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1.
This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.  相似文献   

2.
The chemical structure of lignin, a complex, irregular polymer of phenylpropane units that occurs in plant cell walls, was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The positive ToF-SIMS spectra of lignin isolated from pine and beech wood showed prominent secondary ions possessing guaiacyl (at m/z 137 and 151) or syringyl (at m/z 167 and 181) rings, which are the basic building units of lignin polymer. This shows that ToF-SIMS is a useful tool for lignin structural analysis. The peaks at m/z 137 and 167 were assigned as the C6-C1 ion, and the peaks at m/z 151 and 181 may be double-component, the C6-C1 ion and the C6-C2 ion. We confirmed the characteristic guaiacyl ions using a synthetic lignin model compound, dehydrogenation polymer (DHP), which was formed by polymerizing of unlabeled and deuterium-labeled coniferyl alcohols. The formation mechanism of the main secondary ions was deduced by labeling specific positions of coniferyl alcohols with a stable isotope to study the relationship between chemical structure and secondary ion formation in ToF-SIMS.  相似文献   

3.

Background

A key barrier that limits the full potential of biological processes to create new, sustainable materials and fuels from plant fibre is limited enzyme accessibility to polysaccharides and lignin that characterize lignocellulose networks. Moreover, the heterogeneity of lignocellulosic substrates means that different enzyme combinations might be required for efficient transformation of different plant resources. Analytical techniques with high chemical sensitivity and spatial resolution that permit direct characterization of solid samples could help overcome these challenges by allowing direct visualization of enzyme action within plant fibre, thereby identify barriers to enzyme action.

Results

In the current study, the high spatial resolution (about 30 nm) of scanning transmission X-ray microscopy (STXM), and the detection sensitivity (ppm) of time-of-flight secondary ion mass spectrometry (ToF-SIMS), were harnessed for the first time to investigate the progression of laccase, cellulase and xylanase activities through wood samples, and to evaluate complementary action between lignin-modifying and polysaccharide-degrading enzymes. In particular, complementary insights from the STXM and ToF-SIMS analyses revealed the key role of laccase in promoting xylanase activity throughout and between plant cell walls.

Conclusions

The spatial resolution of STXM clearly revealed time-dependent progression and spatial distribution of laccase and xylanase activities, whereas ToF-SIMS analyses confirmed that laccase promoted protein penetration into fibre samples, leading to an overall increase in polysaccharide degradation. Spectromicroscopic visualizations of plant cell wall chemistry allowed simultaneous tracking of changes to lignin and polysaccharide contents, which provides new possibilities for investigating the complementary roles of lignin-modifying and carbohydrate-active enzymes.
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4.
Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.  相似文献   

5.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides a method for the detection of native and exogenous compounds in biological samples on a cellular scale. Through the development of novel ion beams the amount of molecular signal available from the sample surface has been increased. Through the introduction of polyatomic ion beams, particularly C(60), ToF-SIMS can now be used to monitor molecular signals as a function of depth as the sample is eroded thus proving the ability to generate 3D molecular images. Here we describe how this new capability has led to the development of novel instrumentation for 3D molecular imaging while also highlighting the importance of sample preparation and discuss the challenges that still need to be overcome to maximise the impact of the technique.  相似文献   

6.
Fatty acids are central to brain metabolism and signaling, but their distributions within complex brain circuits have been difficult to study. Here we applied an emerging technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS), to image specific fatty acids in a favorable model system for chemical analyses of brain circuits, the zebra finch (Taeniopygia guttata). The zebra finch, a songbird, produces complex learned vocalizations under the control of an interconnected set of discrete, dedicated brain nuclei 'song nuclei'. Using ToF-SIMS, the major song nuclei were visualized by virtue of differences in their content of essential and non-essential fatty acids. Essential fatty acids (arachidonic acid and docosahexaenoic acid) showed distinctive distributions across the song nuclei, and the 18-carbon fatty acids stearate and oleate discriminated the different core and shell subregions of the lateral magnocellular nucleus of the anterior nidopallium. Principal component analysis of the spectral data set provided further evidence of chemical distinctions between the song nuclei. By analyzing the robust nucleus of the arcopallium at three different ages during juvenile song learning, we obtain the first direct evidence of changes in lipid content that correlate with progression of song learning. The results demonstrate the value of ToF-SIMS to study lipids in a favorable model system for probing the function of lipids in brain organization, development and function.  相似文献   

7.
Fatty liver or steatosis is a frequent histopathological change. It is a precursor for steatohepatitis that may progress to cirrhosis and in some cases to hepatocellular carcinoma. In this study we addressed the in situ composition and distribution of biochemical compounds on tissue sections of steatotic liver using both synchrotron FTIR (Fourier transform infrared) and ToF-SIMS (time of flight secondary ion mass spectrometry) microspectroscopies. FTIR is a vibrational spectroscopy that allows investigating the global biochemical composition and ToF-SIMS lead to identify molecular species in particular lipids. Synchrotron FTIR microspectroscopy demonstrated that bands linked to lipid contribution such as -CH3 and -CH2 as well as esters were highly intense in steatotic vesicles. Moreover, a careful analysis of the -CH2 symmetric and anti-symmetric stretching modes revealed a slight downward shift in spectra recorded inside steatotic vesicles when compared to spectra recorded outside, suggesting a different lipid environment inside the steatotic vesicles. ToF-SIMS analysis of such steatotic vesicles disclosed a selective enrichment in cholesterol as well as in diacylglycerol (DAG) species carrying long alkyl chains. Indeed, DAG C36 species were selectively localized inside the steatotic vesicles whereas DAG C30 species were detected mostly outside. Furthermore, FTIR detected a signal corresponding to olefin (C = C, 3000-3060 cm−1) and revealed a selective localization of unsaturated lipids inside the steatotic vesicles. ToF-SIMS analysis definitely demonstrated that DAG species C30, C32, C34 and C36 carrying at least one unsaturated alkyl chain were selectively concentrated into the steatotic vesicles. On the other hand, investigations performed on the non-steatotic part of the fatty livers have revealed important changes when compared to the normal liver. Although the non-steatotic regions of fatty livers exhibited normal histological aspect, IR spectra demonstrated an increase in the lipid content and ToF-SIMS detected small lipid droplets corresponding most likely to the first steps of lipid accretion.  相似文献   

8.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is potentially well placed to contribute to metabolomic analysis while bringing the added benefit of high resolution, label free imaging. The focused ion beams used to desorb species from the sample can be focused below 1 μm allowing chemical imaging on a sub-cellular scale. In this study we test the capability of ToF-SIMS to generate mass spectrometry and MSMS spectra from a set of standard metabolites that can be compared with open access metabolite databases containing ESI-CID MSMS spectra. The influence of the chemical environment, the matrix effect, on the observed mass spectra is assessed using a mixed metabolite sample and the data discussed in terms of compound identification and quantification. Radical ions and small fragment ions seem to be less sensitive to ion suppression or enhancement and may provide a route to quantification. Understanding such parameters will be key for the successful application of the technique for in situ metabolomics with ToF-SIMS.  相似文献   

9.
In the mid-1980s, two versions of Timm's original immersion sulfide silver method were published. The authors used immersion of tissue in a sulfide solution as opposed to Timm, who used immersion of tissue blocks in hydrogen sulfide-bubbled alcohol. The autometallography staining resulting from the "sulfide only immersion" was not particularly impressive, but the significance of this return to an old approach became obvious when Wenzel and co-workers presented their approach in connection with introduction by the Palmiter group of zinc transporter 3 (ZnT3). The Wenzel/Palmiter pictures are the first high-resolution, high-quality pictures taken from tissues in which free and loosely bound zinc ions have been captured in zinc-sulfur nanocrystals by immersion. The trick was to place formalin-fixed blocks of mouse brains in a solution containing 3% glutaraldehyde and 0.1% sodium sulfide, ingredients used for transcardial perfusion in the zinc-specific NeoTimm method. That the NeoTimm technique results in silver enhancement of zinc-sulfur nanocrystals has been proved by proton-induced X-ray multielement analyses (PIXE) and in vivo chelation with diethyldithiocarbamate (DEDTC). The aims of the present study were (a) to make the immersion-based capturing of zinc ions in zinc-sulfur nanocrystals work directly on sections and slices of fixed brain tissue, (b) to work out protocols that ensure zinc specificity and optimal quality of the staining, (c) to apply "immersion autometallography" (iZnSAMG) to other tissues that contain zinc-enriched (ZEN) cells, and (d) to make the immersion approach work on unfixed fresh tissue.  相似文献   

10.
Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 μg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.  相似文献   

11.
The softwood degrading white-rot fungus, Phanerochaete carnosa, was investigated for its ability to degrade two coniferous woods: balsam fir and lodgepole pine. P. carnosa grew similarly on these wood species, and like the hardwood-degrading white-rot fungus Ceriporiopsis subvermispora, P. carnosa demonstrated selective degradation of lignin, as observed by Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Lignin degradation across cell walls of decayed pine samples was also evaluated by ToF-SIMS and was shown to be uniform. This study illustrates softwood lignin utilization by a white-rot fungus and reveals the industrial potential of the lignocellulolytic activity elicited by this fungus.  相似文献   

12.
The technique for the preparation of biological tissue sections developed for Electron Probe Microanalysis has been adapted for ToF-SIMS analysis of mouse GV stage oocytes. GV-oocyte sample preparation involves the following steps: plunge freezing, freeze drying, impregnation in an embedding medium, and section cutting. Molecule-specific images of the distribution of molecules in a single oocyte have been obtained with the described technique and ToF-SIMS analysis. The ToF-SIMS analysis data show that the efficient lateral image resolution is approximately 1 μm. Hence, ToF-SIMS enables us to study the distribution of chemical substances in relation to the morphological data obtained by scanning electron microscopy or conventional light microscopy.  相似文献   

13.
9 male volunteers took part in the experiment. They were divided in two groups. 5 volunteers (control group) have been in "dry" immersion for 7 days. 4 volunteers (stimulated group) in addition to "dry" immersion were treated with artificial support stimulation. We investigated the number of muscle fibers with the disruptions of sarcolemmal dystrophin and serum creatine kinase levels. 7-day "dry" immersion does not change the mean number of muscle fibers with dystrophin disruptions, it leads to significant decrease of serum creatine kinase levels and does not influence on the sensitivity of sarcolemma to injury. Artificial support stimulation does not influence on these parameters.  相似文献   

14.
To obtain a fundamental understanding of the population behaviour of Acidithiobacillus ferrooxidans at chalcopyrite and pyrite surfaces, the early stage attachment behaviour and biofilm formation by this bacterium on chalcopyrite (CuFeS2) and pyrite (FeS2) were studied by optical microscopy, Raman spectroscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS) and electron backscatter diffraction (EBSD). The results indicate there was no significant difference in selectivity of bacterial attachment between chalcopyrite and pyrite. However, the result of ToF-SIMS analysis suggests that the surface of the pyrite was covered more extensively by biofilm than that of the chalcopyrite, which may indicate more extracellular polymeric substances (EPS) formation by bacterial cells growing on pyrite. EBSD and optical image analysis indicated that selectivity of bacterial attachment to chalcopyrite was not significantly affected by crystal orientation. The results also suggest that the bacterial population in defective areas of chalcopyrite was significantly higher than on the polished surfaces.  相似文献   

15.
The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format (1). Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique (2). This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion (3-5). Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction (6). HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials (5,3).  相似文献   

16.
To determine the role of the support-proprioceptive factor in the functioning of the vestibular system, in particular the static torsional otolith-cervical-ocular reflex (OCOR), comparative OCOR studies with videooculography recording were performed after a 7-day "dry" horizontal immersion (16 immersion subjects) and after a prolonged (126 to 195 days) exposure to weightlessness (14 ISS cosmonauts). For the first time it was demonstrated that minimization of the support and propripceptive afferentation may results in an inversion or absence of the static torsional OCOR and the development of a positional nystagmus with an inverted reflex. A comparative OCOR data analysis of cosmonauts and immersion subjects has revealed similarity of responses. However, changes in OCOR after immersion were noted in only 60% of subjects, while after space fight, 90% of cosmonauts showed them. Post-flight changes were more frequent, marked and long-lasting.  相似文献   

17.
Three‐photon microscopy excited at the 1700‐nm window (roughly covering 1600‐1840 nm) is especially suitable for deep‐brain imaging in living animals. To match the brain refractive index, D2O has been exclusively used as the immersion medium. However, the hygroscopic property of D2O leads to a decrease of transmittance of the excitation light and as a result a decrease in three‐photon signals over time. Solutions such as replacing D2O from time to time, wrapping both the objective lens and the immersion D2O, and sealing D2O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2O for deep‐brain imaging. Excited at 1600 nm, our comparative deep‐brain imaging using both D2O and silicone oil immersion show that silicone oil immersion yields 17% higher three‐photon signal in third‐harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three‐photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long‐span three‐photon brain imaging excited at the 1700‐nm window.   相似文献   

18.
The ability of micropatterned surfaces to modulate cell behavior is combined with the well-known angiogenic property of the hyaluronan-Cu (II) complex. Hyaluronan-Cu (II) microstripes 100 and 25 mum wide on aminosilanised glass substrates were fabricated by photoimmobilization following two different methods: i.e., method I consisting in the photoimmobilization of the Hyal-Cu (II) complex; and method II based on the photoimmobilization of Hyal followed by the coordination with Cu (II). The chemistry and topography of the fabricated micropatterned samples were investigated by ATR FT-IR, atomic absorption, AFM, SEM, and ToF-SIMS. ATR FT-IR analysis demonstrated that hyaluronan conjugated with a photoreactive moiety was able to coordinate Cu (II) ions and that the photoimmobilization process was successful, as indicated by the intensity decrease of the IR band of the azidic group after the photoreaction. AFM and SEM images showed that reproducible Hyal-Cu (II) microstructures with both chemical and topographical heterogeneities have been obtained by the two preparation methods. The distribution of copper on the fabricated Hyal-Cu (II) microstructures has been investigated by ToF-SIMS. In both ToF-SIMS images and spectra, on Hyal-Cu (II) microstructures prepared by method I, the Cu peak (63 m/z) was detected only on the Hyal-Cu (II) microstripes, while on Hyal-Cu (II) microstructures prepared by method II, the Cu peak showed the same intensity both on the Hyal-Cu (II) microstripes and on the aminosilanised glass substrate, in agreement with the higher amount of Cu revealed by atomic absorption. The influence of Hyal-Cu (II) micropatterned surfaces on BAEC and LEC, in terms of migration and adhesion, has been analyzed. The results obtained indicate that Hyal-Cu (II) influences BAEC behavior inducing cell migration, while it is devoid of any effect on LEC.  相似文献   

19.
Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput "omics"-scale screening of molecular interactions by nonexpert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of nonsynthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors.  相似文献   

20.
Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.  相似文献   

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