RESUMEN
The pioneering contributions of Ondrej Krivanek to the development of electron energy loss spectrometers, energy filters, and detectors for transmission and scanning transmission electron microscopes have provided researchers with indispensible tools across a wide range of disciplines in the physical sciences, ranging from condensed matter physics, to chemistry, mineralogy, materials science, and nanotechnology. In addition, the same instrumentation has extended its reach into the life sciences, and it is this aspect of Ondrej Krivanek's influential contributions that will be surveyed here, together with some personal recollections. Traditionally, electron microscopy has given a purely morphological view of the biological structures that compose cells and tissues. However, the availability of high-performance electron energy loss spectrometers and energy filters offers complementary information about the elemental and chemical composition at the subcellular scale. Such information has proven to be valuable for applications in cell and structural biology, microbiology, histology, pathology, and more generally in the biomedical sciences.
Asunto(s)
Disciplinas de las Ciencias Biológicas/instrumentación , Energía Filtrada en la Transmisión por Microscopía Electrónica/métodos , Espectroscopía de Pérdida de Energía de Electrones/métodos , Electrones , Humanos , Nanotecnología/instrumentaciónRESUMEN
BACKGROUND: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. RESULTS: The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. CONCLUSIONS: The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. GENERAL SIGNIFICANCE: Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Neoplasias de la Mama/metabolismo , Clatrina/metabolismo , Doxorrubicina/metabolismo , Portadores de Fármacos , Endocitosis , Ácido Fólico/metabolismo , Magnetismo/métodos , Nanopartículas de Magnetita , Nanomedicina/métodos , Polietilenglicoles/química , Neoplasias del Cuello Uterino/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Caveolas/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Doxorrubicina/química , Doxorrubicina/farmacología , Endosomas/metabolismo , Femenino , Ácido Fólico/química , Células HeLa , Humanos , Lisosomas/metabolismo , Células MCF-7 , Nanopartículas de Magnetita/química , Microscopía Confocal , Microscopía Electrónica de Transmisión de Rastreo , Microscopía de Fluorescencia por Excitación Multifotónica , Espectroscopía de Pérdida de Energía de Electrones , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Mosquitoes (Diptera: Culicidae) represent a key threat for millions of people worldwide, since they act as vectors for devastating parasites and pathogens. In this scenario, eco-friendly control tools against mosquito vectors are a priority. Green synthesis of silver nanoparticles (AgNP) using a cheap, aqueous leaf extract of Anisomeles indica by reduction of Ag(+) ions from silver nitrate solution has been investigated. Bio-reduced AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction analysis (XRD). The acute toxicity of A. indica leaf extract and biosynthesized AgNP was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both the A. indica leaf extract and AgNP showed dose dependent larvicidal effect against all tested mosquito species. Compared to the leaf aqueous extract, biosynthesized AgNP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 31.56, 35.21 and 38.08 µg/mL, respectively. Overall, this study firstly shed light on the mosquitocidal potential of A. indica, a potential bioresource for rapid, cheap and effective AgNP synthesis.
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Culicidae , Insectos Vectores , Insecticidas , Lamiaceae/química , Nanopartículas del Metal , Extractos Vegetales , Animales , Dengue/prevención & control , Dengue/transmisión , Encefalitis Japonesa/prevención & control , Encefalitis Japonesa/transmisión , Malaria/prevención & control , Malaria/transmisión , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Hojas de la Planta/química , Plata , Organismos Libres de Patógenos Específicos , Espectrofotometría Ultravioleta , Espectroscopía de Pérdida de Energía de Electrones , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
The valence state, hybridization and electronic band structure of charge ordered AlV2O4 are investigated by measuring the electron energy loss spectra (EELS) and performing band structure calculations using the WIEN2k code. White line ratio and O K edges of V2O5, VO2, V2O3 and AlV2O4, obtained using electron energy loss spectroscopy, are analysed specifically to probe systematically the VO6 octahedra in all of them. The systematic decrease of the L2 intensity and the O K edge intensity from V(5+) in V2O5 to AlV2O4 indicates a progressive increase in the occupancy of the hybridized states, which is corroborated by the absence of a transition from O 1s to hybridized 2t(2g). Band structure calculations on the parent charge frustrated cubic phase and the charge ordered rhombohedral phase clearly document a band gap in the charge ordered state. From the structural information obtained after convergence and the spectroscopic information from EELS, it appears that partial orbital occupancy may lead to a deviation from an integral valence state on all the vanadium in this exotic charge ordered spinel system.
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Óxido de Aluminio/química , Electrones , Espectroscopía de Pérdida de Energía de Electrones , Vanadio/químicaRESUMEN
Silicon supplementation has been shown to play an important role in skeleton development, however, the potential role that silicon plays in mediating bone formation, and an understanding of where it might localise in the resulting bone tissue remain elusive. An improved understanding of these processes could have important implications for treating pathological mineralisation. A key aspect of defining the role of silicon in bone is to characterise its distribution and coordination environment, however, there is currently almost no information available on either. We have combined a sample-preparation method that simultaneously preserved mineral, ions, and the extracellular matrix (ECM) with secondary ion mass spectroscopy (SIMS) and electron energy-loss spectroscopy (EELS) to examine the distribution and coordination environment of silicon in murine osteoblasts (OBs) in an in vitro model of bone formation. SIMS analysis showed a high level of surface contamination from polydimethysiloxane (PDMS) resulting from sample preparation. When the PDMS was removed, silicon compounds could not be detected within the nodules either by SIMS or by energy dispersive X-ray spectroscopy (EDX) analysis. In comparison, electron energy-loss spectroscopy (EELS) provided a powerful and potentially widely applicable means to define the coordination environment and localisation of silicon in mineralising tissues. We show that trace levels of silicon were only detectable from the mineral deposits located on the collagen and in the peripheral region of mineralised matrix, possibly the newly mineralised regions of the OB nodules. Taken together our results suggest that silicon plays a biological role in bone formation, however, the precise mechanism by which silicon exerts its physicochemical effects remains uncertain. Our analytical results open the door for compelling new sets of EELS experiments that can provide detailed and specific information about the role that silicates play in bone formation and disease.
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Silicatos/química , Animales , Calcio/metabolismo , Línea Celular , Ratones , Osteoblastos/citología , Silicio/metabolismo , Espectrometría de Masa de Ion Secundario , Espectrometría por Rayos X , Espectroscopía de Pérdida de Energía de ElectronesRESUMEN
Over the past decades there have been significant advances in transmission electron microscopy for biological applications, including in energy filtering and spectrum imaging, which are techniques based on the principles of electron energy loss spectroscopy. These imaging modalities allow quantitative mapping of specific chemical elements with high sensitivity and spatial resolution. This chapter describes the experimental and computational procedures for elemental mapping in two dimensions as well as a more recent extension to three dimensions, which can reveal quantitative distributions of elements in cells on a macromolecular scale.
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Biología/métodos , Elementos Químicos , Espectroscopía de Pérdida de Energía de Electrones/métodos , Animales , Caenorhabditis elegans/citología , ADN/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Imagenología Tridimensional , Microscopía Electrónica de Transmisión/instrumentación , Fósforo/metabolismo , Estadística como Asunto , Timocitos/citología , Timocitos/ultraestructuraRESUMEN
We report the discovery in Lake Cuitzeo in central Mexico of a black, carbon-rich, lacustrine layer, containing nanodiamonds, microspherules, and other unusual materials that date to the early Younger Dryas and are interpreted to result from an extraterrestrial impact. These proxies were found in a 27-m-long core as part of an interdisciplinary effort to extract a paleoclimate record back through the previous interglacial. Our attention focused early on an anomalous, 10-cm-thick, carbon-rich layer at a depth of 2.8 m that dates to 12.9 ka and coincides with a suite of anomalous coeval environmental and biotic changes independently recognized in other regional lake sequences. Collectively, these changes have produced the most distinctive boundary layer in the late Quaternary record. This layer contains a diverse, abundant assemblage of impact-related markers, including nanodiamonds, carbon spherules, and magnetic spherules with rapid melting/quenching textures, all reaching synchronous peaks immediately beneath a layer containing the largest peak of charcoal in the core. Analyses by multiple methods demonstrate the presence of three allotropes of nanodiamond: n-diamond, i-carbon, and hexagonal nanodiamond (lonsdaleite), in order of estimated relative abundance. This nanodiamond-rich layer is consistent with the Younger Dryas boundary layer found at numerous sites across North America, Greenland, and Western Europe. We have examined multiple hypotheses to account for these observations and find the evidence cannot be explained by any known terrestrial mechanism. It is, however, consistent with the Younger Dryas boundary impact hypothesis postulating a major extraterrestrial impact involving multiple airburst(s) and and/or ground impact(s) at 12.9 ka.
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Sedimentos Geológicos/química , Geología , Meteoroides , Modelos Teóricos , Carbono/análisis , Carbón Orgánico/análisis , Europa (Continente) , Groenlandia , Historia Antigua , Lagos/química , Magnetismo , México , Microscopía Electrónica de Rastreo , Nanodiamantes/análisis , América del Norte , Polen/fisiología , Hollín/análisis , Espectroscopía de Pérdida de Energía de Electrones , Temperatura , Factores de Tiempo , Difracción de Rayos XRESUMEN
Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacterium's physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.
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Hierro/metabolismo , Lactobacillus/metabolismo , Carne/microbiología , Espectrometría de Masa de Ion Secundario/métodos , Espectroscopía de Pérdida de Energía de Electrones/métodos , Catalasa/metabolismo , Hemo/metabolismo , Transferrina/metabolismoRESUMEN
Boron Neutron Capture Therapy (BNCT) is based on the ability of the stable isotope 10B to capture neutrons, which leads to a nuclear reaction producing an alpha- and a 7Li-particle, both having a high biological effectiveness and a very short range in tissue, being limited to approximately one cell diameter. This opens the possibility for a highly selective cancer therapy. BNCT strongly depends on the selective uptake of 10B in tumor cells and on its distribution inside the cells. The chemical properties of boron and the need to discriminate different isotopes make the investigation of the concentration and distribution of 10B a challenging task. The most advanced techniques to measure and image boron are described, both invasive and non-invasive. The most promising approach for further investigation will be the complementary use of the different techniques to obtain the information that is mandatory for the future of this innovative treatment modality.