Imaging surfaces of nano‐scale roughness by atomic force microscopy with carbon nanotubes as tips: a comparative study

Accurate knowledge of the nanoroughness of surfaces is crucial for many applications related to optics, electronics or tribology. Although atomic force microscopy (AFM) can image surfaces with a nanometre spatial resolution, the finite size of standard tips means that pores, pits or grooves with dimensions similar to or smaller than the tip apex will not be accurately imaged. Furthermore, standard tips are made of silicon or silicon nitride and are prone to wear. Mitigation may arise from the availability of AFM tips with a carbon nanotube (CNT) at their foremost end. This study compares the imaging performance of ultrasharp Si tips, CNT AFM tips prepared by a Langmuir‐Blodgett (LB) technique, and of CNT AFM tips prepared by a chemical vapour deposition (CVD) technique. The free length of the CNT AFM tips is in the range 80–200 and 600–750 nm, respectively. A polycrystalline niobium film surface is imaged that shows nanoroughness. The measurements demonstrate that CNT AFM tips allow excellent imaging if the scan parameters are adjusted very carefully. Nevertheless, in some cases distortions are found. The measured average grain diameter is 19.9 ± 3.6 nm in the case of a CNT AFM tip made by the LB technique, and 18.0 ± 3.3 nm in the case of a CNT AFM tip made by CVD. In addition to cross‐sections of topography images, also the power spectral density (PSD) is analyzed. An empirical approach for the readout of the characteristic length is suggested that involves the first derivative of the decadic logarithm of the PSD. Copyright © 2011 John Wiley & Sons, Ltd.     

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.     

Cholesteric liquid crystals: Image contrast in the TEM

Bright-field image contrast of thermotropic cholesteric liquid crystalline materials in the transmission electron microscope (TEM) is investigated. Possible sources of contrast for these systems are discussed in terms of their molecular anisotropy. A cholesteric side-chain liquid crystalline compound was investigated with TEM, low voltage high resolution scanning electron microscopy (LVHRSEM), and atomic force microscopy (AFM) to determine the origin of the strong contrast observed in these systems using bright-field transmission electron microscopy. Initial contrast of thin microtomed sections, as viewed with TEM low dose techniques and an image intensifier, was much weaker than observed under normal viewing conditions. The periodic steady state contrast typically observed for these materials is the result of beam damage. Furthermore, the surface of microtomed samples (parallel to the cholesteric helical axis) is corrugated with a periodicity of 1/2 the pitch due to a preferred fracture path in the glassy cholesteric state. AFM profile analysis shows an average peak to valley height of approximately 20-25 nm. AFM of free surfaces from aligned films also indicates a corrugation with a periodicity equal to 1/2 the pitch with substantially smaller average corrugation depths. TEM indicates a series of +1/2 and -1/2 disclination lines at the surface due to a rotation of the preferred helix direction parallel to the surface, consistent with previously reported models.     

Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica

The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36 kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.     

Computer simulations of atomic force microscopy: crystalline polymers and the effects of surface contaminants

The atomistic dynamics of the interaction of an atomic force microscopic (AFM) probe with a crystalline polyethylene surface was examined by using the molecular dynamics method. The results show that the internal dynamics of the polymer crystal is such that rapid relaxation occurs, providing for a large amount of structural reversibility and making it possible to perform nondestructive AFM experiments. However, surface and/or AFM tip defects or contaminants (such as those which can be induced by polar molecules adsorbed on the surface), can result in significant perturbations in the AFM images produced, causing large and sharp structures to appear on the surface topology. A rationale of the mechanisms responsible for the image distortions is presented, and a relationship to defects observed in AFM and STM experiments is given.     

Fusion of 2-D SIMS Images Using the Wavelet Transform

 Secondary ion mass spectroscopy (SIMS) is a powerful method for element distribution examination of conducting and semi-conducting surfaces at high spatial resolution and with a high sensitivity. Routine surface analysis produces about 8 to 15 images in a short time, each of which displays the intensity distribution of one mass, thus generating a multispectral SIMS image. Formation of occlusions, segregations, and the overall location of the elements relative to each other, are difficult to recognise when looking at n separate 2-D images. Image fusion is a process whereby images obtained from various sensors, or at different moments of time, or under different conditions, are combined together to provide a more complete picture of the object under investigation. The process of combining SIMS images may be viewed as an attempt to compensate for the inherent effect of SIMS to channel the information obtained from the sample into different images, corresponding to different element phases. The wavelet transform is a powerful method for fusion of images. This work covers the use of wavelet based fusion algorithms on multispectral SIMS images, evaluating the performance of different wavelet based fusion rules on different type of image systems and comparing the results to conventional fusion techniques. An aim of this study is to increase the information, i.e. the number of masses, which can be merged into one image in order to enhance the perception and interpretation of the SIMS surface images.     

Fabrication of a Biocompatible Mica/Gold Surface for Tip-Enhanced Raman Spectroscopy

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.     

Implications of the contact radius to line step (CRLS) ratio in AFM for nanotribology measurements

Investigating the mechanisms of defect generation and growth at surfaces on the nanometer scale typically requires high-resolution tools such as the atomic force microscope (AFM). To accurately assess the kinetics and activation parameters of defect production over a wide range of loads (F(z)), the AFM data should be properly conditioned. Generally, AFM wear trials are performed over an area defined by the length of the slow (L(sscan)) and fast scan axes. The ratio of L(sscan) to image resolution (res, lines per image) becomes an important experimental parameter in AFM wear trials because it defines the magnitude of the line step (LS = L(sscan)/res), the distance the AFM tip steps along the slow scan axis. Comparing the contact radius (a) to the line step (LS) indicates that the overlap of successive scans will result unless the contact radius-line step ratio (CRLS) is < or =(1)/(2). If this relationship is not considered, then the scan history (e.g., contact frequency) associated with a single scan is not equivalent at different loads owing to the scaling of contact radius with load (a proportional variant F(z)(1/3)). Here, we present a model in conjunction with empirical wear tests on muscovite mica to evaluate the effects of scan overlap on surface wear. Using the Hertz contact mechanics definition of a, the CRLS model shows that scan overlap pervades AFM wear trials even under low loads. Such findings indicate that simply counting the number of scans (N(scans)) in an experiment underestimates the full history conveyed to the surface by the tip and translates into an error in the actual extent to which a region on the surface is contacted. Utilizing the CRLS method described here provides an approach to account for image scan history accurately and to predict the extent of surface wear. This general model also has implications for any AFM measurement where one wishes to correlate scan-dependent history to image properties as well as feature resolution in scanned probe lithographies.     

AFM and low-pressure argon adsorption analysis of geometrical properties of phyllosilicates

The distribution of edge and basal surface areas of phyllosilicate particles is an essential parameter for understanding the interaction mechanisms at solid/gas or solid/liquid interfaces. Among the techniques proposed to determine the geometrical heterogeneities of flat solids, low-pressure argon adsorption and AFM analysis are the most promising to derive the weight-averaged values of specific surface areas. A series of publications have recently been dedicated to the combination of both methods showing the correlation between the two approaches. As obtaining a large set of high-resolution AFM images is time-consuming, it is necessary to test the ability of AFM routine analysis to derive surface areas and aspect ratio systematically and statistically, with all possible experimental and instrumental artefacts. In the present study, the expected agreement was found between AFM and argon adsorption determination for total, basal, and edge-specific surface areas of nonswelling clay minerals, except for one kaolinite, which is very heterogeneous in size. In addition, it was observed that for a given sample, individual particles present similar shapes, whatever their size, making it possible to derive a statistical relationship between AFM basal and total surface areas. On the basis of the obtained results, recommendations are given to derive accurate edge, basal, and total specific surface areas of phyllosilicates by combining conventional gas adsorption (nitrogen BET) and routine AFM techniques.     

Formation of microcrystals, micropuddles, and other spatial inhomogenieties in surface reactions under ambient conditions: an atomic force microscopy study of water and nitric acid adsorption on MgO(100) and CaCO3(104)

In this study, atomic force microscopy (AFM) is used to image freshly cleaved MgO(100) and CaCO3(104) as these surfaces undergo reaction with water and nitric acid under ambient conditions of temperature, pressure, and relative humidity. The reaction of water and nitric acid results in the formation of hydroxylated and nitrated surfaces, respectively. It is clear from the AFM images that there are spatial inhomogenieties and surface features that form on micrometer and nanometer length scales as these reactions proceed. These features, which include hillocks, patches, microcrystallites, and micropuddles, are due to surface and phase segregation as a result of facile ion mobility in the presence of adsorbed water. In addition, instabilities and oscillations in the AFM images provide an indication of liquid formation and the deliquescence (i.e., a solid to liquid-phase transition) of nitrate salts as a function of relative humidity.     

聚甲基丙烯酸甲酯单分子膜的原子力学显微镜研究

报道了利用原子力显微镜（ＡＦＭ）技术对聚甲基丙烯酸甲酯（ＰＭＭＡ）单分子膜进行分子结构水平的观察研究．由ＡＰＭ图像中得到的ＰＭＭＡ单体所占面积与用π－Ａ曲线中换算出液面上ＰＭＭＡＬａｕｇｍｕｉｒ膜中单体的面积符合得很好．从ＡＦＭ图像中可见ＰＭＭＡＬＢ膜中ＰＭＭＡ的线型碳链是与档板（ｂａｒｒｉｅｒ）平行的紧密排列，与ＬＢ提膜方向相垂直．并由此对ＰＭＭＡＬＢ膜的形成过程进行分析．此外，还观察到了在Ｓｉ基底上ＰＭＭＡ直链的卷绕伸直排列形态，初步分析了其形成原因．     

Micro‐thermal analysis of polymers: current capabilities and future prospects

The current state of development of micro‐thermal analysis (micro‐TA) and related techniques are briefly reviewed. Results for a PET/epoxy resin composite and a bilayer polymer film are given as illustrations. Details are given of a new interface that enables the micro‐TA unit to be placed inside a conventional FTIR spectrometer to carry out photothermal IR microscopy. New results are presented for a micro‐pyrolysis‐mass spectroscopy technique. The limitations of the current instrumentation are discussed in terms of the overriding problem being one of spatial resolution. Images obtained using pulsed force mode AFM with a high‐resolution heated tip indicate the scope for future development of this technique. The possibility of even higher spatial resolution with other forms of probe are discussed along with the potential for imaging micro‐pyrolysis time of flight mass spectroscopy and even tomography. It is concluded that these methods offer excellent prospects for characterising a wide range of polymer systems.     

Chemical and constitutional influences in the self-assembly of functional supramolecular hydrogen-bonded nanoscopic fibres

A new series of secondary amides bearing long alkyl chains with pi-electron-donor cores has been synthesized and characterised, and their self-assembly upon casting at surfaces has been studied. The different supramolecular assemblies of the materials have been visualized by using atomic force microscopy (AFM) and transmission electron microscopy (TEM). It is possible to obtain well-defined fibres of these aromatic core molecules as a result of the hydrogen bonds between the amide groups. Indeed, by altering the alkyl-chain lengths, constitutions, concentrations and solvent, it is possible to form different rodlike aggregates on graphite. Aggregate sizes with a lower limit of 6-8 nm width have been reached for different amide derivatives, while others show larger aggregates with rodlike morphologies which are several micrometers in length. For one compound that forms nanofibres, doping was performed by using a chemical oxidant, and the resulting layer on graphite was shown to exhibit metallic-like spectroscopy curves when probed with current-sensing AFM. This technique also revealed current maps of the surface of the molecular material. Fibre formation not only takes place on the graphite surface: nanometre scale rods have been imaged by using TEM on a grid after evaporation of solutions of the compounds in chloroform. Molecular modelling proves the importance of the hydrogen bonds in the generation of the fibres, and indicates that the constitution of the molecules is vital for the formation of the desired columnar stacks, results that are consistent with the images obtained by microscopic techniques. The results show the power of noncovalent bonds in self-assembly processes that can lead to electrically conducting nanoscale supramolecular wires.     

Does the nanometre scale topography of titanium influence protein adsorption and cell proliferation?

To investigate the influence of titanium films with nanometre scale topography on protein adsorption and cell growth, three different model titanium films were utilized in the present study. The chemical compositions, surface topographies and wettability were investigated by using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and water contact angle measurement, respectively. The films share the same surface chemistry but exhibit different topographies on a nanometre scale. Thus, they act as model systems for biological studies regarding surface topography effects. The films were obtained by varying the deposition rate and the film thickness, respectively. These films displayed nanometre scale surface roughness (root mean square roughness, R_rms) from 2 to 21 nm over areas of 50 μm × 50 μm, with different grain sizes at their surfaces. Albumin and fibrinogen adsorption on these model titanium films were performed in this study. Bicinchoninic acid assay was employed to determine the amount of adsorbed protein on titanium film surfaces. No statistically significant differences, however, were observed for either albumin or fibrinogen adsorption between the different groups of titanium films. No statistically significant influence of surface roughness on osteoblast proliferation and cell viability was detected in the present study.     

Some recent developments in SPM of crystalline polymers

Some recent advances in the application of atomic force microscopy to crystalline polymers are detailed. Ultra‐high resolution imaging of crystal surfaces, combined with the analysis of computer generated Connolly surfaces, enables the unambiguous identification of features on the cellulose crystal surface at near‐atomic resolution. The electronic enhancement of the quality factor of the cantilever when tapping in liquids enables a considerable improvement in force sensitivity to be obtained, allowing the fully saturated surface of an isotactic polystyrene gel to be imaged under the solvating molecule, at nanometre resolution. A series of experiments are detailed in which processes such as crystallization, crystal thickening and crystal deformation are followed in situ, in real time, providing significant new insights into long standing problems in polymer science.     

Application of atomic force microscopy to particle sizing

AFM has been applied for studying morphology and size distribution of nanometer-sized particles adsorbed on flat surfaces. For the quantitative evaluation of the images an algorithm for automatical particle detection and volume calculation has been developed. In this way a large number of particles can be automatically evaluated in order to derive size distributions or surface coverages. The method has been successfully applied to determine size distributions of environmental aerosol particles collected with an 11-stage low pressure impactor. The first four stages with average aerodynamic equivalent diameters (aed) ranging from 21 to 170 nm were investigated. The calculated aed values were in good agreement with the predicted aed for each stage. Additionally, it could be shown that sampling conditions and storage time affect the derived size distributions. Furthermore, AFM has been applied as reference method for conventional particle sizing techniques. For this purpose technologically relevant powders as titanium oxide powder and tungsten carbide powder were investigated using AFM and the results were compared with conventional techniques such as high resolution SEM and a light scattering method. The derived cumulative size distributions were in good agreement. The results clearly show that AFM constitutes a convenient tool for size determination, not only for ultrafine particles exploiting the high resolving power, but also in the case of larger particles. Received: 2 September 1998 / Revised: 19 October 1998 / Accepted: 22 October 1998     

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.     

Chemical Analysis of Inorganic and Organic Surfaces and Thin Films by Static Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

By using mass spectrometry to analyze the atomic and molecular secondary ions that are emitted from a solid surface when bombarded with ions, one obtains detailed information about the chemical composition of the surface. A time-of-flight mass spectrometer is especially suitable for the analysis of secondary ions because of its high transmission, high mass resolution, and ability to detect ions of different masses simultaneously. By using a finely focused primary ion beam it is also possible to analyze microareas and generate surface images with a lateral resolution of 0.1 μm or less. Static time-of-flight secondary ion mass spectrometry (TOF-SIMS) allows monolayer imaging and local analysis of monolayers with high sensitivity, a wide mass range, high mass resolution, and high lateral resolution. Besides information on elements and isotopes, the technique yields direct information on the molecular level and can also be used to analyze surface species of high molecular mass that are thermally unstable and cannot be vaporized. The method can be applied to practically all types of materials and sample forms, including insulators in particular. In this article the basic principles of TOF-SIMS are explained, and its analytical capabilities for both large area and imaging applications are illustrated by examples. These include silicon surfaces (both uniform and structured), thermally unstable organic molecules on surfaces, synthetic polymers, and synthetically prepared molecular surface films, particles, and fibers. Emitted neutral particles can also be analyzed by postionization with a laser, and the possibilities of this technique are discussed.     

Mapping the placement of oligonucleotide molecules using scanning probe microscopy

The successful development of novel bio-inspired devices requires the ability to place specific biomolecules on a substrate with nanometre precision, in such a way so that their bioactivity is retained. A method is required that can verify this bio-modification. Scanning probe microscopy (SPM) can image and probe a surface in a liquid environment with nanometre resolution. Using short chain complementary oligonucleotides as the bioactive molecules we have modified continuous and patterned gold substrates and SPM probes. We demonstrated that the attached oligonucleotides retained their biological activity after surface attachment with a hybridization interaction force that varies between 50 and 400pN as measured by SPM force measurements. Finally, the position of the attached oligonucleotides was determined with nanometre resolution. Thus we have demonstrated the capabilities of SPM in the application of the development of substrates and templates suitable for forming the basis of novel and innovative devices.     

Different interactions between the two sides of purple membrane with atomic force microscope tip

Atomic force microscopy (AFM) is known to be capable of measuring local surface charge density based on the DLVO model. However, it has failed to distinguish charge density difference between the extracellular and cytoplasmic sides of purple membrane (PM) in previous studies. In this paper, tapping-mode AFM with thioglycolate-modified tips was used to image PM in buffers of different salt concentrations. When imaged in 25 mM KCl buffer, the topography of membranes appeared to be of two different types, one flat and the other domelike. Such a difference was not observed in buffers of high salt concentrations. This suggests that the topography variation results from differences in electrostatic interaction between the AFM tip and the different membrane surfaces. With images of papain-digested PM and high-resolution images of membrane surface structure, we proved that the membrane surfaces with flat topography were on the extracellular side while the surfaces with domelike topography were on the cytoplasmic side. Hence, this provides a straightforward method to distinguish the two sides of PM without the requirement of high-resolution imaging. Force-distance curves clearly demonstrated the different tip-sample interactions. The force curves recorded on the extracellular side of PM were consistent with the DLVO model, so its surface charge density can be estimated well. However, the curves recorded on the cytoplasmic side had a much longer decay length, which is supposed to be relevant to the flexibility of the C-terminus of bacteriorhodopsin (bR).