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PubMed2006

Synthesis of PNIPAM-co-MBAA copolymer nanotubes with composite control.

We report the preparation of copolymer, poly(N-isopropylacrylamide)-co-(N,N'-methylenebisacrylamide) (PNIPAM-co-MBAA) nanotubes with different composite ratios through surface-initiated atom transfer radical polymerization using a porous anodic aluminum oxide membrane as a template. It is found that the tubular wall strongly depends on the monomer concentration. The composite-induced changes in dimension and shape of nanotubes, especially the wall thickness, were studied by scanning electron microscopy, transmission electron microscopy, atom force microscopy, FT-IR, and gel permeation chromatography. The PNIPAM-co-MBAA nanotubes exhibit a high flexibility and stability.

Journal Article↗

PubMed1999

Imaging of single DNA molecule: applications to high-resolution genomic studies.

Single molecule analysis of DNA has revealed new insights into its structural and physical properties. The application of new methods for manipulating and visualizing DNA has resulted in important advances in high-resolution physical mapping of the genome and quantitative cytogenetic studies of genomic abnormalities (Lichter 1997). Studies of single molecules of DNA have employed a variety of approaches including electron microscopy, atomic force microscopy, scanning-tunneling microscopy and fluorescence microscopy. A number of new technologies have recently been developed to exploit fluorescence microscopy's full potential for genomic analysis and the fine mapping of subtle genetic alterations. In the case of the latter application, particular emphasis has been placed on developing new methods for stretching DNA for high-resolution fluorescence in-situ hybridization studies. We have recently described a process called molecular combing according to which single DNA molecules bound by their extremities to a solid surface are uniformly stretched and aligned by a receding air/water interface (Bensimon et al. 1994). In the following, we will review recent developments concerning molecular combing and discuss its current and potential applications for the high-resolution mapping of the human genome, the detection and quantification of subtle genomic imbalances and the positional cloning of disease-related genes.

Chromosome Aberrations↗

PubMed1997

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies.

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK1 cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144. 15-37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K+, Na+ and Ca2+). Structurally damaged cells revealed lower K+ and higher Na+ and Ca2+ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.

Animals↗

PubMed2005

Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel.

The molecular mechanism of cell death induced by AGS 115 and EFDAC, sesquiterpene analogs of paclitaxel, was investigated in human breast cancer MCF-7 cells. The study was carried out using laser scanning cytometry, homeostatic confocal microscopy, atomic force microscopy and electron microscopy. AGS 115 and EFDAC exhibited a microtubule-stabilizing effect as confirmed by a significant increase in alpha-tubulin aggregation. Both paclitaxel analogs also induced death in MCF-7 cells. Evaluation of biochemical and morphological features suggested that the major form of programmed cell death induced by AGS 115 and EFDAC was autophagy. This was confirmed by MAP I LC3 expression and the ultrastructural pattern revealed by electron microscopy. Surface images of cells undergoing autophagy showed that, unlike during apoptosis, the dimensions remained unchanged, but the surface of the cell was deformed. The occurrence of apoptosis was confirmed by the efflux of Smac/DIABLO from mitochondria, caspase-7 activation and DNA loss, and did not exceed 9.7%. Therefore, AGS 115 and EFDAC appear to be promising candidates for further investigation in anti-cancer therapy.

Animals↗

PubMedDate not supplied

DNA binding to fluorescent ruthenium species released from calcium phosphate/nanoporous silicon structures.

This work centers on an analysis of calf thymus DNA binding to emissive Ru complexes which diffuse from biocompatible calcium phosphate/nanoporous silicon films. These nanostructures were characterized by scanning electron microscopy, atomic force microscopy, energy dispersive X-ray analysis, and infrared vibrational spectroscopy. In terms of polynucleotide binding, three different systems were analyzed: (1) an aqueous solution of Ru(phen)(3)2+ (a control); (2) surface-adsorbed Ru(phen)(3)2+ onto undoped calcium phosphate/porous Si/Si in aqueous solution; (3) as-prepared and annealed Ru(phen)3(2+)-doped calcium phosphate/porous Si structures in water. For films with fluorescent Ru originally embedded throughout the film, biphasic diffusion character is found; such behavior is attributed to DNA binding to both surface-bound Ru(phen)(3)2+ and species which originate from deeper regions of the film.

Animals↗

PubMed2005

Nanoscale fabrication by nonconventional approaches.

An overview of three major nonconventional approaches in nanofabrication, scanning probe microscopy lithography, self-assembly, and imprint lithography, is presented. Typically, these nonconventional approaches are emerging technologies based on simple principles with potential cost-effective manufacturability, as compared to those conventional processes that are widely used and highly developed for making microelectronic circuits. Following the introduction of nonconventional technologies and their significances in nanofabrication, the details of each approach are presented. The lithographic applications of scanning probe microscopy, which involves three major variations: scanning tunneling microscopy, atomic force microscopy, and scanning near-field optical microscopy, are first evaluated with emphasis on their abilities in making structures with subnanoscale resolutions. The principles and processes for each technique are presented while their differences are also discussed. For the second approach, self-assembly, which uses a bottom-up fabrication strategy, is reported starting with an introduction of its basic principle. Self-assembly, with and without externally controlled forces for patterning nanoscale structures, is then examined. The associated principles and procedures of key assembling processes are presented. The third one, imprint lithography, is addressed with an emphasis on its recent progress and challenges. The nanolithographic abilities of different techniques developed using the general imprinting principle are examined. Finally, concluding remarks are provided to summarize the major technologies studied and to recommend the scopes for technology improvement and future research.

Crystallization↗

PubMed2005

Nanoscale characterization of carbazole-indole copolymers modified carbon fiber surfaces.

Polycarbazole, carbazole and indole containing copolymers were electrochemically coated onto carbon fiber. The resulting polymers and copolymers were characterized by scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. Characterization of the thin polymer films were performed on the polymer-coated surface of the carbon fiber. Therefore, the results obtained could elucidate the relationship between the initial feed monomer ratio, the resulting polymer/copolymer film morphology and the surface structure formed. The thickness increase (in diameter) was 0.3 and 0.9 microm, for two different composition of carbazole/indole on the carbon fiber. The carbon fibers coated with copolymer thin films were from 6.5 to 8.2 microm in diameter (from AFM measurement).

Carbazoles↗

PubMed2006

Fabrication and characterization of microstructures with optical quality surfaces in fused silica glass using femtosecond laser pulses and chemical etching.

We present a study of the sidewall surface quality inside microchannels fabricated in fused silica glass by femtosecond laser pulses and chemical etching. Multiple combinations of laser exposure and etching solution parameters were examined. Results of scanning electron microscopy, atomic force microscopy, and optical reflection analyses of the surfaces are presented. The results obtained demonstrate the feasibility of optical quality surface fabrication, which in turn demonstrates the feasibility of fabricating complex integrated devices containing microfluidic channels and optical waveguides in the glass substrates.

Journal Article↗

PubMed2005

Microbiologically influenced corrosion: looking to the future.

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).

Alloys↗

PubMed2006

[Construction of multifunctonal nano-delivery system crossing blood brain barrier].

OBJECTIVE: To construct multifunctonal nano-delivery system crossing the blood brain barrier (BBB). METHODS: The magnetic nanoparticles were preprared with O-carboxylmethylated chitosan (O-CMC) and conjugated with a peptide sequence from the human immunodeficiency virus 1-tat protein and transferrin (Tf), and anti-tumor drug methotrexate (MTX), and thus constructed a O-CMC magnetic nanoparticles carrier system conjugating with Tat and Tf (O-MNPs-Tat-Tf) that combines multiple functions including crossing BBB, magnetism, receptor-mediated dual targets and anti-tumor capabilities. The appearance, diameter, and magnetism of MTX-O-MNPs-Tat-Tf carrier system were characterized with transmission electronic microscopy, atomic force microscopy and vibrating samples magnetometer. The cytotoxicity of MTX-loaded O-MNPs-Tat-Tf was investigated with C6 glioma cells. The ability of O-MNPs-Tat-Tf crossing BBB was investigated in rats by single photon emission computed tomography. RESULTS: The mean particle diameter was 75 nm, along with good anti-tumor property. The multi-functioned carrier system successfully crossed the BBB in rat. CONCLUSION: The establishment of MTX-O-MNPs-Tat-Tf carrier model implies a promising future for its application in therapy of cerebral diseases.

Blood-Brain Barrier↗

PubMed1997

Protein architecture: assembly of ordered films by means of alternated adsorption of oppositely charged macromolecules.

Methods of making molecularly ordered protein films are reviewed with special reference to the recently developed technique of protein multilayer assembly by alternated adsorption of opposite-charge polyions. This method has been applied for linear and branched polyions, DNA, polynucleotides, proteins, viruses and clay nanoplates. This provides good prospects for biomolecular architecture. Quartz crystal microbalance, X-ray and neutron reflectivity, scanning electron microscopy, atomic force microscopy and UV-absorbance data are used to analyze the film structure. Multilayer buildup by alternation of polyions and 16 different charged proteins is discussed. In most cases, enzymes in the films retained their activity. Protein/ceramic nanoplates consisting of alternated montmorillonite clay and glucose oxidase layers electrostatically linked by polycations were also assembled. Protein layers can be arranged according to specific biological activity. Consecutive enzymic reactions were performed in anisotropic protein layers prepared with precise control of distances between the active layers (1-50 nm). Film superlattices containing ordered layers of more than one protein were constructed using myoglobin, lysozyme, peroxidase, glucoamylase, glucose oxidase and catalase. Glucoamylase, glucose oxidase/peroxidase catalyze the starch-glucose-H2020 reaction. The reaction products and nonreacting starch were separated by filtration when the substrate solution passed through the multienzyme films assembled on a filter. Formation of alternate outermost layers (of opposite charge or opposite specificity) at every adsorption cycle is the key point of the layer-by-layer assembly. Multilayers were obtained by alternated adsorption of concanavalin A and glycogen (or streptavidin and biotinylated polylysine) were designed using their biospecific interaction. Protein films are of extreme interest as novel biologically active materials.

Adsorption↗

PubMed2002

Surface micromechanics of ultrahigh molecular weight polyethylene: Microindentation testing, crosslinking, and material behavior.

The wear behavior of ultrahigh molecular weight polyethylene (UHMWPE) is critical to the success of total joint replacements. Recent attempts to modify the wear behavior of UHMWPE by processing, in particular, crosslinking UHMWPE have shown promise to increase wear resistance, but concerns persist regarding other mechanical properties. It is also unclear what specific surface mechanical properties govern the wear resistance seen in these materials. The goal of this study was to demonstrate a custom-built surface mechanical test system and method that measures the micromechanical response of microtomed UHMWPE surfaces to depth-sensing microindentation tests. The surface structure of these UHMWPE materials was also examined using scanning electron microscopy and atomic force microscopy. A custom designed microindentation test system assessed the microindentation behavior of three UHMWPE resins: 1. Hylamertrade mark, 2. GUR-1020 CMS, and 3. Marathontrade mark-a lightly crosslinked material. The effects of material and indentation depth were studied. Microindentation tests were performed with indentation depths ranging from 2 to 45 microm. Four different measurements of surface micromechanical behavior were obtained including the surface modulus, microhardness, hysteresis energy (irreversible work done to the sample per unit cycle) and its associated energy dissipation factor, and loading slope. Statistically significant differences in each of these parameters were found for each material. Generally, Hylamer had the largest values for these parameters, followed by the GUR resin and then the Marathon. Surface modulus was independent of depth of testing and found to be 651 MPa for Marathon, 738 MPa for GUR, and 1015 MPa for Hylamer (Modulus for bulk UHMWPE is 540 MPa for Hylamer, 620 for GUR, and 1380 for Hylamer). The microhardness varied between 67 and 162 MPa depending on material and depth of testing. Surface structural characterization shows that the microtoming process for surface preparation generated distinct surface features that varied between materials. Intermittent drawn ribbons of polymer with oriented crystals were observed in both scanning electron microscopy and atomic force microscopy. The surface density and size of these features were characteristic of the materials with the Hylamer having the fewest, but largest ribbons, followed by GUR and then Marathon.

Biocompatible Materials↗

PubMed1998

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM.

There is a substantial body of information indicating that 18-methyleicosanoic acid (18-MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer beta-layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid-containing outer beta-layer and the delta-layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18-MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non-covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the delta-layer, the proteinaceous epicuticle and outer beta-layers of the CCMC. The delta-layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross-sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer beta-layer was softer than the epicuticle and the delta-layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm-3) at 25 degrees C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18-MEA from the surface.

Animals↗

PubMed2006

Ultrathin film deposition by liquid CO2 free meniscus coating-uniformity and morphology.

Ultrathin organic films of sucrose octaacetate (SOA) were deposited on 12.5 cm diameter silicon wafer substrates using high-pressure free meniscus coating (hFMC) with liquid CO2 (l-CO2) as a coating solvent. The dry film thickness across the wafer and the morphology of deposited films were characterized as a function of coating conditions-withdrawal velocity, solution concentration, and evaporation driving force (deltaP). When no evaporation driving force was applied (deltaP = 0), highly uniform films were deposited with thickness in the range of 8-105 angstroms over the entire concentration range (3-11 wt%). Uniform films were also obtained at low concentrations (3-5 wt%) with a low evaporation driving force (deltaP = 0.0138 MPa). However, films deposited at medium to high concentrations (7-11 wt%) were thicker (110-570 angstroms) and less uniform, with larger nonuniformities at higher applied evaporation driving forces. Optical microscopy and atomic force microscopy (AFM) were used to characterize film morphology including drying defects and film roughness. Films deposited without evaporation had no apparent drying defects and very low root-mean-square (RMS) roughness (1.4-3.8 angstroms). Spinodal-like dewetting morphologies including holes with diameters in the range of 100-300 nm, and surface undulations were observed in films deposited at medium concentration (7 wt%) and low deltaP (0.0138-0.0276 MPa). At higher concentrations and higher evaporative driving forces, spinodal-like dewetting morphologies disappeared but concentric ring defect structures were observed with diameters in the range 20-125 microm. The film thickness and morphology of SOA films deposited from 1-CO2 hFMC were compared to those deposited from toluene and acetone under normal dip coating. Films deposited from l-CO2 hFMC were much thinner, more uniform, and exhibited much fewer drying defects and lower RMS roughness.

Carbon Dioxide↗

PubMed1996

Atomic (scanning) force microscopy in cardiovascular research.

The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The AFM images by using a sharp cantilever tip to sense the repulsive and attractive forces between the tip and the sample surface. The force of interaction is kept constant while raster scanning, resulting in images of the surface contours with molecular and, on hard inorganic surfaces, even atomic resolution. Movement of the cantilever in the Z plane is detected by a laser beam reflected off the cantilever to a photodiode system, a piezotube allows an X and Y raster, and a three-dimensional image results. Its capabilities include: (1) the three-dimensional imaging of membranes and biomolecules with molecular and submolecular resolution; (2) such imaging not only of dry specimens but of specimens in a physiologic solution, thereby allowing the investigation of dynamic processes in both viable biomolecules and living cells; (3) the sensing of charge and intermolecular interaction forces; (4) the chemical or biochemical modification of the cantilever tip, which allows the identification of specific structures and the measurement of specific interactions (e.g., a ligand-receptor interaction); (5) nanometer control of the position and force of the cantilever, which, in turn, allows the physical manipulation of biomolecules, the dissection of biological structures (e.g., the separation of one gap junctional hemichannel from its neighbor, thereby revealing normally inaccessible surfaces), the delivery of ligands, drugs, or other materials to specific locations, and the precise measurement of interacting forces at specific sites; and (6) the modification of the apparatus by adding complementary methodologies (e.g., magnetic resonance imaging, fluorescence microscopy, confocal microscopy, and perhaps electrophysiology). AFM, however, is only now being applied to biological research, many technical and methodologic problems exist, and a number of them are considered in this review. Little work has been done in cardiovascular research and the purpose of this review is to introduce this new and exciting approach to investigation.

Animals↗

PubMed2006

Role of crystal surface adhesion in kidney stone disease.

PURPOSE OF REVIEW: Atomic force microscopy has been used recently to characterize the adhesion force between selected calcium oxalate crystal surfaces and biologically relevant chemical groups attached to the atomic force microscopy probe tip. These measurements have permitted comparisons of the adhesion properties of different, well defined crystal faces, as well as determination of the influence of solution-phase macromolecules on adhesion. These studies have produced new insight into the specific chemical interactions that regulate kidney stone formation. RECENT FINDINGS: The adhesion force measurements have demonstrated that the large hexagonal (100) face of calcium oxalate monohydrate is the most adhesive. In contrast, the large (101) face of calcium oxalate dihydrate is the least adhesive. Carboxylate and amidinium groups on the atomic force microscopy tip exhibit equivalently large adhesion at a given crystal face, implicating specific binding to crystal surface lattice ions. Solution-phase macromolecules modulate adhesion in a face-selective manner, dependent on their chemical structures. SUMMARY: The low adhesion force for calcium oxalate dihydrate predicts a decreased ability of these crystals to aggregate or attach to cells, and correlates with the relative absence of calcium oxalate dihydrate in kidney stones. These measurements provide new understanding of the macromolecular regulation of crystal aggregation and attachment to cells in stone formation.

Adhesiveness↗

PubMed2002

Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.

Animals↗