RESUMO
Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.
Assuntos
Cor , Lisossomos/metabolismo , Melanossomas/fisiologia , Organelas/fisiologia , Pigmentos Biológicos/fisiologia , Pele/metabolismo , Aranhas/fisiologia , Animais , Endossomos/metabolismoRESUMO
Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP-2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP-2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP-RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP-Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP-Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP-Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.
Assuntos
Corantes Fluorescentes , Fótons , Microscopia de Fluorescência , Mitocôndrias , Imagem Óptica , Raios XRESUMO
The impressive progress in the performance of synchrotron radiation sources is nowadays driven by the so-called `ultimate storage ring' projects which promise an unprecedented improvement in brightness. Progress on the detector side has not always been at the same pace, especially as far as soft X-ray 2D detectors are concerned. While the most commonly used detectors are still based on microchannel plates or CCD technology, recent developments of CMOS (complementary metal oxide semiconductor)-type detectors will play an ever more important role as 2D detectors in the soft X-ray range. This paper describes the capabilities and performance of a camera equipped with a newly commercialized backside-illuminated scientific CMOS (sCMOS-BSI) sensor, integrated in a vacuum environment, for soft X-ray experiments at synchrotron sources. The 4â Mpixel sensor reaches a frame rate of up to 48â framesâ s-1 while matching the requirements for X-ray experiments in terms of high-intensity linearity (>98%), good spatial homogeneity (<1%), high charge capacity (up to 80â ke-), and low readout noise (down to 2â e- r.m.s.) and dark current (3â e- per second per pixel). Performance evaluations in the soft X-ray range have been carried out at the METROLOGIE beamline of the SOLEIL synchrotron. The quantum efficiency, spatial resolution (24â line-pairsâ mm-1), energy resolution (<100â eV) and radiation damage versus the X-ray dose (<600â Gy) have been measured in the energy range from 40 to 2000â eV. In order to illustrate the capabilities of this new sCMOS-BSI sensor, several experiments have been performed at the SEXTANTS and HERMES soft X-ray beamlines of the SOLEIL synchrotron: acquisition of a coherent diffraction pattern from a pinhole at 186â eV, a scattering experiment from a nanostructured Co/Cu multilayer at 767â eV and ptychographic imaging in transmission at 706â eV.
RESUMO
A new multi-platform freeware has been developed for the processing and reconstruction of scanning multi-technique X-ray imaging and tomography datasets. The software platform aims to treat different scanning imaging techniques: X-ray fluorescence, phase, absorption and dark field and any of their combinations, thus providing an easy-to-use data processing tool for the X-ray imaging user community. A dedicated data input stream copes with the input and management of large datasets (several hundred GB) collected during a typical multi-technique fast scan at the Nanoscopium beamline and even on a standard PC. To the authors' knowledge, this is the first software tool that aims at treating all of the modalities of scanning multi-technique imaging and tomography experiments.
Assuntos
Software , Processamento de Imagem Assistida por Computador , Radiografia , Síncrotrons , Tomografia Computadorizada por Raios X , Raios XRESUMO
The Nanoscopium 155â m-long beamline of Synchrotron Soleil is dedicated to scanning hard X-ray nanoprobe techniques. Nanoscopium aims to reach ≤100â nm resolution in the 5-20â keV energy range for routine user experiments. The beamline design tackles the tight stability requirements of such a scanning nanoprobe by creating an overfilled secondary source, implementing all horizontally reflecting main beamline optics, applying high mechanical stability equipment and constructing a dedicated high-stability building envelope. Multi-technique scanning imaging and tomography including X-ray fluorescence spectrometry and spectro-microscopy, absorption, differential phase and dark-field contrasts are implemented at the beamline in order to provide simultaneous information on the elemental distribution, speciation and sample morphology. This paper describes the optical concept and the first measured performance of the Nanoscopium beamline followed by the hierarchical length-scale multi-technique imaging experiments performed with dwell times down to 3â ms per pixel.
RESUMO
The release of lanthanides (Ln) into the environment has increased in recent decades due to their expanding applications in society. Studying their toxicity in aquatic ecosystems is urgent and challenging, with contradictory evidence presented in the literature. This study compared the biodistribution of La and Gd in Daphnia magna exposed to sub-chronic conditions and developed the first Toxicokinetic-Toxicodynamic (TKTD) model for these lanthanides with this model crustacean. D. magna were initially exposed for 7 days to concentrations close to the LC50 of La (2.10 mg L-1) and Gd (1.70 mg L-1). After exposure, half of the live daphnids were introduced in a clean media to allow depuration over 24 h, while the other organisms were directly prepared for synchrotron imaging measurements. Synchrotron X-ray fluorescence analysis revealed that metal distribution in the organisms was similar for both La and Gd, predominantly localized in the intestinal tract, even after the depuration process. These results indicate that ingested metal can adversely affect organisms under sub-chronic exposure conditions, highlighting the importance of using nominal concentrations as a more suitable indicator of metal bioavailability for risk assessment. The General Unified Threshold Model of Survival (GUTS) TKTD framework, in its reduced form (GUTS-RED), was developed for La and Gd using dissolved and nominal concentrations. D. magna were exposed for 7 days to concentrations from 0.5 to 5 mg L-1 of La or Gd and mortality monitored daily. The mechanistic model revealed a faster toxicokinetics for La than Gd and a higher toxicity for Gd than La in the organism. This study confirmed, despite similar chemical properties, the variation in both toxicity and toxicokinetics between these two metals.
Assuntos
Elementos da Série dos Lantanídeos , Poluentes Químicos da Água , Animais , Daphnia magna , Raios X , Daphnia , Ecossistema , Síncrotrons , Distribuição Tecidual , Imagem Óptica , Poluentes Químicos da Água/toxicidadeRESUMO
Indo-Pacific tropical island streams are home to freshwater pipefish (Microphis spp., Syngnathidae). Otoliths were used to uncover life history traits in four species, including a New Caledonian endemic. All four species present the same methodological challenge: their otoliths are small, fragile and mute for growth marks using basic observation tools. Strontium (Sr) is calcium substituent in the mineral lattice, driven by salinity conditions, and thus useful to study diadromous migrations. Synchrotron-based scanning X-ray fluorescence 2D high-resolution mapping allowed us to tackle the global and hyperfine strontium (Sr) distribution. We developed analytical imaging processes to retrieve biological information from otoliths from the data generated via synchrotron analysis. We uncovered plasticity in the life cycle: all species were amphidromous, apart from some freshwater residents from New Caledonia. Understanding life cycle modalities is crucial to categorize species distribution limits and to implement adapted conservation measures, especially when endemic species are at stake. 2D fine-scale images outlined the heterogeneity of Sr distribution: in addition to the trivial Sr incorporation driven by environmental ionic conditions, there is an unusual mosaic arrangement of Sr distribution and we hypothesize that biological control, especially growth during the early life stages, may sometimes overrule stoichiometry. This shows that it is worth studying otolith formation and element integration at imbricated scales, and our methods and results provide a strong basis for future works and prospects in otolith science.
RESUMO
Deciphering the fossil record of cyanobacteria is crucial to understand their role in the chemical and biological evolution of the early Earth. They profoundly modified the redox conditions of early ecosystems more than 2.4 Ga ago, the age of the Great Oxidation Event (GOE), and provided the ancestor of the chloroplast by endosymbiosis, leading the diversification of photosynthetic eukaryotes. Here, we analyze the morphology, ultrastructure, chemical composition, and metals distribution of Polysphaeroides filiformis from the 1040-1006 Ma Mbuji-Mayi Supergroup (DR Congo). We evidence trilaminar and bilayered ultrastructures for the sheath and the cell wall, respectively, and the preservation of Ni-tetrapyrrole moieties derived from chlorophyll in intracellular inclusions. This approach allows an unambiguous interpretation of P. filiformis as a branched and multiseriate photosynthetic cyanobacterium belonging to the family of Stigonemataceae. It also provides a possible minimum age for the emergence of multiseriate true branching nitrogen-fixing and probably heterocytous cyanobacteria.
RESUMO
A distributed fast-acquisition system for synchronized multi-technique experiments is presented, in which the collection of metadata and the asynchronous merging of large data volumes from multiple detectors are managed as part of the data collection process. This fast continuous scanning scheme, named FLYSCAN, enables measurement of microscopy data on a timescale of milliseconds per pixel. Proof-of-principle multi-technique experiments, namely scanning X-ray fluorescence spectrometry combined with absorption, differential phase contrast and dark-field imaging, have been performed on biological and geological samples.
Assuntos
Microscopia de Varredura por Sonda/métodos , Espectrometria por Raios X/métodos , Síncrotrons , Animais , Diagnóstico por Imagem , Fósseis , França , Microscopia de Varredura por Sonda/instrumentação , Mariposas/ultraestrutura , Síncrotrons/instrumentaçãoRESUMO
While our awareness of the toxicity of rare earth elements to aquatic organisms increases, our understanding of their direct interaction and accumulation remains limited. This study describes the acute toxicity of lanthanum (La) and gadolinium (Gd) in Daphnia magna neonates and discusses potential modes of action on the basis of the respective patterns of biodistribution. Ecotoxicological bioassays for acute toxicity were conducted and dissolved metal concentrations at the end of the tests were determined. The results showed a significant difference in nominal EC50 (immobility) between La (>30 mg L-1) and Gd (13.93 (10.92 to 17.38) mg L-1). Daphnids that were then exposed to a concentration close to the determined EC50 of Gd (15 mg L-1, nominal concentration) for 48 h and 72 h were studied by synchrotron micro and nano-X-ray fluorescence to evaluate the biodistribution of potentially accumulated metals. X-ray fluorescence analyses showed that La was mainly found in the intestinal track and appeared to accumulate in the hindgut. This accumulation might be explained by the ingestion of solid La precipitates formed in the media. In contrast, Gd could only be detected in a small amount, if at all, in the intestinal tract, but was present at a much higher concentration in the tissues and became more pronounced with longer exposure time. The solubility of Gd is higher in the media used, leading to higher dissolved concentrations and uptake into tissue in ionic form via common metal transporting proteins. By studying La and Gd biodistribution in D. magna after an acute exposure, the present study has demonstrated that different uptake pathways of solid and dissolved metal species may lead to different accumulation patterns and toxicity.
Assuntos
Metais Terras Raras , Poluentes Químicos da Água , Animais , Gadolínio/toxicidade , Lantânio/toxicidade , Lantânio/metabolismo , Daphnia , Distribuição Tecidual , Metais Terras Raras/toxicidade , Metais/metabolismo , Poluentes Químicos da Água/análiseRESUMO
Scientists use otoliths to trace fish life history, especially fish migrations. Otoliths incorporate signatures of individual growth and environmental use. For many species, distinct increment patterns in the otolith are difficult to discern; thus, questions remain about crucial life history information. To unravel the history of such species, we use synchrotron-based scanning X-ray fluorescence. It allows the mapping of elements on the entire otolith at a high spatial resolution. It gives access to precise fish migration history by tagging landmark signature for environmental transition and it also characterises localised growth processes at a mineral level. Freshwater pipefish, which are of conservation concern, have otoliths that are small and fragile. Growth increments are impossible to identify and count; therefore, there is a major lack of knowledge about their life history. We confirm for the first time, by mapping strontium that the two tropical pipefish species studied are diadromous (transition freshwater/marine/freshwater). Mapping of other elements uncovered the existence of different migratory routes during the marine phase. Another major breakthrough is that we can chemically count growth increments solely based on sulphur signal as it is implicated in biomineralization processes. This novel method circumvents reader bias issues and enables age estimation even for otoliths with seemingly untraceable increments. The high spatial resolution elemental mapping methods push back limits of studies on life traits or stock characterisation.
Assuntos
Membrana dos Otólitos , Síncrotrons , Animais , Raios X , Fluorescência , PeixesRESUMO
The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140â eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL. The X-ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging-plate detector and then measured with both the XPAD3S-CdTe and the XPAD3S-Si, a silicon photon-counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit-cell parameter determination and the measurement of high-quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X-ray diffraction opened up by this type of technology are discussed.
Assuntos
Cristalografia por Raios X/métodos , Muramidase/química , Animais , Calibragem , Galinhas , Fótons , Síncrotrons/instrumentaçãoRESUMO
Non-invasive multi-scale and multimodal 3D characterization of heterogeneous or hierarchically structured intact mesoscale samples is of paramount importance in tackling challenging scientific problems. Scanning hard X-ray tomography techniques providing simultaneous complementary 3D information are ideally suited to such studies. However, the implementation of a robust on-site workflow remains the bottleneck for the widespread application of these powerful multimodal tomography methods. In this paper, we describe the development and implementation of such a robust, holistic workflow, including semi-automatic data reconstruction. Due to its flexibility, our approach is especially well suited for on-the-fly tuning of the experiments to study features of interest progressively at different length scales. To demonstrate the performance of the method, we studied, across multiple length scales, the elemental abundances and morphology of two complex biological systems, Arabidopsis plant seeds and mouse renal papilla samples. The proposed approach opens the way towards routine multimodal 3D characterization of intact samples by providing relevant information from pertinent sample regions in a wide range of scientific fields such as biology, geology, and material sciences.
Assuntos
Imageamento Tridimensional , Tomografia Computadorizada por Raios X , Animais , Imageamento Tridimensional/métodos , Camundongos , Cintilografia , Tomografia Computadorizada por Raios X/métodos , Fluxo de TrabalhoRESUMO
The acquisition of photosynthesis is a fundamental step in the evolution of eukaryotes. However, few phototrophic organisms are unambiguously recognized in the Precambrian record. The in situ detection of metabolic byproducts in individual microfossils is the key for the direct identification of their metabolisms. Here, we report a new integrative methodology using synchrotron-based X-ray fluorescence and absorption. We evidence bound nickel-geoporphyrins moieties in low-grade metamorphic rocks, preserved in situ within cells of a ~1 Gyr-old multicellular eukaryote, Arctacellularia tetragonala. We identify these moieties as chlorophyll derivatives, indicating that A. tetragonala was a phototrophic eukaryote, one of the first unambiguous algae. This new approach, applicable to overmature rocks, creates a strong new proxy to understand the evolution of phototrophy and diversification of early ecosystems.
Assuntos
Clorofila/química , Clorófitas/ultraestrutura , Complexos de Coordenação/química , Fósseis , Fotossíntese/fisiologia , Evolução Biológica , Clorofila/história , Clorófitas/anatomia & histologia , Clorófitas/classificação , Clorófitas/fisiologia , República Democrática do Congo , Ecossistema , Células Eucarióticas , Sedimentos Geológicos/análise , História Antiga , Microscopia Eletrônica de Transmissão , Níquel/química , Filogenia , Células Vegetais/fisiologia , Células Vegetais/ultraestrutura , Tetrapirróis/química , Espectroscopia por Absorção de Raios XRESUMO
Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined.
Assuntos
Cristalografia por Raios X/métodos , Substâncias Macromoleculares/química , Síncrotrons/instrumentação , Muramidase/química , Pressão , Raios XRESUMO
Alzheimer's disease (AD) is the most common cause of dementia, costing about 1% of the global economy. Failures of clinical trials targeting amyloid-ß protein (Aß), a key trigger of AD, have been explained by drug inefficiency regardless of the mechanisms of amyloid neurotoxicity, which are very difficult to address by available technologies. Here, we combine two imaging modalities that stand at opposite ends of the electromagnetic spectrum, and therefore, can be used as complementary tools to assess structural and chemical information directly in a single neuron. Combining label-free super-resolution microspectroscopy for sub-cellular imaging based on novel optical photothermal infrared (O-PTIR) and synchrotron-based X-ray fluorescence (S-XRF) nano-imaging techniques, we capture elemental distribution and fibrillary forms of amyloid-ß proteins in the same neurons at an unprecedented resolution. Our results reveal that in primary AD-like neurons, iron clusters co-localize with elevated amyloid ß-sheet structures and oxidized lipids. Overall, our O-PTIR/S-XRF results motivate using high-resolution multimodal microspectroscopic approaches to understand the role of molecular structures and trace elements within a single neuronal cell.
RESUMO
Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 µM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62 % of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labeled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and for the resulting neurotoxic effects.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Urânio/metabolismo , Linhagem Celular , Neurônios Dopaminérgicos/química , Humanos , Compostos Organometálicos/metabolismo , Espectrometria por Raios X , Síncrotrons , Urânio/análiseRESUMO
XPAD3S is a single-photon-counting chip developed in collaboration by SOLEIL Synchrotron, the Institut Louis Néel and the Centre de Physique de Particules de Marseille. The circuit, designed in the 0.25 microm IBM technology, contains 9600 square pixels with 130 microm side giving a total size of 1 cm x 1.5 cm. The main features of each pixel are: single threshold adjustable from 4.5 keV up to 35 keV, 2 ms frame rate, 10(7) photons s(-1) mm(-2) maximum local count rate, and a 12-bit internal counter with overflow allowing a full 27-bit dynamic range to be reached. The XPAD3S was hybridized using the flip-chip technology with both a 500 microm silicon sensor and a 700 microm CdTe sensor with Schottky contacts. Imaging performances of both detectors were evaluated using X-rays from 6 keV up to 35 keV. The detective quantum efficiency at zero line-pairs mm(-1) for a silicon sensor follows the absorption law whereas for CdTe a strong deficit at low photon energy, produced by an inefficient entrance layer, is measured. The modulation transfer function was evaluated and it was shown that both detectors present an ideal modulation transfer function at 26 keV, limited only by the pixel size. The influence of the Cd and Te K-edges of the CdTe sensor was measured and simulated, establishing that fluorescence photons reduce the contrast transfer at the Nyquist frequency from 60% to 40% which remains acceptable. The energy resolution was evaluated at 6% with silicon using 16 keV X-rays, and 8% with CdTe using 35 keV X-rays. A 7 cm x 12 cm XPAD3 imager, built with eight silicon modules (seven circuits per module) tiled together, was successfully used for X-ray diffraction experiments. A first result recently obtained with a new 2 cm x 3 cm CdTe imager is also presented.
RESUMO
A conjugate of a Mn-based superoxide dismutase mimic with a Re-based multimodal probe 1[combining low line] was studied in a cellular model of oxidative stress. Its speciation was investigated using Re and Mn X-fluorescence. Interestingly, 1[combining low line] shows a distribution different from its unconjugated analogue but a similar concentration in mitochondria and a similar bioactivity.