AFM Topographs Research Articles

β-(1 → 3)(1 → 6)glucan from Schizophyllum commune 227E.32: High yield production via glucose/xylose co-metabolization

A co-metabolization of xylose and glucose by Schizophyllum commune 227E.32 wild mushroom for exopolysaccharide (EPS) production is presented. Cultivations performed with S. commune 227E.32 at different xylose concentrations demonstrated that the concentration of 50 g·L−1 of xylose achieved the highest EPS production, around 4.46 g·L−1. Scale-up in a stirred tank reactor (STR) was performed. 10 % inoculum showed the highest cost/benefit ratio regarding sugar conversion and EPS production (Y P/S = 0.90 g·g−1), achieving 1.82 g·L−1 of EPS. Isolation, purification, and characterization were conducted with EPS produced in flasks and STR. GC–MS analysis showed glucose as main monosaccharide constituents for both isolates. 13C NMR and HSQC-edited showed that both EPS isolated consisted of a β-D-Glcp (1 → 3) main chain, partially substituted at O-6 with nonreducing β-D-Glcp ends on every third residue, similar to β-D-glucan isolated from S. commune basidiomes known as schizophyllan (SPG). The Mw was determined by GPC to 1.5 × 106 Da (flasks) and 1.1 × 106 Da (STR). AFM topographs revealed a semi-flexible appearance of the β-D-glucan, consistent with the triple helical structures adopted by SPG and overall contour length consistent with a high molar mass.

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs.

Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes 'magnification' of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.

IR filtering properties of TiAlN/Cu/TiAlN coatings

In this paper, to explore the potential infrared filtering applications of magnetron sputtered TiAlN/Cu/TiAlN coatings, we investigated the optical, morphological and surface topographical properties using UV/Vis/NIR spectrometry, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy, respectively. From IR Spectroscopy analysis, it is observed that glass/TiAlN/Cu/TiAlN structure with 10 nm thick Cu layer showed highest visible transmittance of 78% and lowest IR transmittance of 22% at 600 nm and 2500 nm wavelength, respectively. In additon the AFM topographs further indicate that the inner TiAlN seeding layer could reduce the average roughness of Cu layer to 3 nm as compared to 8 nm without seeding layer. Moreover, outer TiAlN layer demonstrates very good environmental and corrosion protection property. Based on the results and analysis, it is proposed that TiAlN/Cu/TiAlN can be used as an efficient IR reflective material with high visible transmittance.

DockAFM: benchmarking protein structures by docking under AFM topographs

Proteins can adopt a variety of conformations. We present a simple server for scoring the agreement between 3D atomic structures and experimental envelopes obtained by atomic force microscopy. Three different structures of immunoglobulins (IgG) or blood coagulation factor V activated were tested and their agreement with several topographical surfaces was computed. This approach can be used to test structural variability within a family of proteins. DockAFM is available at http://biodev.cea.fr/dockafm.

Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered “Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum” were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm3 volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm3) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm3). Reflecting the contributions of thousands of pigment–pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000–2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.

Electron Conduction and Photocurrent Generation of a Light-Harvesting/Reaction Center Core Complex in Lipid Membrane Environments.

To reveal the structure-function relationship of membrane proteins, a membrane environment is often used to establish a suitable platform for assembly, functioning, and measurements. The control of the orientation of membrane proteins is the main challenge. In this study, the electron conductivity and photocurrent of a light-harvesting/reaction center core complex (LH1-RC) embedded in a lipid membrane were measured using conductive atomic force microscopy (C-AFM) and photoelectrochemical analysis. AFM topographs showed that LH1-RC molecules were well-orientated, with their H-subunits toward the membrane surface. Rectified conductivity was observed in LH1-RC under precise control of the applied force on the probe electrode (<600 pN). LH1-RC embedded in a membrane generated photocurrent upon irradiation when assembled on an electrode. The observed action spectrum was consistent with the absorption spectrum of LH1-RC. The control of the orientation of LH1-RC by lipid membranes provided well-defined conductivity and photocurrent.

Imaging of the Xenopus laevis oocyte plasma membrane in physiological-like conditions by Atomic Force Microscopy

Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy ~AFM! to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiologicallike conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

Structural Information, Resolution, and Noise in High-Resolution Atomic Force Microscopy Topographs

AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 Å (AqpZ), 12 Å (AQP0), 13 Å (AQP2), and 20 Å (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and “blurs” structural details.

Substrate effects in poly(ethylene glycol) self-assembled monolayers on granular and flame-annealed gold

Poly(ethylene glycol) (PEG) self-assembled monolayers (SAMs) are surface coatings that efficiently prevent nonspecific adhesion of biomolecules to surfaces. Here, we report on SAM formation of the PEG thiol CH 3O(CH 2CH 2O) 17NHCO(CH 2) 2SH (PEG 17) on three types of Au films: thermally evaporated granular Au and two types of Au films from hydrogen flame annealing of granular Au, Au(111), and Au silicide. The different Au surfaces clearly affects the morphology and mechanical properties of the PEG 17 SAM, which is shown by AFM topographs and force distance curves. The two types of SAMs found on flame-annealed Au were denoted “soft” and “hard” due to their difference in stiffness and resistance to scratching by the AFM probe. With the aim of nanometer scale patterning of the PEG 17, the SAMs were exposed by low energy (1 kV) electron beam lithography (EBL). Two distinctly different types of behaviour were observed on the different types of SAM; the soft PEG 17 SAM was destroyed in a self-developing process while material deposition was dominant for the hard PEG 17 SAM.

Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes

We have studied the mechanical properties of encapsulated Saccharomyces cerevisiae yeast cells by performing AFM force measurements. Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte layers and mechanically trapped into a porous membrane. Coated and uncoated cells in presence/absence of bud scars, i.e. scars resulting from previous budding events, have been investigated. No significant differences between encapsulated and bare cells could be inferred from AFM topographs. On the other hand, investigation on the system elasticity through the acquisition and analysis of force curves allowed us to put in evidence the differences in the mechanical properties between the hybrid cell/polyelectrolyte system and the uncoated cells. Analysis of the curves contact region indicates that the polyelectrolyte coating increases the system rigidity. Quantitative evaluation of the cell rigidity through the Hertz-Sneddon model showed that coated cells are characterized by a Young's modulus higher than the value obtained for uncoated cells and similar to the value observed on the bud scar region of uncoated cells.

Structural properties of polyC–scleroglucan complexes

Successive changes of solvent conditions can be used to dissociate and reassociate the triple-helical structure of (1,3)-beta-D-glucans. Ultramicroscopic techniques have revealed a blend of circular and other structures following renaturation. When this solvent exchange process is carried out in the presence of certain polynucleotides, the process creates a novel macromolecular complex. Here, we use size exclusion chromatography (SEC) to study such (1,3)-beta-D-glucan-polynucleotide complexes. Online multi-angle laser-light scattering (MALLS) and refractive index (RI) detectors allowed determination of molecular weight and radius of gyration of the molecules. An ultraviolet (UV) detector allowed specific detection of the polynucleotide. The poly-cytidylic acid (poly C) shifted to coelution with the linear fraction of the scleroglucan following the renaturation of poly C-scleroglucan blends, indicating that poly C is incorporated in linear, but not in circular, structures of scleroglucan. This conclusion was consistent with AFM topographs that revealed a decreased fraction of circular structures upon addition of poly C during the renaturation process. The combined information about radius of gyration (R(g)) and molecular weight (M(w)) allowed us to conclude that the poly C-scleroglucan complexes are more dense and have a higher persistence length than linear scleroglucan triple helixes. The experimentally determined mass per unit length was used as a basis for elucidating possible molecular arrangements within the poly C-scleroglucan complex.

Investigation of the Microstructure of SiC-Zn Nanocomposites by Microscopic Methods: SEM, AFM and TEM

SiC-Zn nanocomposites with about 20% volume fraction of metal were fabricated by infiltration process under the pressure of 2-8 GPa and at the temperature of 400_1000oC. SiC nanopowders used in the experiments formed loosely agglomerated chains of single crystal nanoparticles. The dimension of the agglomerates was in the micrometer range, the mean grain size was up to tens of nanometers. Microstructural investigations of the nanocomposites were performed at a different resolution levels using scanning, transmission electron microscopy and atomic force microscopy techniques (SEM, TEM, AFM, respectively). SEM observations indicate a presence of nano-dispersed, uniform (on the micrometer scale) mixture of two phases. TEM observations show that distribution of SiC and Zn nanocrystallites is uniform on the nanometer scale. High-resolution TEM images demonstrate an existence of thin (on the order of tens of Angstroms) Zn layers separating SiC grains. AFM images of the mechanically polished samples show a smooth surface with the roughness on the order of the SiC grain size (10-30 nm). After ion etching of some samples the AFM topographs show surface irregularities: periodically spaced hillocks 50-100 nm in height. The size of the SiC grains remains equal to that of the initial powder crystallites. The size of the Zn grains varies in the range of 20-100 nm depending on the initial SiC grain size and the composite fabrication conditions.

Variable LH2 stoichiometry and core clustering in native membranes of Rhodospirillum photometricum.

The individual components of the photosynthetic unit (PSU), the light-harvesting complexes (LH2 and LH1) and the reaction center (RC), are structurally and functionally known in great detail. An important current challenge is the study of their assembly within native membranes. Here, we present AFM topographs at 12 A resolution of native membranes containing all constituents of the PSU from Rhodospirillum photometricum. Besides the major technical advance represented by the acquisition of such highly resolved data of a complex membrane, the images give new insights into the organization of this energy generating apparatus in Rsp. photometricum: (i) there is a variable stoichiometry of LH2, (ii) the RC is completely encircled by a closed LH1 assembly, (iii) the LH1 assembly around the RC forms an ellipse, (iv) the PSU proteins cluster together segregating out of protein free lipid bilayers, (v) core complexes cluster although enough LH2 are present to prevent core-core contacts, and (vi) there is no cytochrome bc1 complex visible in close proximity to the RCs. The functional significance of all these findings is discussed.

Cr3+/COO-Complexation Induced Aggregation of Gelatin in Dilute Solution

The Cr3+/COO- complexation induced aggregation of linear gelatin chains in aqueous solutions under different conditions, such as the Cr3+ and gelatin concentrations, was investigated by a combination of static and dynamic laser light scattering (LLS). The results reveal a competition between the intrachain and interchain complexation. For a given gelatin concentration, there exists a “critical” Cr3+ concentration at which the interchain complexation became dominant. The intensity I(q) of the light scattered from the resultant aggregates was scaled to the scattering vector q as I(q) ∝ q-dq with dq = 1.61−1.84, suggesting that the aggregation was a diffusion-limited process. The variation of the Cr3+ concentration had less influence on the structure of the resultant aggregates. On the other hand, the decrease of the gelatin concentration led to an increase of dq from 1.61 to 2.04, indicating that the aggregation changed from a diffusion-limited process to a reaction-limited one. Precipitation is independent of the size of the aggregates but closely related to the gelatin concentration as well as the Cr3+/gelatin molar ratio. Two-dimensional AFM topographs of the aggregates revealed a clear growing fractal, supporting the results observed by LLS.

スパッタ銅膜の表面ラフネス成長の原子間力顕微鏡による観察

Scaling analysis of the surface roughness has been performed on copper films prepared by DC magnetron sputter deposition. The deposition was performed at Ar gas pressures of 2 and 10 Pa. Under both pressures, deposition rates were almost the same and were 1.1 nm/s. Self-affine parameters α and β were determined from a series of AFM topographs of films deposited for various periods (30180 min). At 10 Pa, the roughness parameter α was 0.60.7 and the dynamical parameter β was 0.9, while at 2 Pa, where the surface roughness grew more modestly, α was 0.50.7 and β was 0.6. The difference also appeared in the correlation lengthξ. The correlation length became larger as the deposition proceeded at 10 Pa, in accordance with the roughness evolution theory. On the other hand, it stayed almost constant at 2 Pa. The effect of gas pressure on the surface roughness is discussed.