Image-EELS: Simultaneous recording of multiple electron energy-loss spectra from series of electron spectroscopic images

A new approach for element microanalysis with energy-filtering transmission electron microscopy (EFTEM) is presented which was accomplished with the CEM 902 electron microscope (Zeiss, Germany). This method is called Image-EELS, because it is a synthesis of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). Series of energy-filtered images at increasing energy losses are recorded from one area with a TV camera. In a second step the intensity of selected regions in the image stack is measured with an image analysis system and plotted as a function of the energy loss. Thus many spectra from different objects can be calculated from one image series and compared with each other. The spatial resolution of EELS is considerably enhanced, the noise is decreased because many pixels from irregular objects are integrated, and the information from ESI can be analysed as a function of the energy loss.     

The use of mass spectrometry to study amyloid-β peptides

Amyloid-β peptide (Aβ) varies in size from 39 to 43 amino acids and arises from sequential β- and γ-secretase processing of the amyloid precursor protein. Whereas the non-pathological role for Aβ is yet to be established, there is no disputing that Aβ is now widely regarded as central to the development of Alzheimer's disease (AD). The so named "amyloid cascade hypothesis" states that disease progression is the result of an increased Aβ burden in affected areas of the brain. To elucidate the Aβ role in AD, many analytical approaches have been proposed as suitable tools to investigate not only the total Aβ load but also many other issues that are considered crucial for AD, such as: (i) the aggregation state in which Aβ is present; (ii) its interaction with other species or metals; (iii) its ability to induce oxidative stress; and (iv) its degradative pathways. This review provides an insight into the use of mass spectrometry (MS) in the field of Aβ investigation aimed to assess its role in AD. In particular, the different MS-based approaches applied in vitro and in vivo that can provide detailed information on the above-mentioned issues are reviewed. Moreover, the advantages offered by the MS methods over all the other techniques are highlighted, together with the recent developments and uses of combined analytical approaches to detect and characterize Aβ.     

Electron spectroscopy imaging to study ELNES at a nanoscale

Series of energy‐filtered TEM images have been acquired with very narrow energy slit using a post‐column energy filter. This allowed us to reconstruct spectra with an energy resolution estimated to 2 eV, and a spatial resolution in the order of 0.5 nm. In that way, fine structures of the N‐K edge in AlN/GaN heterostructures have been investigated and compared to EELS spectra. The fine structure in the two nitrides is very sensitive to the local environment. Very good agreement between ESI and EELS spectra was found. Moreover, this technique allowed analysis of the AlN/GaN interface at a nanoscale. The second example is an application of the technique to construct bonding maps. In this case, maps differentiating AlN nanoprecipitates with either the cubic or the hexagonal phase were created.     

Demonstration of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata with electron energy-loss spectroscopy and electron spectroscopic imaging

The subcellular distribution of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata has been analysed. Using two cytochemical methods, phosphate precipitation and a combined oxalate-pyroantimonate technique, electron energy-loss spectroscopy and electron spectroscopic imaging have been applied for elemental analysis. Both precipitation techniques revealed a high calcium content in the melanosomes of both species. Calcium was also located in the vicinity of collagen fibrils and in the plasma membrane.     

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.     

ESI in situ analyses of extrachromosomal RNP granules

Observation of unstained ultrathin sections of salivary gland cells of Chironomus thummi and C. tentans, by means of electron spectroscopic imaging (ESI), has revealed that phosphorus is distributed in two types of granular structures in the nucleoplasm of these cells. In addition to a specific type of premessenger ribonucleoprotein (RNP) particle, known as the Balbiani ring (Br) granule, ESI revealed a new type of phosphorus-rich small granular component. Examination of unstained sections by energy-filtering transmission electron microscopy (EFTEM) offers the opportunity of obtaining the signal from the specimen alone, thus avoiding the possible contributions of heavy metals present in any staining product.     

The incorporation of metal cations into polymer backbones: An important consideration in the interpretation of ESI-MS spectra

Poly(acrylic acid) (polyAA), poly(N-isopropylacrylamide) (polyNIPAAm) and poly(diisopropyl vinyl phosphonate) (polyDISP) samples were subjected to electrospray ionisation-mass spectrometry (ESI-MS) analysis employing quadrupole-ion trap as well as high resolution quadrupole time-of-flight (Q-TOF) detectors, and the resulting spectra screened for the detection of polymers in which metal cations have become incorporated into the chain backbone following deprotonation reactions during the ESI process. Adducts formed from such polymer-metal salts were detected in spectra produced from each of the samples under investigation, indicating that the formation, ionisation and detection of polymer-metal salts can be readily achieved under typical ESI-MS experimental conditions. Specifically (under conditions of MeOH containing 0.1 mM sodium acetate) polyAA is most susceptible to the incorporation of metal ions (up to 3 replacements), followed by polyNIPAAm with two replacements and polyDISP with one replacement. The results obtained from the present investigation clearly demonstrate that for synthetic polymers capable of undergoing deprotonation under ESI conditions, the potential detection of polymer-metal salt adducts must be considered during spectral data interpretation to avoid erroneous species assignments.     

Preparation of quercetin glucuronides and characterization by HPLC–DAD–ESI/MS

Quercetin is the main flavonol in the human diet, and the most commonly used in studies of biological activity. The major circulating forms of quercetin found in the human plasma after consumption of food containing distinct quercetin glycosides are glucuronides and sulfates. In this work quercetin glucuronides have been obtained from green beans (quercetin 3-glucuronide) and by enzymic synthesis (quercetin 4′-glucuronide) using a modification of the method described by Plumb et al. (Methods in polyphenol analysis, The Royal Society of Chemistry, Cambridge, pp 187, 2003) so as to improve the original low yields of that methodology. The method finally optimised got yields of 19% in the preparation of quercetin 4′-glucuronide, which allows its further isolation for their use in biological assays. In addition, quercetin 3′-glucuronide, 3-glucuronide and a diglucuronide were synthesised with lower yields. The compounds prepared have been employed to perform assays in order to obtain data for their identification by HPLC coupled to photodiode array detection and tandem mass spectrometry. It was observed that the analysis by HPLC–ESI/MS/MS could allow the identification of different quercetin glucuronides based on the presence of some minor key MS² fragments.     

Technological properties and non-enzymatic browning of white lupin protein enriched spaghetti

Spaghetti was prepared by replacing semolina with different amounts of lupin protein, in order to increase the protein content. A detailed investigation of the rheological properties of the dough and the cooking quality of pasta was performed in comparison to standard semolina spaghetti. Moreover, the effect of the addition of lupin protein on non-enzymatic browning was evaluated by measuring ε-furoylmethyllysine (furosine) and 5-hydroxymethyl-2-furancarboxaldehyde (HMF), which are considered useful indices of semolina quality and pasta processing conditions. Dried spaghetti fortified with 5% of lupin protein isolate has a colour and rheological features comparable with the semolina sample and also the behaviour during cooking results to be satisfactory. As far as the thermal damage is concerned, the furosine values of fortified spaghetti differ only marginally from standard pasta and the percentage lysine loss is quite small (ranging from 12.1% to 15.7%).