Addressable graphene encapsulation of wet specimens on a chip for optical, electron, infrared and X-ray based spectromicroscopy studies

Label-free spectromicroscopy methods offer the capability to examine complex cellular phenomena. Electron and X-ray based spectromicroscopy methods, though powerful, have been hard to implement with hydrated objects due to the vacuum incompatibility of the samples and due to the parasitic signals from (or drastic attenuation by) the liquid matrix surrounding the biological object of interest. Similarly, for many techniques that operate at ambient pressure, such as Fourier transform infrared spectromicroscopy (FTIRM), the aqueous environment imposes severe limitations due to the strong absorption of liquid water in the infrared regime. Here we propose a microfabricated multi-compartmental and reusable hydrated sample platform suitable for use with several analytical techniques, which employs the conformal encapsulation of biological specimens by a few layers of atomically thin graphene. Such an electron, X-ray, and infrared transparent, molecularly impermeable and mechanically robust enclosure preserves the hydrated environment around the object for a sufficient time to allow in situ examination of hydrated bio-objects with techniques operating under both ambient and high vacuum conditions. An additional hydration source, provided by hydrogel pads lithographically patterned in the liquid state near/around the specimen and co-encapsulated, has been added to further extend the hydration lifetime. Note that the in-liquid lithographic electron beam-induced gelation procedure allows for addressable capture and immobilization of the biological cells from the solution. Scanning electron microscopy and optical fluorescence microscopy, as well as synchrotron radiation based FTIR and X-ray fluorescence microscopy, have been used to test the applicability of the platform and for its validation with yeast, A549 human carcinoma lung cells and micropatterned gels as biological object phantoms

Response of Foraminifera to Anthropogenic Nicotine Pollution of Cigarette Butts: An Experimental Approach

The most often dispersed environmental pollutants that are released both directly and indirectly into the environment that may eventually reach aquatic ecosystems and contaminate aquatic biomes are cigarette butts (CBs). Toxicants such as nicotine, dangerous metals, total particulate matter, and recognized carcinogens can be introduced and transported via CBs into aquatic ecosystems. The examination of the effects of synthetic nicotine on three different species of cultured benthic foraminifera was the focus of this study. Three foraminiferal species from three distinct biomineralization pathways were specifically examined for viability and cellular ultrastructure, including the calcareous perforate Rosalina globularis, the calcareous imperforate Quinqueloculina spp., and the agglutinated Textularia agglutinans. The survival rate, cellular stress, and decalcification were used to assess the toxicological effects of synthetic nicotine. We were able to analyze the reaction of major macromolecules and calcium carbonate to this pollutant using FTIR (Fourier Transform Infrared) spectroscopy. High Performance Liquid Chromatography (HPLC) study was performed to increase our understanding of nicotine bioavailability in the medium culture. Different acute experiments were performed at different dates, and all indicated that synthetic nicotine is acutely hazardous to all three cultured foraminiferal taxa at lethal and sublethal concentrations. Each species responded differently depending on the type of shell biomineralization. Synthetic nicotine enhances shell decalcification and affects the composition of cytoplasmic macromolecules such as lipids and proteins, according to the FTIR spectroscopy investigations. The lipid content rose at lethal concentrations, possibly due to the creation of vesicles. The proteins signal evidences general cellular dyshomeostasis. The integration among the acute toxicity assay, synchrotron, and chemical HPLC analyses provided a valuable approach for the assessment of nicotine as a biomarker of exposure to the toxicants associated with smoking and the impact of this emerging and hazardous material on calcifying marine species

Effects of Ionizing Radiation and Long-Term Storage on Hydrated vs. Dried Cell Samples of Extremophilic Microorganisms

A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the “STARLIFE” project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core’s content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage

Soft X-ray induced radiation damage in thin freeze-dried brain samples studied by FTIR microscopy

In order to push the spatial resolution limits to the nanoscale, synchrotron-based soft X-ray microscopy (XRM) experiments require higher radiation doses to be delivered to materials. Nevertheless, the associated radiation damage impacts on the integrity of delicate biological samples. Herein, the extent of soft X-ray radiation damage in popular thin freeze-dried brain tissue samples mounted onto Si3N4 membranes, as highlighted by Fourier transform infrared microscopy (FTIR), is reported. The freeze-dried tissue samples were found to be affected by general degradation of the vibrational architecture, though these effects were weaker than those observed in paraffin-embedded and hydrated systems reported in the literature. In addition, weak, reversible and specific features of the tissue–Si3N4 interaction could be identified for the first time upon routine soft X-ray exposures, further highlighting the complex interplay between the biological sample, its preparation protocol and X-ray probe

RBS, PIXE, Ion-Microbeam and SR-FTIR Analyses of Pottery Fragments from Azerbaijan

The present work is aimed at the investigation of the ceramic bulk and pigmented glazed surfaces of ancient potteries dating back to XIX century A.D. and coming from the charming archeological site located in the Medieval Agsu town (Azerbaijan), a geographic area of special interest due to the ancient commercial routes between China, Asia Minor, and Europe. For the purpose of the study, complementary investigation tools have been exploited: non-destructive or micro-destructive investigation at elemental level by ion beam analysis (IBA) techniques, by using Rutherford Backscattering Spectrometry (RBS), Proton-Induced X-ray Emission (PIXE) spectroscopy and ion-microbeam analysis, and chemical characterization at microscopic level, by means of synchrotron radiation (SR) Fourier transform infrared (FTIR) microspectroscopy. The acquired information reveals useful for the identification of the provenance, the reconstruction of the firing technology, and finally, the identification of the pigment was used as a colorant of the glaze

Unexpected silicon localization in calcium carbonate exoskeleton of cultured and fossil coccolithophores

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals

Differential protein folding and chemical changes in lung tissues exposed to asbestos or particulates

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (\u3bcXRF) and Fourier Transform InfraRed (\u3bcFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. \u3bcXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. \u3bcFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of \u3b2-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects

Effetti dell’inquinamento da plastiche sui foraminiferi bentonici

Le plastiche sono divenuti contaminanti ubiquitari negli ecosistemi marini, d’acqua dolce e terrestri che producono rilevanti impatti sulle specie che in essi vivono. Dal 1950 ad oggi sono stati accumulati nell’ambiente circa 5 miliardi di tonnellate di plastica (Geyer et al., 2017). I meccanismi di interazione tra microplastiche e biosfera nonché gli effetti biochimici delle molecole sintetiche, specialmente sugli organismi eucariotici unicellulari marini, sono scarsamente studiati. In particolare, i foraminiferi bentonici costituiscono una componente fondamentale delle comunità marine e svolgono un ruolo chiave nel funzionamento dell’ecosistema e nei cicli biogeochimici. La loro sensibilità e la rapida risposta allo stress ambientale li rendono efficienti indicatori dei cambiamenti climatici e ambientali attuali e del passato (Schönfeld et al., 2012).Per comprendere meglio l’effetto delle plastiche negli oceani e negli organismi marini, abbiamo valutato l’incorporazione di (bio)polimeri e microplastiche in foraminiferi bentonici utilizzando tecniche di spettromicroscopia ad infrarossi in trasformata di Fourier (μFTIR). In questo studio, abbiamo raccolto ed analizzato spettri ed immagini μFTIR dauna selezione di specie di foraminiferi bentonici: Rosalina globularis cresciuta in colture inquinate con la plastica e Cibicidoides lobatulus, Rosalina bradyi e Textularia bocki raccolti su un frammento di plastica trovato sepolto in un sedimento del fondale del Mar Mediterraneo. In particolare, i foraminiferi provenienti dalle colture sono stati intossicati con molecola di di-2-etilesilftalato (DEHP) allo scopo di valutarne l’incorporazione nel citoplasma. Questo studio ha permesso di documentare: (1) la presenza di microplastiche nel citoplasma e nel guscio agglutinante di T. bocki; (2) segnali di stress ossidativo e di aggregazione proteica nella componente cellulare di C. lobatulus, R. bradyi e T. bocki, ancorati alla busta di plastica; (3) l’incorporazione del DEHP nel citoplasma di R. globularis. Questo studio ha confermato il ruolo chiave svolto dai foraminiferi bentonici come proxy per la valutazione degli effetti dell’inquinamento da microplastiche sia a livello cellulare che di biomineralizzazione confermando l’ingresso delle microplastiche e DEHP nei cicli biogeochimici. Questa indagine ha inoltre dimostrato che la microscopia FTIR è uno strumento efficace per studiare, senza l’utilizzo di marcatori specifici, l’interazione su scala molecolare tra plastica, citoplasma e guscio dei foraminiferi

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): Toward a powerful label-free cell-based assay

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay