Role of the Hyal-Cu (II) complex on bovine aortic and lymphatic endothelial cells behavior on microstructured surfaces

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.     

Label free biochemical 2D and 3D imaging using secondary ion mass spectrometry

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.     

Evaluating the challenges associated with time-of-fight secondary ion mass spectrometry for metabolomics using pure and mixed metabolites

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.     

Small molecule analysis and imaging of fatty acids in the zebra finch song system using time-of-flight-secondary ion mass spectrometry

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.     

Polymer microarrays for high throughput discovery of biomaterials

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).     

A scanning near-field optical microscope approach to biomolecule patterning

In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.     

Time-of-flight secondary ion mass spectrometry: characterisation of stainless steel surfaces immersed in natural seawater.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been employed to study the biofouling of stainless steel samples immersed in seawater. The aim of these characterisations was to understand the initial mechanisms of biomolecule adsorption for relatively short immersion times (from 0 to 24 h).The results show that: (i) there were unavoidable sample "precontaminations" on the surfaces, despite precaution during their preparation and manipulation (washing, drying and storing); (ii) the major peaks detected were the substrate ones whatever the immersion time [However, some organic (nitrogen and oxygen containing) and inorganic secondary ions appeared and grew with the immersion time.]; (iii) the surface contaminations, the nonuniformity of the adsorbed material so as and bacteria have been clearly observed by high-lateral resolution molecular ToF-SIMS mapping.     

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.     

Addressing individual paramagnetic molecules through ESN-STM

We present our recent efforts in the build-up of a room temperature electron spin noise scanning tunnelling microscope (ESN-STM) designed for ultra-thin molecular films investigation. We describe here the first results obtained with this system on a commercial paramagnetic molecule deposited on Au(1 1 1) surfaces, namely 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further we briefly present our ongoing work on the preparation and characterization of a new class of samples for this instrumentation. These are based on the self-assembling on surfaces of functionalized organic radicals. We suggest here a complete procedure to assess a good candidate molecule for ESN-STM experiments through X-band CW-ESR, standard STM investigations and ToF-SIMS analysis.     

Positive detection of mycoplasma contamination by the whole-mount preparation of cell cultures for transmission electron microscopy.

Low-level mycoplasma contamination of cell cultures is difficult to recognize with presently available techniques. This report describes the adaptation of the whole-mount technique, usually used for scanning microscopy, for transmission electron microscopy. The differentiation between microvilli and the equal-sized filamentous mycoplasma is based on the differential density obtained by the use of the method described. This method allows positive identification of mycoplasma and reduces the preparation time and the time necessary for scanning the preparation.     

Atomic force microscopy imaging of dried or living bacteria.

Atomic force microscopy (AFM) was used to obtain micrographs of dried bacteria in air, and of living ones in their culture medium. Images of dried bacteria were very similar to images obtained elsewhere by the much more complicated cryoetching preparation technique for transmission electron microscopy. Living bacteria were immobilized on a poly-L-lysine film, and directly observed in their culture medium at a resolution unattainable by any other technique applicable to living material. The images were similar to those obtained in scanning electron microscopy where the specimen must be fixed, dried and coated with conductive material, and as a result, no longer viable.     

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

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.     

Surface morphology and chemistry of<Emphasis Type="Italic"> Prunus laurocerasus</Emphasis> L. leaves: a study using X-ray photoelectron spectroscopy,time-of-flight secondary-ion mass spectrometry,atomic-force microscopy and scanning-electron microscopy

The surface properties of the plant cuticle play a crucial role in plant–pathogen interactions and the retention and penetration of agriculturally important chemicals. This paper describes the use of X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), tapping-mode atomic force microscopy (TM-AFM) and scanning electron microscopy (SEM) to determine surface-specific chemical and material properties of the adaxial surface of Prunus laurocerasus L. leaves. XPS data, derived from the uppermost few nanometres (<10 nm) of the leaf surface, were consistent with the wax components and functionality known to be present within the waxes. ToF-SIMS provided molecular speciation from the outermost monolayer of the leaf surface, indicating the importance of a family of acetates with chain lengths ranging from C₂₀ to C₃₄. The presence of alkanes with C₂₉ and C₃₁ chain lengths was also confirmed. SEM and TM-AFM topography images revealed a textured granular surface, while simultaneously recorded AFM phase images revealed heterogeneous material properties at the nanoscale. The relevance of these data to plant cuticle development, allelochemistry and agrochemical delivery is discussed.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)^{7, 5, 9}, developed and hosted in the authors'' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)^{1, 4, 8}, vascular networks (India ink)^{1, 4}, and cell body distribution (Nissl)³. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain⁶, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research¹⁰; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.     

A direct observation of bacterial coverage and biofilm formation by Acidithiobacillus ferrooxidans on chalcopyrite and pyrite surfaces

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.     

Surface composition and morphology of starch,amylose, and amylopectin films

The surfaces of solution-cast films of starch, amylose, and amylopectin were examined with scanning electron microscopy (SEM), atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface topography visualized by SEM showed that amylopectin films were very smooth whereas amylose and starch films were rougher. It appears that crystallinity or phase separation in the bulk of the film affects the surface topography. AFM showed that the outmost surfaces of all films were covered with small protrusions, 15-35 nm wide and 1-4 nm high. Studies with ESCA revealed the presence of 3-8% nitrogen on the surfaces. ToF-SIMS indicated that the nitrogen originates from protein because ionic fragments from amino acids and the peptide backbone were found. Extracts from the top surface layer of the starch film showed protein bands in gel electrophoresis (SDS-PAGE) around 60 kDa, which is in the same molecular weight range as the biosynthesizing enzyme GBSS I present in starch granules. The proteins apparently phase separated during film formation and migrated to the surface, resulting in an extensive enrichment of proteins in the film surface, where about 8% of the protein is present in the top 0.01% of the film. We believe that the protrusions observed with AFM could be one or a few proteins aggregated side by side.     

A physico-chemical and biological study of novel chitosan-chloroquinoline derivative for biomedical applications

This paper describes an elegant cross-linking technique for the preparation of chitosan-chloroquinoline derivative by using a greener technique. Chitosan solution in aqueous acetic acid was treated with 2-chloroquinoline-3-carbaldehyde solution to form hydrogel; the resulting hydrogel was subjected to solvent exchange. Combining the results of FTIR and XRD confirmed that 2-chloroquinoline-3-carbaldehyde have been reacted to chitosan. The morphology of the derivative was investigated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The thermal stability of the derivative was examined by thermogravimetric analysis (TGA). The photoluminescence (PL) spectra of chitosan-chloroquinoline derivative show red-shifted emission maximum. The microbiological screening has demonstrated the antimicrobial activity of the derivative against bacteria viz. Staphylococcus aureus, Escherichia coli and Candida albicans. The obtained results showed that the chitosan-chloroquinoline derivative might be a promising candidate for novel antimicrobial agents for biomedical applications.