Atomic Force Microscopy Topographs Research Articles

Studies on structural, dielectric and charge transport properties of PVA:LiBr solid polymer electrolyte films

AbstractIn the present study, solid polymer electrolytes (SPEs) based on poly (vinyl alcohol) (PVA) doped with lithium bromide (LiBr) were prepared by solution casting method. Fourier transform infrared spectroscopy results affirm the complexation of LiBr with PVA. X‐ray diffraction results exhibit the increase of amorphous nature of the polymer electrolytes, which is also observed in scanning electron microscopy images and atomic force microscopy topographs. Thermogravimetric analysis thermographs endorse the increase of thermal stability of the polymer due to doping. Dielectric studies exhibit non‐Debye nature of the polymer electrolytes. Conductivity spectra reveal the maximum ionic conductivity (1.15 × 10−4 S/cm) for 20 wt% LiBr/PVA electrolyte at ambient temperature. Impedance analysis reveals the decrease of ionic relaxation in the polymer electrolytes and the studied transport properties of the electrolyte show that the major contribution to the conduction in this polymer electrolyte is ions.

Protein fibril length in cerebrospinal fluid is increased in Alzheimer’s disease

Alzheimer’s disease (AD) associated proteins exist in cerebrospinal fluid (CSF). This paper evidences that protein aggregate morphology distinctly differs in CSF of patients with AD dementia (ADD), mild cognitive impairment due to AD (MCI AD), with subjective cognitive decline without amyloid pathology (SCD) and with non-AD MCI using liquid-based atomic force microscopy (AFM). Spherical-shaped particles and nodular-shaped protofibrils were present in the CSF of SCD patients, whereas CSF of ADD patients abundantly contained elongated mature fibrils. Quantitative analysis of AFM topographs confirms fibril length is higher in CSF of ADD than in MCI AD and lowest in SCD and non-AD dementia patients. CSF fibril length is inversely correlated with CSF amyloid beta (Aβ) 42/40 ratio and CSF p-tau protein levels (obtained from biochemical assays) to predict amyloid and tau pathology with an accuracy of 94% and 82%, respectively, thus identifying ultralong protein fibrils in CSF as a possible signature of AD pathology.

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.

Membrane organization of photosystem I complexes in the most abundant phototroph on Earth.

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light (HL), low-light (LL) and nutrient-poor ecological niches. We used atomic force microscopy (AFM) to image the different photosystem I (PSI) membrane architectures of the MED4 (HL) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (LL) and SS120 (LL) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. AFM topographs and structural modelling revealed a series of specialised PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the LL MIT9313 is organised into a tightly-packed pseudo-hexagonal lattice that maximises harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly two-fold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economise on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

Sawtooth structure formation under nonlinear-regime ion bombardment

Linear-regime Ar+ bombardment of Si produces symmetrical ripple structures at ion incidence angles above 45° measured off-normal (Madi 2009 J. Phys.: Condens. Matter 21). In the nonlinear regime, new behaviors emerge. In this paper, we present experimental results of ion bombardment that continues into the nonlinear regime until pattern saturation at multiple ion incidence angles, showing the evolution of their grazing incidence small-angle x-ray scattering (GISAXS) spectra as well as atomic force microscopy topographs of the final, saturated structures. Asymmetric structures emerge parallel to the direction of the projected ion beam on the sample surface, constituting a height asymmetry not found in the linear regime. We then present simulations of surface height evolution under ion bombardment using a nonlinear partial differential equation developed by Pearson and Bradley (2015 J. Phys.: Condens. Matter 27 015010). We present simulated GISAXS spectra from these simulations, as well as simulated scattering from a sawtooth structure using the FitGISAXS software package (Babonneau 2010 J. Appl. Crystallogr. 43 929–36), and compare the simulated spectra to those observed experimentally. We find that these simulations reproduce many features of the sawtooth structures, as well as the nearly-flat final GISAXS spectra observed experimentally perpendicular to the sawtooth structures. However, the model fails to reproduce the final GISAXS spectra observed parallel to the sawtooth structures.

Modification of the atomic structure and surface relief in recrystallized molybdenum foils tensile strained in ultrahigh vacuum

Changes in the atomic structure and surface morphology of a recrystallized molybdenum foil under the action of uniaxial tension in ultrahigh vacuum have been studied by low -energy electron diffraction (LEED) and atomic-force microscopy (AFM). It is established that uniaxial tension of the foil surface, which consists of separate blocks of dominating (100) face with 1 × 1 structure, leads to a local destruction of this face and the rotation of blocks. AFM topographs of various regions on the surface of fractured samples have been obtained. The variation of the LEED patterns agrees well with the evolution of the surface relief measured by AFM.

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.

Investigation of photosynthetic membrane structure using atomic force microscopy

Photosynthetic processes, including light capture, electron transfer, and energy conversion, are not only ensured by the activities of individual photosynthetic complexes but also substantially determined and regulated by the composition and assembly of the overall photosynthetic apparatus at the supramolecular level. In recent years, atomic force microscopy (AFM) has matured as a unique and powerful tool for directly assessing the supramolecular assembly of integral membrane protein complexes in their native membrane environment at submolecular resolution. This review highlights the major contributions and advances of AFM studies to our understanding of the structure of the bacterial photosynthetic machinery and its regulatory arrangement during chromatic adaptation. AFM topographs of other biological membrane systems and potential future applications of AFM are also discussed.

Higher order structure of short immunostimulatory oligonucleotides studied by atomic force microscopy

Immunostimulatory CpG-DNA activates the innate immune system by binding to Toll-like receptor 9. Structurally different CpG-containing oligonucleotides trigger a different type of immune response while activating the same receptor. We therefore investigated the higher order structure of two different classes of immunostimulatory CpG-DNA. Class A, which contains a partly self-complementary sequence and poly-G ends, forms duplexes and nanoparticles in salt solution, while class B, which does not contain these features and is purely linear, does not form a duplex or nanoparticles. Results obtained here by high-resolution atomic force microscopy of classes A and B CpG-DNA, reflect these differences in secondary structure. Detailed structural analysis of the atomic force microscopy topographs is presented for two different sample preparation methods.

Compacting effect of BBR3464, a new-generation trisplatinum anticancer agent, on DNA

BBR3464 is a trinuclear platinum compound of formula [{ trans-PtCl(NH 3) 2} 2-μ- trans-Pt(NH 3) 2{NH 2(CH 2) 6NH 2} 2] 4+. It is a new-generation platinum chemotherapeutic agent that exhibits cytotoxicity at ten to thousand times lower dose limit compared to the well-known platinum drug cisplatin, in cisplatin-sensitive as well as in cisplatin-resistant cells. DNA is thought to be the primary cellular target of BBR3464. In this work, we have applied high-resolution atomic force microscopy (AFM) for the first time, to obtain direct information on BBR3464-induced structural changes of DNA. It is found that the DNA molecules get compacted after treatment with BBR3464, for the drug:DNA molar ratio and the drug treatment period of 0.01 and 48 h, respectively. These values of molar ratio and incubation period have been obtained previously, as a result of biochemical optimization studies carried out for achieving maximum drug effects. The DNA structural changes, as observed in AFM topographs, have been correlated to the bulk level spectroscopic information. A remark on the significance of BBR3464-induced DNA compaction with respect to the available AFM reports on DNA modification by cisplatin has been made.

Intermittent contact mode AFM investigation of native plasma membrane of Xenopus laevis oocyte

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 +/- 0.2 nm (mean +/- SE, n = 263) and 5.4 +/- 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.

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.

Atomic Force Microscopy Studies of Native Photosynthetic Membranes

In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes for quinones to diffuse freely. Measurement of the intercomplex distances between adjacent LH2 rings of Phaeospirillum molischianum has permitted the first calculation of the separation of bacteriochlorophyll a molecules in the native ICM. A recent AFM analysis of the organization of green plant photosystem II (PSII) in grana thylakoids revealed the protruding oxygen-evolving complex, crowded together in parallel alignment at three distinct levels of stacked membranes over the lumenal surface. The results also confirmed that PSII-LHCII supercomplexes are displaced relative to one another in opposing grana membranes.

Structure and stability of polynucleotide-(1,3)-β- D-glucan complexes

The structure and stability of complexes formed between (1,3)- β- D -glucans and the polynucletide poly(C) are investigated using differential scanning calorimetry (DSC), atomic force microscopy (AFM) and size exclusion chromatography (SEC). DSC revealed the melting transition of the complexes at 55 °C and an endothermic transition for renatured scleroglucan in the interval 30–40 °C. AFM topographs support the interpretations that the latter transition is due to melting of associated polymer sequences positioned in between structural defects in renatured triplexes. The complexes form between poly(C) and single stranded or renatured scleroglucan, but not with annealed scleroglucan. The complex formation thus seems to require single strands, but the short single stranded stretches present in renatured scleroglucan are sufficient, and full dissociation into single strands is not required. No differences were observed between complexes formed by adding poly(C) to renatured triple helical or denatured single stranded (1,3)- β- D -glucans. Implications of this insight on proposed structural models are discussed.

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This “fix-and-look” approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the “fix-and-look” approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.

Structural effects of nogalamycin, an antibiotic antitumour agent, on DNA

The structural changes of DNA, induced by the antitumour antibiotic nogalamycin, have been studied by atomic force microscopy (AFM). The transformation in the tertiary structure of 4361 bp long plasmid pBR322 DNA, after incubation with nogalamycin at 37 °C, has been monitored at the single molecule level. The AFM topographs of free DNA and the DNA–nogalamycin complex, incubated for 6, 12, 24, 36 and 48 h, reveal a gradual change from the circular supercoiled form having strand crossovers to the more compact plectonemic superhelix. With increasing incubation time, the extent of plectonemic coiling increases, indicating increasing level of drug binding via intercalative mode. Supportive evidences are obtained from the CD and UV–vis spectroscopic studies. To our knowledge, this is the first report on an AFM imaging study of the effects of nogalamycin, an anthracyclin intercalator, on DNA.

Quantitative analysis of atomic force microscopy topographs of biopolymer multilayers: Surface structure and polymer assembly modes

The morphology of polymer multilayer surfaces has been studied by quantitative analysis of atomic force microscopy (AFM) topographs. Multilayers were prepared for three different systems: chitosan–alginate, poly(L-lysine)-alginate and chitosan–scleraldehyde polymer pairs with up to 10 layer pairs. These utilized either an electrostatic dominated mechanism for the chitosan–alginate and poly(L-lysine)-alginate pairs, or covalent linkage for the chitosan–scleraldehyde pair. The surface structures were studied by evaluating the power spectral density (PSD) obtained from the height autocorrelation function calculated from the AFM topographs. By fitting the averaged PSD to the k-correlation model, information related to surface features such as pore size was extracted. Additionally, the dimensions of pronounced network strands were extracted from the peak at large spatial frequency observed for some of the multilayers. The analysis shows that quantitative information about properties of network like surfaces can be extracted, i.e. development of mean pore sizes of the multilayers prepared using stiff polymers and that such pore size can be tuned by choosing the components of the polymer pairs employed and/or the number of layer pairs.

Influence of Oligoguluronates on Alginate Gelation, Kinetics, and Polymer Organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.

From high-resolution AFM topographs to atomic models of supramolecular assemblies

Atomic force microscopy (AFM) has developed into a powerful tool in membrane biology. AFM features an outstanding signal-to-noise ratio that allows substructures on individual macromolecules to be visualized. Most recently, AFM topographs have shown the supramolecular assembly of the bacterial photosynthetic complexes in native membranes. Here, we have determined the translational and rotational degrees of freedom of the complexes in AFM images of multi-protein assemblies, in order to build realistic atomic models of supramolecular assemblies by docking high-resolution structures into the topographs. Membrane protein assemblies of megadalton size comprising several hundreds of polypeptide chains and pigments were built with Angstrom precision.

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.