reducing bacteria played an intrinsic role in the precipitation of microbial carbonate at all study sites, irrespective of the geological setting. Abundant biomarkers indicative of sulfate reducers include a variety of terminally-branched and mid chain-branched fatty acids as well as mono-O-alkyl glycerol ethers. Isotope evidence for bacterial sulfate reduction is represented by low δ 34S values of pyrite (-43 to -42‰) enclosed in the microbialites and, compared to seawater sulfate, slightly elevated δ 34S and δ 18O values of carbonate-associated sulfate (21.9 to 22.2‰ and 11.3 to 12.4‰, respectively). Microbialite formation took place in anoxic micro-environments, which presumably developed through the fertilization of the reef environment and the resultant accumulation of organic matter including bacterial extracellular polymeric substances (EPS), coral mucus, and marine snow in cavities within the coral framework. ToF-SIMS analysis reveals that the dark layers of laminated microbialites are enriched in carbohydrates, which are common constituents of EPS and coral mucus. These results support the hypothesis that bacterial degradation of EPS and coral mucus within microbial mats favored carbonate precipitation. Because reefal microbialites formed by similar processes in very different geological settings, this comparative study suggests that a volcanic hinterland is not required for microbialite growth. Yet, detrital input derived from the weathering of volcanic rocks appears to be a natural fertilizer, being conductive for the growth of microbial mats, which fosters the development of particularly abundant and thick microbial crusts.

Marine sediments of the lowermost Eocene Stolleklint Clay and Fur Formation of north-western Denmark have yielded abundant well-preserved insects. However, despite a long history of research, in-depth information pertaining to preservational modes and taphonomic pathways of these exceptional animal fossils remains scarce. In this paper, we use a combination of scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), transmission electron microscopy (TEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess the ultrastructural and molecular composition of three insect fossils: a wasp (Hymenoptera), a damselfly (Odonata) and a pair of beetle elytra (Coleoptera). Our analyses show that all specimens are preserved as organic remnants that originate from the exoskeleton, with the elytra displaying a greater level of morphological fidelity than the other fossils. TEM analysis of the elytra revealed minute features, including a multilayered epicuticle comparable to those nanostructures that generate metallic colors in modern insects. Additionally, ToF-SIMS analyses provided spectral evidence for chemical residues of the pigment eumelanin as part of the cuticular remains. To the best of our knowledge, this is the first occasion where both structural colors and chemical traces of an endogenous pigment have been documented in a single fossil specimen. Overall, our results provide novel insights into the nature of insect body fossils and additionally shed light on exceptionally preserved terrestrial insect faunas found in marine paleoenvironments. © 2022 by the authors.

Marine deposits of earliest Eocene age in northern Jutland, Denmark, are renowned for yielding diverse teleost assemblages that have proved central for enhancing our understanding of the early evolution of many extant actinopterygian clades. In this study, we investigate diminutive larval fish fossils from the Stolleklint Clay, Ølst Formation, that retain multiple soft-tissue features preserved as distinct dark-coloured stains. To examine the elemental and molecular composition of these soft parts, we employed a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS). Our analyses revealed that the preserved structures contain chemically identifiable eumelanin intimately associated with densely aggregated microbodies that are morphologically consistent with melanosome organelles. Thus, we conclude that the carbonaceous structures represent traces of originally melanized body parts, including the eyes and peritoneum. Comparable pigmentation patterns are seen in many extant teleost larvae that use semi-transparency as a means of camouflage in pelagic environments, to suggest a similar visual appearance of the Stolleklint Clay fish fossils. This in turn suggests that adaptations for concealment and UV-protection had already evolved by the beginning of the Eocene, notably during a time interval characterized by an extreme greenhouse climate, when the global fish fauna become increasingly modern in composition. © 2021 The Authors. 

Oxidation is the dominating degradation process in most polymers during long term use in nuclear power plant (NPP) applications. Under radiation, oxygen diffusion is known to be detrimental for poly-mers at room temperature and it is accelerated by increased temperature. Diffusion limited oxidation (DLO) has an effect to the heterogeneous oxidation behavior of polymers. [1]. During normal service of NPPs the temperature within the containment can be tens of degrees beyond room temperature. Also radiation levels can vary, depending on e.g. the reactor design, typically being less than 10 Gy/h during normal service [2].

The long-term performance of polymeric materials is obviously best assessed by exposure of test specimens to natural ageing conditions followed by examination of their performance characteristics in terms of changes in physical properties. The testing conditions can be either in-door or out-door envi-ronments, and the first signs of degradation are often detected by testing mechanical properties such as tensile strength and elongation-at-break [3]. Although natural ageing is the most reliable

exposure method for examining long-term performance, its use is restricted due to the extremely long exposure times required to achieve service lifetimes. In order to decrease the testing time significantly, and thereby achieve more rapid prediction of long-term behaviour of polymeric materials, artificial ac-celerated test methods involving harsher environments are used [4]. However, the accelerated ageing conditions cause at least two kinds of problems. Firstly, the chemistry of degradation can be markedly affected when the conditions of artificial ageing are too harsh. One consequence of this is a poor cor-relation with the changes that occur during natural ageing, resulting in misleading performance data. Secondly, all factors used in artificial ageing to induce chemical degradation and obtain observable physical changes must be accelerated to the same degree. However, neither the factors responsible for the ultimate degradation of a more or less complex polymeric system, nor the effects of their inter-relation are known. Consequently, it is essential that the analytical tools used to follow the ageing are capable of detecting and identifying differences between the employed accelerated tests and the natu-ral ageing already at an early stage of degradation. Moreover, sensitive tools can limit the need for high acceleration factors in artificial ageing.

During accelerated ageing DLO effects may cause heterogeneous oxidation of the polymer which usu-ally does not occur to the same extent under normal service conditions at NPP. Combined to DLO ef-fects, understanding the combined effects of ionizing radiation and temperature is essential when evaluating the lifetime of polymeric components during normal service life or in Design Based Accident (DBA) situations.

In order to study the oxygen containing degradation products on the surface compared to the bulk ma-terial a surface sensitive analytical instrument, Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), was applied on EPDM samples. The aim is ultimately to evaluate the possibility to use ToF-SIMS in oxidation profile measurements. For comparisons this study includes differential scanning DSC, and Fourier transform infrared Spectroscopy (FTIR) as well as tensile testing.

Residual melanins have been detected in multimillion-year-old animal body fossils; how-ever, confident identification and characterization of these natural pigments remain challenging due to loss of chemical signatures during diagenesis. Here, we simulate this post-burial process through artificial maturation experiments using three synthetic and one natural eumelanin exposed to mild (100◦C/100 bar) and harsh (250◦C/200 bar) environmental conditions, followed by chemical analysis employing alkaline hydrogen peroxide oxidation (AHPO) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results show that AHPO is sensitive to changes in the melanin molecular structure already during mild heat and pressure treatment (resulting, e.g., in increased C-C cross-linking), whereas harsh maturation leads to extensive loss of eumelanin-specific chemical markers. In contrast, negative-ion ToF-SIMS spectra are considerably less affected by mild maturation conditions, and eumelanin-specific features remain even after harsh treatment. Detailed analysis of ToF-SIMS spectra acquired prior to experimental treatment revealed significant differences between the investigated eumelanins. However, systematic spectral changes upon maturation reduced these dissimilarities, indicating that intense heat and pressure treatment leads to the formation of a com-mon, partially degraded, eumelanin molecular structure. Our findings elucidate the complementary nature of AHPO and ToF-SIMS during chemical characterization of eumelanin traces in fossilized organismal remains. © 2020 by the authors.

Exceptionally preserved Mesozoic feathered dinosaur fossils (including birds) are famous, but recognized from only very few localities worldwide, and are especially rare in the Southern Hemisphere. Here we report an assemblage of non-avian and avian dinosaur feathers from an Early Cretaceous polar (around 70°S) environment in what is now southeastern Australia. The recovered remains incorporate small (10–30 mm long) basal paravian-like tufted body feathers, open-vaned contour feathers, and asymmetrical bird-like wing feathers that possess high-angled barbs with possible remnants of barbicels — amongst the geologically oldest observed to date. Such morphological diversity augments scant skeletal evidence for a range of insulated non-avian theropods and birds inhabiting extreme southern high-latitude settings during the Mesozoic. Although some of these fossil feathers exhibit what may be residual patterning, most are uniformly toned and preserve rod-shaped microbodies, as well as densely-packed microbody imprints on the barbules that are structurally consistent with eumelanosomes. Geochemical analysis detected no identifiable residual biomolecules, which we suspect were lost via hydrolysis and oxidization during diagenesis and weathering. Nevertheless, an originally dark pigmentation can be reasonably inferred from these melanic traces, which like the coloured feathers of modern birds, might have facilitated crypsis, visual communication and/or thermoregulation in a cold polar habitat. 

The study of lipid transfer between lipid membranes is of great interest for the fundamental understanding of this complex and important process and, furthermore, for providing a new avenue for the in situ modification of supported lipid bilayers (SLBs). SLBs are conveniently formed by vesicle spreading onto a solid support, but this method is limited to conditions (i.e., combination of vesicle lipid composition, surface chemical properties, and buffer) such that the vesicles break spontaneously upon adsorption to the surface. Many SLB compositions are not accessible by this approach. In the present study, we give an example of how lipid transfer can be made use of to form lipid layers with striking new features, notably with respect to stability. After lipid transfer between negatively charged POPS small unilamellar vesicles and a positively charged POEPC SLB on TiO2, an SLB is obtained, which, upon exposure to SDS, leaves behind a lipid monolayer. It is shown how this monolayer can be used for creating new SLBs. The several step procedure, bilayer formation, lipid transfer, removal of a lipid monolayer and the reassembly of a bilayer, is monitored in real time by the quartz crystal microbalance with a dissipation (QCM-D) technique, and the lipid composition is analyzed for each step in postpreparation spectroscopic analyses using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Comparison of the measured signal ratios with those of the reference samples containing known fractions of D31-POPS directly shows that the relative concentration of D31-POPS is 50% in the SLB after D31-POPS exchange, significantly higher in the monolayer prepared in situ by SDS rinse, and 20-25% after reassembly of the SLB using POEPC vesicles. The results thus provide unambiguous evidence for extensive lipid transfer between the initial POEPC SLB and D31-POPS vesicles in solution. We suggest that the reassembled SLB has a significant asymmetry between the two leaflets, and we propose that the described method is promising for the in situ preparation of asymmetric SLBs.

Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze-dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze drying ex situ causes the celia to collapse and cover the plasma membrane.

Surface and Interface Analysis

Conditioning films and biofilms forming on surfaces of solid materials exposed to aqueous media play a key role in in the interaction between the geo- and biospheres. In this study, time-of-flight secondary ion mass spectrometry and scanning electron microscopy were used to investigate the time scale, mode of formation, and chemistry of conditioning films and biofilms that formed on Si substrates exposed to aquifer water in the subsurface Äspö Hard Rock Laboratory, SE-Sweden. The detection of fragment ions of amino acids, carbohydrates, and carboxylic acids revealed that different types of organic compounds had adhered to the Si surface already after 10 min of exposure to the aquifer fluids, whereas the attachment of microbial cells was first observed after 1000 min. The organic compounds first formed isolated μm-sized accumulations and subsequently started to distribute on the wafer surface more homogenously. Simultaneously further microorganisms attached to the surface and formed biofilm-like cell accumulations after 3 months of exposure to aquifer water.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface sensitive MS technique that offers a new way of studying lipid biomarkers at the microscopic level, without the need to destroy the physical integrity of the sample by extraction. We applied ToF-SIMS to a cryosection of a microbial mat and compared the results with ToF-SIMS and gas chromatography-MS (GC-MS) analysis of extracts from the same material. A wide range of lipid biomarkers was identified with ToF-SIMS in the microbial mat cryosection. Spectra and ion images revealed that individual biomarkers, including fatty acids, mono-, di- and triacylglycerols, carotenoids and chlorophyll were localized with diatom cells identified as Planothidium lanceolatum using optical microscopy. This diatom species can thus be regarded as a major lipid source within the microbial mat system. The results underpin the idea that ToF-SIMS has the potential to become an important technique for future biomarker studies, in particular for the clear cut assignment of biomarkers to distinctive morphological structures and specific microorganisms within complex biogeochemical samples.

RATIONALE Over the last decade, the high lateral resolution and imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) have increasingly stimulated interest in studying organic molecules in complex environmental materials. However, unlike with the established mass spectrometric techniques, the use of ToF-SIMS in the biogeosciences is still hampered by a lack of reference spectra of the relevant biomarker compounds. Here we present and interpret ToF-SIMS reference spectra of ten different cyclic lipids that are frequently used as biological tracers in ecological, organic geochemical and geobiological studies. METHODS Standard compounds of α,β,β-(20R, 24S)-24-methylcholestane, (22E)-ergosta-5,7,22-trien-3β-ol, 17α(H),21β-(H)-30-norhopane, hope-17(21)-ene, hop-22(29)-ene, 17β(H),21β(H)-bacteriohopane-32,33,34,35-tetrol, 17β(H), 21β(H)-35-aminobacteriohopane-32,33,34-triol, α-tocopherol, β,β-carotene, chlorophyll a, and cryosections of microbial mats and a fungus were analyzed using a ToF-SIMS instrument equipped with a Bi 3+ cluster ion source. RESULTS The spectra obtained from the standard compounds showed peaks in the molecular weight range (molecular ions, protonated and deprotonated molecules, adduct ions) and diagnostic fragment ion peaks in both, positive and negative ion modes. For the cyclic hydrocarbons, however, the positive ion mode spectra typically showed more and stronger characteristic peaks than the negative ion mode spectra. Using real world samples the capability of ToF-SIMS to detect and image selected compounds in complex organic matrices was tested. 17β(H),21β(H)-35- Aminobacteriohopane-32,33,34-triol, carotene and chlorophyll a were successfully identified in cryosections of microbial mats, and the distribution of ergosterol was mapped at μm resolution in a cryosection of a fungus (Tuber uncinatum). CONCLUSIONS This study further highlights the utility of ToF-SIMS for the identification and localization of lipids within environmental samples and as a technique for biomarker-related research in organic geochemistry and geobiology.

The accurate spatial distribution of various lipid species during atherogenesis has remained unexplored. Herein, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to analyze the lipid dis-tribution in human coronary artery cryosections. The images from the TOF-SIMS allowed visualization ofions derived from individual species of cholesterol esters, phospholipids, and triacylglycerols in thecontext of lesion characteristics and severity. In addition, cholesterol-containing crystal-like structureswere seen in high-resolution images of advanced lesions. The ratio of cholesterol fragment ions (m/z385:m/z 369) was found to differentiate unesterified cholesterol from cholesterol esters. This ratiochanged during atherogenesis and in different areas of the lesions, reflecting differences in theaccumulation of the two forms of cholesterol. Thus, atheromas were characterized by accumulation ofcholesterol esters with apolipoprotein B near the intima-media border, whereas in the complicatedlesions, unesterified cholesterol dominated in neovessel-containing areas enriched in glycophorin A.Interestingly, triacylglycerols were found in areas surrounding neovessels and lacking either form ofcholesterol. The lipid composition of the tunica media reflected the alterations observed in the intimallipids, yet being more subtle. The detailed molecular information obtained by TOF-SIMS revealedunanticipated differences in the type and composition of the accumulating lipids in different stages ofatherogenesis, notably the spatial segregation of cholesterol and triglycerides in the advancing lesions.

The holotype (MHM-K2) of the Eocene cheloniine Tasbacka danica is arguably one of the best preserved juvenile fossil sea turtles on record. Notwithstanding compactional flattening, the specimen is virtually intact, comprising a fully articulated skeleton exposed in dorsal view. MHM-K2 also preserves, with great fidelity, soft tissue traces visible as a sharply delineated carbon film around the bones and marginal scutes along the edge of the carapace. Here we show that the extraordinary preservation of the type of T. danica goes beyond gross morphology to include ultrastructural details and labile molecular components of the once-living animal. Haemoglobin-derived compounds, eumelanic pigments and proteinaceous materials retaining the immunological characteristics of sauropsid-specific β-keratin and tropomyosin were detected in tissues containing remnant melanosomes and decayed keratin plates. The preserved organics represent condensed remains of the cornified epidermis and, likely also, deeper anatomical features, and provide direct chemical evidence that adaptive melanism - a biological means used by extant sea turtle hatchlings to elevate metabolic and growth rates - had evolved 54 million years ago. © 2017 The Author(s).

Colour, derived primarily from melanin and/or carotenoid pigments, is integral to many aspects of behaviour in living vertebrates, including social signalling, sexual display and crypsis. Thus, identifying biochromes in extinct animals can shed light on the acquisition and evolution of these biological traits. Both eumelanin and melanin-containing cellular organelles (melanosomes) are preserved in fossils, but recognizing traces of ancient melanin-based coloration is fraught with interpretative ambiguity, especially when observations are based on morphological evidence alone. Assigning microbodies (or, more often reported, their ‘mouldic impressions’) as melanosome traces without adequately excluding a bacterial origin is also problematic because microbes are pervasive and intimately involved in organismal degradation. Additionally, some forms synthesize melanin. In this review, we survey both vertebrate and microbial melanization, and explore the conflicts influencing assessment of microbodies preserved in association with ancient animal soft tissues.We discuss the types of data used to interpret fossil melanosomes and evaluate whether these are sufficient for definitive diagnosis. Finally, we outline an integrated morphological and geochemical approach for detecting endogenous pigment remains and associated microstructures in multimillion-year-old fossils.

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1–3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4–7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Feathers are amongst the most complex epidermal structures known, and they have a well-documented evolutionary trajectory across non-avian dinosaurs and basal birds. Moreover, melanosome-like microbodies preserved in association with fossil plumage have been used to reconstruct original colour, behaviour and physiology. However, these putative ancient melanosomes might alternatively represent microorganismal residues, a conflicting interpretation compounded by a lack of unambiguous chemical data. We therefore used sensitive molecular imaging, supported by multiple independent analytical tests, to demonstrate that the filamentous epidermal appendages in a new specimen of the Jurassic paravian Anchiornis comprise remnant eumelanosomes and fibril-like microstructures, preserved as endogenous eumelanin and authigenic calcium phosphate. These results provide novel insights into the early evolution of feathers at the sub-cellular level, and unequivocally determine that melanosomes can be preserved in fossil feathers.