Confined polymer melts studied by atomic force microscopy

Using an atomic force microscope (AFM) the interaction between an AFM tip and different planar solid surfaces have been measured across a long-chain poly(dimethyl siloxane) (PDMS, M_W = 18,000 g/mol), a short-chain PDMS (M_W = 4200 g/mol), a poly(ethylmethyl siloxane) (PEMS, M_W = 16,800 g/mol), and a diblock copolymer consisting of one PDMS and one PEMS block (PDMS-b-PEMS, M_W = 15,100 g/mol). The interaction changed significantly during the first 10 h after immersing the solids in the polymer melt. This demonstrates that the time scale of structural changes at a solid surface is much slower than in the bulk. On mica and silicon oxide both polymers formed an immobilized “pinned” layer beyond which a monotonically decaying repulsive force was observed. Attractive forces were observed with short-chain PDMS on silicon oxide and PEMS on mica and silicon oxide. On the basal plane of graphite PEMS caused a stable, exponentially decaying oscillatory force.     

Interaction between solid surfaces in a polymer melt studied by atomic force microscopy

Using an atomic force microscope (AFM) the interaction between an AFM tip and a planar silicon oxide surface has been measured across poly(dimethylsiloxane) (PDMS, M_W = 18 000). Due to the small radius of curvature of the AFM tip the hydrodynamic repulsion of the tip was negligible and forces could be measured in equilibrium. This is confirmed by the fact that force-versus-distance curves measured at different approaching velocities were indistinguishable. In equilibrium a repulsive force was observed which could best be described by a power law, F ∝ 1/d^2.5 where d is the distance.     

Determination of solid surface tension from particle-substrate pull-off forces measured with the atomic force microscope

Atomic force microscopy (AFM) is capable of solid surface characterization at the microscopic and submicroscopic scales. It can also be used for the determination of surface tension of solids (gamma) from pull-off force (F) measurements, followed by analysis of the measured F values using contact mechanics theoretical models. Although a majority of the literature gamma results was obtained using either Johnson-Kendall-Roberts (JKR) or Derjaguin-Muller-Toporov (DMT) models, re-analysis of the published experimental data presented in this paper indicates that these models are regularly misused. Additional complication in determination of gamma values using the AFM technique is that the measured pull-off forces have poor reproducibility. Reproducible and meaningful F values can be obtained with strict control over AFM experimental conditions during the pull-off force measurements (low humidity level, controlled and known loads) for high quality substrates and probes (surfaces should be free of heterogeneity, roughness, and contamination). Any probe or substrate imperfections complicate the interpretation of experimental results and often reduce the quality of the generated data. In this review, surface imperfection in terms of roughness and heterogeneity that influence the pull-off force are analyzed based upon the contact mechanics models. Simple correlations are proposed that could guide in selection and preparation of AFM probes and substrates for gamma determination and selection of loading conditions during the pull-off force measurements. Finally, the possibility of AFM measurements of solid surface tension using materials with rough surfaces is discussed.     

Local surface charge dissipation studied using force spectroscopy method of atomic force microscopy

We propose herein a method to study local surface charge dissipation in dielectric films using force spectroscopy technique of atomic force microscopy. By using a normalization procedure and considering an analytical expression of the tip‐sample interaction force, we could estimate the characteristic time decay of the dissipation process. This approach is completely independent of the atomic force microscopy tip geometry and considerably reduces the amount of experimental data needed for the calculation compared with other techniques. The feasibility of the method was demonstrated in a freshly cleaved mica surface, in which the local charge dissipation after cleavage followed approximately a first‐order exponential law with the characteristic time decay of approximately 7–8 min at 30% relative humidity (RH) and 2–3.5 min at 48% RH. Copyright © 2015 John Wiley & Sons, Ltd.     

Melting behavior of lamellae of isotactic polypropylene studied using hot-stage atomic force microscopy

The α- and β-form lamellae of isotactic polypropylene were developed at different temperatures. The melting behaviors of the lamellae were observed in real time at elevated temperatures using a hot-stage atomic force microscopy. The melting behavior of the α-form lamellae was determined by the lamellar defects. For the α-form lamellae developed at different undercoolings, the larger the undercoolings, the relatively higher amount of defect in the lamellae was observed. The lamellae with defects were melted into lamellar segments, and recrystallization took place during the heating process. The β-form lamellae had lower thermal stability, and they melted firstly and separately from that of α-form.     

Atomic force microscopy investigation of asymmetric diblock copolymer morphologies in thin films

Microphase separation and the resulting morphology of asymmetric diblock copolymers of poly(ε-caprolactone) (PCL) in thin films have been investigated by atomic force microscopy. Copolymers consisted of a short block of PCL (M_n∼2500-4500 g/mole) and a longer second block of poly(methyl methacrylate) (PMMA), poly(styrene) (PS) or poly(cyclohexene oxide) (PCHO). Tendency for microphase separation above the glass transition temperature of the second block (PMMA, PS or PCHO) resulted in a pitted morphology on the surface of the thin films. This tendency was strongest for PMMA and weakest for PCHO. The presence of up to 54% PMMA homopolymer in PCL-PMMA block copolymer did not prevent the formation of such pitted morphology on the surface. The effect of the chemical structure of the second block and the possible orientations of the block copolymer molecules in thin films are discussed.     

Visualisation of an ultrafiltration membrane by non-contact atomic force microscopy at single pore resolution

Non-contact atomic force microscopy (AFM) has been used to investigate the furface pore structure of a polyethersulfone ultrafitration membrane of specified molecular weight cut off (MWCO) 25 000 (ES625, PCI Membrane Systems). Excellent images at up to single pore resolution were obtained. This is the first time that AFM images of a membrane at such high resolution have been presented. Analysis of the images gave a mean pore size of 5.1 nm with a standard deviation of 1.1 nm. The results have been compared to previously published studies of membranes of comparable MWCO using contact AFM and electron microscopy. Non-contact AFM is a powerful means of studying the surface pore characteristics of ultrafiltration membranes.     

Intermolecular forces between acetylcholine and acetylcholinesterases studied with atomic force microscopy

With the aid of atomic force microscopy, the intermolecular forces between acetyleholinesterases (AChE) and its natural substrate acetylcholine (ACh) have been studied. Through force spectrum measurement based on imaging of AChE molecules it was found that the attraction force between individual molecule pairs of ACh and AChE was (10±1) pN just before the quaternary ammonium head of ACh got into contact with the negative end of AChE and the decaying distance of attraction was (4±1) nm from the surface of ACHE. The adhesion force between individual ACh and AChE molecule pairs was (25±2) pN, which had a decaying feature of fast-slow-fast (FSF). The attraction forces between AChE and choline (Ch), the quaternary ammonium moiety and hydrolysate of ACh molecule, were similar to those between AChE and ACh. The adhesion forces between AChE and Ch were (20±2) pN, a little weaker than that between ACh and ACHE. These results indicated that AChE had a steering role for the diffusion of ACh toward it and had r     

Changes in the surface structure of purple membrane upon illumination measured by atomic force microscopy

Bacteriorhodopsin (BR) patches with a diameter of 1 to 3 μm were investigated in their native state by atomic force microscopy (AFM) in buffer solution. The patches were immobilized deposited and investigated on mica in 150 mM KCl and 10 mM Tris-buffer at pH 8. Under this buffer condition they adsorb preferred with their extracellular side to the solid support mica. The structure of the two-dimensional light adapted crystals was resolved with an imaging force of about 100 pN up to a resolution of 13 Å. The topography of the surface gets smoother if an imaging force of 1000 pN was applied indicating that protruding structures are compressed. Upon illumination with white light, during imaging with a force of 200 pN, the surface structure of the BR lattice changed. The force- and light-induced structural changes were reversible.     

Chemical force microscopy: applications in surface characterisation of natural hydroxyapatite

Detailed mapping of surface chemistry with nanometer resolution has application throughout the physical and life sciences. The atomic force microscope (AFM) has provided a tool that, when using functionalised probes, is capable of providing chemical information with this level of spatial resolution. Here, we describe the technique of chemical force microscopy (CFM) and demonstrate the sensitivity of the technique using chemical force titrations against pH. We describe in detail the specific application of mapping the surface charge on natural hydroxyapatite from skeletal tissue and show that this new information leads to a better understanding of the binding of matrix proteins to the mineral surface.     

Effect of alkaline treatment on cellulose supramolecular structure studied with combined confocal Raman spectroscopy and atomic force microscopy

The aim of this work was to investigate the morphological and structural changes associated with mercerization of cellulose fibres with combined confocal Raman and atomic force microscopy (AFM). During mercerization the alkali induces a change in polymorphic lattice from cellulose I to II. This was observed by confocal Raman spectroscopy from cellulose samples treated with 10, 15 and 25% aqueous sodium hydroxide solution. AFM images from the same samples illustrated that microfibrils were swollen and more granular in cellulose II than in cellulose I. Raman spectral images in plane and depth directions showed that the polymorphous cellulose structure was uniform throughout the cell wall, whereas the microfibril orientation varied between fibre cell wall layers. The changes in microfibril orientation on the sample surfaces were confirmed by AFM images measured from the same sample position.     

Dissolution of the barite (001) surface by the chelating agent DTPA as studied with non-contact atomic force microscopy

DTPA (diethylenetriaminepentaacetic acid) is a chelating agent widely used for removal of barium sulfate (barite) scale in the petroleum industry. In this paper we report ex-situ investigations of barite dissolution in deionized water and in 0.18 M DTPA aqueous solutions. Non-contact atomic force microscopy (NC-AFM) was used to observe dissolution on the BaSO₄ (001) cleavage surface. Dissolution was carried out at room temperature in a 10 ml reactor. Each sample was first etched in solution and dried before examination by NC-AFM. Dissolution on the BaSO₄ (001) surface took place via development of etch pits. In deionized water, triangular etch pits were observed on the (001) terraces at room temperature. And, zigzag shaped etch pits were found at the edges of steps. In DTPA solutions, etch pits on the (001) terraces were observed and these became deeper and longer with increasing time. The geometry of these etch pits was trapezoidal, and/or trapezohedral. To explain this characteristic morphology caused by dissolution we suggest that the active sites of one DTPA molecule bind to two or three Ba²⁺ cations exposed on the (001) surface.     

Surface microstructure of bitumen characterized by atomic force microscopy

Bitumen, also called asphalt binder, plays important roles in many industrial applications. It is used as the primary binding agent in asphalt concrete, as a key component in damping systems such as rubber, and as an indispensable additive in paint and ink. Consisting of a large number of hydrocarbons of different sizes and polarities, together with heteroatoms and traces of metals, bitumen displays rich surface microstructures that affect its rheological properties. This paper reviews the current understanding of bitumen's surface microstructures characterized by Atomic Force Microscopy (AFM). Microstructures of bitumen develop to different forms depending on crude oil source, thermal history, and sample preparation method. While some bitumens display surface microstructures with fine domains, flake-like domains, and dendrite structuring, ‘bee-structures’ with wavy patterns several micrometers in diameter and tens of nanometers in height are commonly seen in other binders. Controversy exists regarding the chemical origin of the ‘bee-structures’, which has been related to the asphaltene fraction, the metal content, or the crystallizing waxes in bitumen. The rich chemistry of bitumen can result in complicated intermolecular associations such as coprecipitation of wax and metalloporphyrins in asphaltenes. Therefore, it is the molecular interactions among the different chemical components in bitumen, rather than a single chemical fraction, that are responsible for the evolution of bitumen's diverse microstructures, including the ‘bee-structures’. Mechanisms such as curvature elasticity and surface wrinkling that explain the rippled structures observed in polymer crystals might be responsible for the formation of ‘bee-structures’ in bitumen. Despite the progress made on morphological characterization of bitumen using AFM, the fundamental question whether the microstructures observed on bitumen surfaces represent its bulk structure remains to be addressed. In addition, critical technical challenges associated with AFM characterization of bitumen surface structures are discussed, with possible solutions recommended. For future work, combining AFM with other chemical analysis tools that can generate comparable high resolution to AFM would provide an avenue to linking bitumen's chemistry to its microscopic morphological and mechanical properties and consequently benefit the efforts of developing structure-related models for bituminous materials across the different length scales.     

Imaging of reconstituted purple membranes by atomic force microscopy

The organization of bacteriorhodopsin (bR) within reconstituted purple membranes (RPM) was examined using atomic force microscopy (AFM). Five reconstituted species were examined: RPM 3 (bR/native polar lipids/dimyristoylphosphatidylcholine (DMPC) in a 1:9:14 molar ratio), RPM 4 (bR/native polar lipids in a 1:7 molar ratio), RPM 5 (bR/native polar lipids/1,2-di-O-phytanyl-sn-glycerol in a 1:3.5:6.1 molar ratio), RPM 6 (bR/native polar lipids/1,2-di-O-phytanyl-sn-glycero-3-phosphocholine in a 1:3.5:4.9 molar ratio), and RPM 7 (bR/native polar lipids/1,2-diphytanoyl-sn-glycero-3-[phospho-l-serine] in a 1:3.5:4.6 molar ratio). RPM 3 patches adsorbed onto mica exhibit domains of crystallized bR trimers arranged in a hexagonal packing structure, similar to those found in native purple membrane (NPM). These domains are enclosed by DMPC-rich regions. RPM 4 patches were observed to have larger domains of crystallized bR, with trimer orientation 30° different from that found in NPM. The bR-rich domains are enclosed by a large, protein-free, lipid-rich region. The topography of RPM 5 was difficult to resolve as the surface had no discernable patterns or structure. The topographies of RPM 6 and 7 were similar to that found in RPM 3 in that higher domains were formed within the patch adsorbed onto mica. They may contain protein-rich regions, but clear images of protein arrangement could not be obtained using AFM. This may be a result of imaging limitations or of the lack of organization of bR within these domains.     

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose–response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.     

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy

The combination of alternating electric fields with nanometer‐sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real‐time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements.     

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Intercalation-induced changes in DNA supercoiling observed in real-time by atomic force microscopy

Atomic force microscopy (AFM) has been employed to observe in real-time and in an aqueous environment the process of ethidium bromide induced supercoiling in individual DNA plasmid molecules. Image data reveal both the onset and the progressive presence of plectonemic DNA supercoiling. In addition, significant molecular motion of the surface adsorbed DNA is observed. These data illustrate the potential of AFM in the time-resolved study of biomolecular processes, and hence, provide new insights into biomolecular structure and function.     

Study of the surface morphology of polyisobutylene-based block copolymers by atomic force microscopy

This paper reports the investigation of the nanostructured surface morphology of linear polystyrene-block-polyisobutylene-block-polystyrene (SIBS) triblock copolymers and novel arborescent SIBS block copolymers by Atomic Force Microscopy (AFM) in the tapping mode. Thin films spin coated from toluene onto silicon wafers were studied. The nanostructured morphology of the block copolymers varied with the hard polystyrene (PS) and soft polyisobutylene (PIB) segment composition, ranging from spherical to lamellar nanometer-sized discreet PS phases dispersed in a continuous PIB matrix. Annealing the samples resulted in well developed/ordered structures. The arborescent blocks had irregularly distributed PS phases in the PIB matrix. Annealing had a dramatic effect on the morphology which still remained irregular. Three-dimensional AFM image and section analysis indicated the presence of a height difference between PIB (high-lying plateaus or hills) and PS (low-lying plateaus or valleys) in the block copolymers, which became more prominent during annealing. It is theorized that the rubbery PIB chains are able to relax, thereby protruding from the surface, anchored by the physically crosslinked PS phases.     

Characterization of human cells exposed to deltamethrin by means of Raman microspectroscopy and atomic force microscopy

Raman spectroscopy is a powerful technique for studying cellular biochemistry. In fact, each toxic chemical induces biochemical changes related to the own action mechanism. In this investigation Raman microspectroscopy has been used, in correlation with atomic force microscopy images, to detect biochemical and structural damages occurring in cultured human cells as a consequence of deltamethrin exposure. Cultured human keratinocyte cells have been exposed at increasing concentrations of deltamethrin from 10⁻³ M to 10⁻⁶ M for 24 h. A viability test indicated that the cytotoxic dose corresponds to exposure at deltamethrin solution for 24 h with the chemical concentration between 10⁻⁴ M and 2.5 10⁻⁴ M. The compared analysis of Raman spectra and AFM images allows to state that an evident damage occurs in the plasmatic membrane and it is already detectable after exposure of keratinocytes at the lowest investigated deltamethrin concentration (10⁻⁶ M). The most important modifications are related to the breakdown of CH₂ bonds of lipidic chains, whereas proteineous bonds are less involved in the deltamethrin action. On the whole, cellular damage starts after exposure to deltamethrin doses well lower than that established as cytotoxic.