RESUMO
Peppers (Capsicum spp.) are well known for their ability to cause an intense burning sensation when eaten. This organoleptic response is triggered by capsaicin and its analogs, collectively called capsaicinoids. In addition to the global popularity of peppers as a spice, there is a growing interest in the use of capsaicinoids to treat a variety of human ailments, including arthritis, chronic pain, digestive problems, and cancer. The cellular localization of capsaicinoid biosynthesis and accumulation has previously been studied by fluorescence microscopy and electron microscopy, both of which require immunostaining. In this work, ToF-SIMS has been used to image the distribution of capsaicinoids in the interlocular septum and placenta of Capsicum chinense (Scotch Bonnet peppers). A unique cryo-ToF-SIMS instrument has been used to prepare and analyze the samples with minimal sample preparation. Samples were frozen in liquid propane, cryosectioned in vacuum, and analyzed without exposure to ambient pressure. ToF-SIMS imaging was performed at -110 °C using a Bi3 (+) primary ion beam. Molecular ions for capsaicin and four other capsaicinoids were identified in both the positive and negative ToF-SIMS spectra. The capsaicinoids were observed concentrated in pockets between the outer walls of the palisade cells and the cuticle of the septum, as well as in the intercellular spaces in both the placenta and interlocular septum. This is the first report of label-free direct imaging of capsaicinoids at the cellular level in Capsicum spp. These images were obtained without the need for labeling or elaborate sample preparation. The study demonstrates the usefulness of ToF-SIMS imaging for studying the distribution of important metabolites in plant tissues.
Assuntos
Capsaicina/análogos & derivados , Capsaicina/análise , Capsicum/química , Imagem Óptica/métodos , Espectrometria de Massa de Íon Secundário/métodos , HumanosRESUMO
One of the main goals of microbial ecologists is to assess the contribution of distinct bacterial groups to biogeochemical processes, e.g. carbon cycling. Until very recently, it was not possible to quantify the uptake of a given compound at single cell level. The advent of nano-scale secondary-ion mass spectrometry (nanoSIMS), and its combination with halogen in situ hybridization (HISH) opened up this possibility. Despite its power, difficulties in cell identification during analysis of environmental samples might render this approach challenging for certain applications. A pilot study, designed to quantify the incorporation of phytoplankton-derived carbon by the main clades of heterotrophic aquatic bacteria (i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes), is used to exemplify and suggest potential solutions to these technical difficulties. The results obtained indicate that the main aquatic bacterial clades quantitatively differ in the incorporation of algae-derived organic matter. From the methodological point of view, they highlight the importance of the concentration of the target cells, which needs to be sufficient to allow for a rapid mapping under the nanoSIMS. Moreover, when working with highly productive waters, organic and inorganic particles pose a serious problem for cell recognition based on HISH-SIMS. In this work several technical suggestions are presented to minimize the above mentioned difficulties, including alternatives to improve the halogen labeling of the cells and proposing the use of a combination of FISH and HISH along with a mapping system. This approach considerably enhances the reliability of cell identification and the speed of the subsequent nanoSIMS analysis in such complex samples.
Assuntos
Bactérias/metabolismo , Carbono/metabolismo , Estuários , Hibridização In Situ/métodos , Fitoplâncton/metabolismo , Espectrometria de Massa de Íon Secundário/métodosRESUMO
A skin sample from a South-Andean mummy dating back from the XI(th) century was analyzed using time-of-flight secondary ion mass spectrometry imaging using cluster primary ion beams (cluster-TOF-SIMS). For the first time on a mummy, skin dermis and epidermis could be chemically differentiated using mass spectrometry imaging. Differences in amino-acid composition between keratin and collagen, the two major proteins of skin tissue, could indeed be exploited. A surprising lipid composition of hypodermis was also revealed and seems to result from fatty acids damage by bacteria. Using cluster-TOF-SIMS imaging skills, traces of bio-mineralization could be identified at the micrometer scale, especially formation of calcium phosphate at the skin surface. Mineral deposits at the surface were characterized using both scanning electron microscopy (SEM) in combination with energy-dispersive X-ray spectroscopy and mass spectrometry imaging. The stratigraphy of such a sample was revealed for the first time using this technique. More precise molecular maps were also recorded at higher spatial resolution, below 1 µm. This was achieved using a non-bunched mode of the primary ion source, while keeping intact the mass resolution thanks to a delayed extraction of the secondary ions. Details from biological structure as can be seen on SEM images are observable on chemical maps at this sub-micrometer scale. Thus, this work illustrates the interesting possibilities of chemical imaging by cluster-TOF-SIMS concerning ancient biological tissues.
Assuntos
Derme/química , Epiderme/química , Múmias , Espectrometria de Massa de Íon Secundário/métodos , Cálcio/química , Chile , Colágeno/química , Humanos , Queratinas/química , Lipídeos/química , Microscopia , PeleRESUMO
Although previous studies have demonstrated that TOF-SIMS is a powerful method for the characterization of adsorbed proteins due to its specificity and surface sensitivity, it was unclear from earlier work whether the differences between proteins observed on uniform flat surfaces were large enough to facilitate clear image contrast between similar proteins in small areas on topographically complex samples that are more typical of biological tissues. The goal of this study was to determine whether Bi(3) (+) could provide sufficiently high sensitivity to provide clear identification of the different proteins in an image. In this study, 10 µm polystyrene microspheres were adsorbed with one of three different proteins, human serum albumin (HSA), bovine serum albumin (BSA), and hemoglobin. Spheres coated with HSA were then mixed with spheres coated with either BSA (a very similar protein) or hemoglobin (a dramatically different protein), and deposited on silicon substrates. Fluorescent labeling was used to verify the SIMS results. With maximum autocorrelation factors (MAF) processing, images showed clear contrast between both the very different proteins (HSA and hemoglobin) and the very similar proteins (HSA and BSA). Similar results were obtained with and without the fluorescent labels. MAF images were calculated using both the full spectrum and only characteristic amino acid fragments. Although better image contrast was obtained using the full spectrum, differences between the spheres were still evident when only the amino acid fragments were included in the analysis, suggesting that we are truly observing differences between the proteins themselves. These results demonstrate that TOF-SIMS, with a Bi(3) (+) primary ion, is a powerful technique for characterizing interfacial proteins not only on large uniform surfaces, but also with high spatial resolution on the topographically complex samples typical in biological analysis.
Assuntos
Adsorção , Hemoglobinas/análise , Albumina Sérica/análise , Espectrometria de Massa de Íon Secundário/métodos , Propriedades de Superfície , Animais , Bismuto/química , Bovinos , Humanos , Íons/químicaRESUMO
This paper reports a method for the analysis of secondary metabolites stored in glandular trichomes, employing negative ion 'chip-based' nanospray tandem mass spectrometry. The analyses of glandular trichomes from Lychnophora ericoides, a plant endemic to the Brazilian 'cerrado' and used in traditional medicine as an anti-inflammatory and analgesic agent, led to the identification of five flavonoids (chrysin, pinocembrin, pinostrobin, pinobanksin and 3-O-acetylpinobanksin) by direct infusion of the extracts of glandular trichomes into the nanospray ionisation source. All the flavonoids have no oxidation at ring B, which resulted in a modification of the fragmentation pathways compared with that of the oxidised 3,4-dihydroflavonoids already described in the literature. The absence of the anti-inflammatory and antioxidant di-C-glucosylflavone vicenin-2, or any other flavonoid glycosides, in the glandular trichomes was also demonstrated. The use of the 'chip-based' nanospray QqTOF apparatus is a new fast and useful tool for the identification of secondary metabolites stored in the glandular trichomes, which can be useful for chemotaxonomic studies based on metabolites from glandular trichomes.
Assuntos
Asteraceae/química , Flavonoides/análise , Espectrometria de Massa de Íon Secundário/métodos , Espectrometria de Massas em Tandem/métodos , Flavonoides/química , Procedimentos Analíticos em Microchip , Folhas de Planta/químicaRESUMO
The metabolic activities of microbial mats have likely regulated biogeochemical cycling over most of Earth's history. However, the relationship between metabolic activity and the establishment of isotopic geochemical gradients in these mats remains poorly constrained. Here we present a parallel microgeochemical and microbiological study of micron-scale sulfur cycling within hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico. Dissolved sulfide within the mats was captured on silver discs and analyzed for its abundance and delta(34)S isotopic composition using high-resolution secondary ion mass spectrometry (nanoSIMS). These results were compared to sulfide and oxygen microelectrode profiles. Two-dimensional microgeochemical mapping revealed well-defined laminations in sulfide concentration (on scales from 1 to 200 microm), trending toward increased sulfide concentrations at depth. Sulfide delta(34)S decreased from approximately +10 per thousand to -20 per thousand in the uppermost 3 mm and oscillated repeatedly between -10 per thousand and -30 per thousand down to a depth of 8 mm. These variations are attributed to spatially variable bacterial sulfate reduction within the mat. A parallel examination of the spatial distribution of known sulfate-reducing bacteria within the family Desulfobacteraceae was conducted using catalyzed reporter deposition fluorescence in situ hybridization. Significant concentrations of Desulfobacteraceae were observed in both oxic and anoxic zones of the mat and occurred in several distinct layers, in large aggregates and heterogeneously dispersed as single cells throughout. The spatial distribution of these microorganisms is consistent with the variation in sulfide concentration and isotopic composition we observed. The parallel application of the methodologies developed here can shed light on micron-scale sulfur cycling within microbially dominated sedimentary environments.
Assuntos
Sedimentos Geológicos/microbiologia , Hibridização in Situ Fluorescente/métodos , Espectrometria de Massa de Íon Secundário/métodos , Bactérias Redutoras de Enxofre/metabolismo , Enxofre/metabolismo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Ecossistema , Sedimentos Geológicos/química , México , Microeletrodos , Isótopos de Enxofre/análise , Isótopos de Enxofre/metabolismoRESUMO
A espectrometria de massa de íons secundários (SIMS) permite a detecção rápida de elementos estáveis ou radioativos, bem como o cálculo de seu percentual isotópico. Ademais, essa técnica possibilita a localização de radioisótipos, à níveis de traços, em amostras biológicas. Neste trabalho procurou-se estudar a utilização dessa metodologia na detecção de urânio natural à baixa concentração. Estudos sobre a preparação de amostras e limites de detecção foram também realizados.