Developments for inverted atomic force microscopy

Atomic force microscopy (AFM) has been used to study a wide range of systems. Chemically and biologically modified probes have extended AFM by coupling chemical and biological information with the physical measurements. In an effort to further expand the capabilities of modified AFM probes, previous studies investigated the use of an inverted AFM design (i-AFM), wherein a microfabricated tip array is used to image a cantilever-supported sample. This report details developments in cantilever and tip array fabrication which are aimed at improving the applicability and performance of this i-AFM design. Using an epoxy-based procedure, commercial cantilevers were modified with a series of standard substrates, including template-stripped gold, highly oriented pyrolytic graphite, and mica. The samples on these cantilevers were imaged with i-AFM, and lateral force images are obtained. This paper demonstrates the first use of i-AFM for measuring friction.     

Scaling-index method as an image processing tool in scanning-probe microscopy

The scaling-index method (SIM) is a novel tool for image processing in scanning-probe microscopy. Originating from the theory of complex systems, the SIM can be used in order to extract structural information from arbitrary data sets. This method can readily be applied to the analysis of digital atomic-force microscopy (AFM) images. Especially for biomedical diagnostics, where genetic material is investigated by various microscopic methods, a reliable image segmentation based on the SIM algorithm is helpful. As a first application, AFM-images of GTG-banded human metaphase chromosomes (with G bands obtained by Trypsin using Giemsa) are compared with micrographs from conventional light microscopy by means of a scaling-index analysis. While the grey-level distributions of the optical and the AFM-images are largely different from each other, the scaling-index images are remarkably similar. Using this method, a fingerprint of an image can be produced which helps in the classification and interpretation of the measured data.     

Morphology and amine accessibility of (3-aminopropyl) triethoxysilane films on glass surfaces

3-Aminopropyl) triethoxysilane (APTES) is commonly used to functionalize glass substrates because it can form an amine-reactive film that is tightly attached to the surface. In this study, we investigated the morphology and chemical reactivity of APTES films prepared on glass substrates using common deposition techniques. Films were prepared using concentrated vapor-phase deposition, dilute vapor-phase deposition, anhydrous organic-phase deposition and aqueous-phase deposition. All films were annealed, or cured, at 150 degrees C. The morphology of the films was quantified by fluorescence and by atomic force microscopy (AFM). The optical equivalent of the AFM images was computed and then used to directly compare optical and AFM images. Reactive amine density was determined by a picric acid assay and by a method that employed N-succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP) cross-linked rhodamine. Fluorescence and AFM images showed that silane films prepared from dilute vapor-phase and aqueous-phase deposition were more uniform and had fewer domains than those deposited by the other methods. The ratio of picric acid-accessible amino groups to SPDP cross-linked rhodamine-accessible groups varied with the preparation method, suggesting reactant size-dependent difference in amine accessibility. We found a larger number of accessible amino groups on films prepared by vapor-phase deposition than on those prepared from solution deposition. The dilute vapor-phase deposition technique produced relatively few domains, and it should be a good choice for bioconjugation applications. There were appreciable differences in the films produced by each method. We suggest that these differences originate from differences in film rearrangement during annealing.     

The role of substrates on the structural,optical, and morphological properties of zno nanotubes prepared by spray pyrolysis

ZnO films were deposited onto glass, ITO coated glass, and sapphire substrate by spray pyrolysis, and subsequently annealed at the same temperature of 400°C for 3 h. The role of substrate on the properties of ZnO films was investigated. The structural and optical properties of the films were investigated by X‐ray diffractometer (XRD) and photoluminescence (PL) spectrophotometer, respectively. The surface morphology of the nanostructured ZnO film was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Crystallographic properties revealed that the ZnO films deposited on sapphire and ITO substrates exhibit a strong c‐axis orientation of grains with hexagonal wurtzite structure. Extremely high UV emission intensity was determined in the film on ITO. The different luminescence behaviors was discussed, which would be caused by least value of strain in the film. Films grown on different substrates revealed differences in the morphology. ZnO films on ITO and sapphire substrates revealed better morphology than that of the film on glass. AFM images of the films prepared on ITO show uniform distribution of grains with large surface roughness, suitable for application in dye sensitized solar cells. Microsc. Res. Tech. 77:211–215, 2014. © 2013 Wiley Periodicals, Inc.     

Gloss phenomena and image analysis of atomic force microscopy in molecular and cell biology

Proper sample preparation, scan setup, data collection and image analysis are key factors in successful atomic force microscopy (AFM), which can avoid gloss phenomena effectively from unreasonable manipulations or instrumental defaults. Fresh cleaved mica and newly treated glass cover were checked first as the substrates for all of the sample preparation for AFM. Then, crystals contamination from buffer was studied separately or combined with several biologic samples, and the influence of scanner, scan mode and cantilever to data collection was also discussed intensively using molecular and cellular samples. At last, images treatment and analysis with off‐line software had been focused on standard and biologic samples, and artificial glosses were highly considered for their high probability. SCANNING 31: 49–58, 2009. © 2009 Wiley Periodicals, Inc.     

Micropatterning of bacteria on two-dimensional lattice protein surface observed by atomic force microscopy

In this study, we characterized the two-dimensional lattice of bovine serum albumin (BSA) as a chemical and physical barrier against bacterial adhesion, using fluorescence microscopy and atomic force microscopy (AFM). The lattice of BSA on glass surface was fabricated by micro-contact printing (muCP), which is a useful way to pattern a wide range of molecules into microscale features on different types of substrates. The contact-mode AFM measurements showed that the average height of the printed BSA monolayer was 5-6nm. Escherichia coli adhered rapidly on bare glass slide, while the bacterial adhesion was minimized on the lattices in the range of 1-3mum(2). Especially, the bacterial adhesion was completely inhibited on a 1mum(2) lattice. The results suggest that the anti-adhesion effects are due by the steric repulsion forces exerted by BSA.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Design and capabilities of an experimental setup based on magnetron sputtering for formation and deposition of size-selected metal clusters on ultra-clean surfaces

The design and performance of an experimental setup utilizing a magnetron sputtering source for production of beams of ionized size-selected clusters for deposition in ultra-high vacuum is described. For the case of copper cluster formation the influence of different source parameters is studied and analyzed. Size-selected clusters are deposited on substrates and the efficiency of an electrostatic quadrupole mass selector is tested. Height analysis using atomic force microscopy (AFM) demonstrates relative standard size deviations of 7%-10% for the particles of various sizes between 6 nm and 19 nm. Combined analysis by AFM and transmission electron microscopy reveals that the clusters preserve almost spherical shape after the deposition on amorphous carbon substrates. Supported nanoparticles of a few nanometres in diameter have crystalline structure with a face-centered cubic (fcc) lattice.     

Scanning probe microscopy studies of cereal seed storage protein structures

Scanning probe microscopes (SPMs) share a number of common features which give the techniques advantages over conventional light and electron microscopy. First, high resolution, up to the atomic level, is possible in certain cases, and second, they are nondestructive, requiring no staining or coating and the images can be obtained in the hydrated state or under water. Scanning probe microscopes, particularly scanning tunnelling microscopes (STM) and atomic force microscopes (AFM), have been used to study food-related systems, ranging from relatively large structures such as starch granules to the organisation of secondary structures in proteins and the interaction of proteins. The seed storage proteins (gluten) of wheat are responsible for the viscous and elastic properties of wheat doughs that allow them to be used for a wide range of different food products. Using AFM and STM, images of individual and groups of proteins have been obtained in both the dry and hydrated states. The ability to work in liquid environments allows the conformation of proteins to be determined under conditions approaching “native.” The AFM and STM have been used to image both gliadins and glutenins and to study their aggregative behaviour in relation to gluten and dough systems.     

Atomic force microscopy applied to study macromolecular content of embedded biological material

We demonstrate that atomic force microscopy represents a powerful tool for the estimation of structural preservation of biological samples embedded in epoxy resin, in terms of their macromolecular distribution and architecture. The comparison of atomic force microscopy (AFM) and transmission electron microscopy (TEM) images of a biosample (Caenorhabditis elegans) prepared following to different types of freeze-substitution protocols (conventional OsO4 fixation, epoxy fixation) led to the conclusion that high TEM stainability of the sample results from a low macromolecular density of the cellular matrix. We propose a novel procedure aimed to obtain AFM and TEM images of the same particular organelle, which strongly facilitates AFM image interpretation and reveals new ultrastructural aspects (mainly protein arrangement) of a biosample in addition to TEM data.     

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three‐dimensional topographical information provided by the AFM could be used to better interpret the two‐dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.     

Polystyrene spheres on mica substrates: AFM calibration, tip parameters and scan artefacts

Atomic force microscopy (AFM), in various versions, has had major impact as a surface structural and spectroscopic tool since its invention in 1986. At its present state of development, however, the interpretation of AFM images is limited by the current state of methodologies for calibration over the wide dynamic range of magnification. Also, the parameters of individual tips, as well as the generic characteristics of different kinds of tips, affect both the quality of the images and their interpretation. Finally, the very nature of the tip-to-surface interaction will generate artefacts, in addition to those associated with tip shape, which need to be fully understood by the practitioners of force microscopy. This project seeks to address and shed light on some of these issues. Polystyrene beads deposited on mica substrates form hexagonal close-packed layers. The unit cell parameters are suitable for calibration of the AFM in the lateral plane, while the perpendicular spacing of the layers is appropriate for calibration along the vertical axis. Using different size fractions, it is straightforward to determine the extents of linearity, orthogonality, thermal and instrumental drifts over distances from 100 nm to tens of micrometres. The present results show that the methodologies for contact mode operation can be adapted to noncontact modes. It is known that an AFM image arises from a convolution of surface topography and tip shape, and is mediated by the interaction. In principle it is possible to carry out a deconvolution, if we have complete knowledge about two of the three elements (i.e. tip, surface and interaction). In practice we rarely have the requisite information. Prominent artefacts will occur when the characteristic parameters of the tip are comparable to those of the surface topography, and/or if there is a variable strength, or extent of localization, of the interaction. The present results demonstrate artefacts due to effects of geometry as well as interaction.     

Ag掺杂对TiO2纳米薄膜结构及摩擦学性能的影响

采用溶胶凝胶法在普通载玻片上制备了TiO2和Ag/TiO2纳米结构薄膜,利用X射线衍射仪(XRD)、X光电子能谱(XPS)、原子力显微镜(AFM)及UMT-2摩擦试验机,考察了Ag掺杂量对薄膜组成结构、表面形貌及摩擦学性能的影响。实验结果表明,Ag掺杂量对TiO2薄膜表面形貌和减摩抗磨性能产生重要影响,低掺杂时Ag自润滑性能对薄膜摩擦性能的增强作用占主导,而高掺杂时其对薄膜的影响主要表现为恶化表面,从而导致摩擦性能下降。本研究测试条件下,掺杂量为5.0%(摩尔分数)时具有最佳的耐磨寿命和最低的摩擦因数。     

Characterization of polymer thin films by phase-sensitive acoustic microscopy and atomic force microscopy: a comparative review

The potential of phase-sensitive acoustic microscopy (PSAM) for characterizing polymer thin films is reviewed in comparison to atomic force microscopy (AFM). This comparison is based on results from three-dimensional vector contrast imaging and multimodal imaging using PSAM and AFM, respectively. The similarities and differences between the information that can be derived from the AFM topography and phase images, and the PSAM phase and amplitude micrographs are examined. In particular, the significance of the PSAM phase information for qualitative and quantitative characterization of the polymer films is examined for systems that generate surface waves, and those that do not. The relative merits, limitations and outlook of both techniques, individually, and as a complementary pair, are discussed.     

Correction for piezoelectric creep in scanning probe microscopy images using polynomial mapping

We describe a method for using polynomial mapping to correct scanning probe microscope images for distortion due to piezoelectric creep. Because such distortion varies from image to image, this method can be used when the actual locations of some features within an image are known absolutely, or in a series of images in which the actual locations of some features are known not to vary. While the general case of polynomial mapping of degree N requires the determination of 2(N+ 1)2 matrix elements by regression, we find that by understanding the mechanism by which piezoelectric creep distorts scanning probe microscope images, we can fix most of these coefficients at 0 or 1 a priori, leaving only 2(N+ 1) coefficients to be determined by regression. We describe our implementation of this strategy using the Interactive Data Language (IDL) programming language, and demonstrate our technique on a series of atomic force microscopy (AFM) images of diblock copolymer microdomains. Using our simplified scheme, we are able to reduce the effects of distortion in an AFM image from 5% of the scan width to a single pixel, using only five reference points.     

极紫外多层膜基底表面粗糙度综合表征技术

研究用不同清洗方法对硅基底表面粗糙度的影响,首先使用原子力显微镜(AFM)直接测量硅片表面粗糙度;然后,利用直流磁控溅射方法,在相同制备工艺条件下镀制M o/S i多层膜,使用X射线衍射仪(XRD)对多层膜二级衍射峰进行摇摆曲线扫描;最后,利用同步辐射测量多层膜的反射率,间接表征基底的粗糙度。结果表明,超声清洗后镀制的多层膜反射率最高,结论与AFM,XRD等表征方法一致。     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

A large-sample atomic force microscope observing in both air and liquid

A large-sample atomic force microscope (AFM) that allows high resolution observation in both air and liquid has been developed. With a unique beam tracking method, laser beam is capable of reflecting off the same spot on the AFM cantilever throughout raster scan over the entire scan area, either operating in air or in liquid environment. Incorporating the stand-alone AFM probe unit with an automated large sample stage, wide-scan-range imaging can be realized with high resolution and slight distortion. In addition, an image stitching method is utilized to build a broad merged image with range up to millimeters while keeping nanometer order resolution. By using a large-volume liquid bath, large and massive sample can be observed in liquid with this AFM system. Several typical experiments have been carried out to demonstrate the imaging ability and stability of this AFM. Topographic structures of gold pattern on a glass substrate are scanned at two different places on the same specimen surface. The porosity of a sheet of filter paper is then characterized in both air and water. Finally, larger-area AFM image of anodic aluminum oxide template in oxalic acid is on spot obtained by merging several individually scanned images together. Experiments show that this AFM system can offer high resolution and wide range AFM images even for large samples with remarkable capabilities in various environments.