Atomic force microscopy for ultrafiltration membrane imaging

Atomic force microscopy (AFM) can reveal nanometer-scale structure of samples without the sample preparation techniques that involve dehydration. This is particularly important for hydrophilic organic materials. An asymmetric polysulfone ultrafiltration membrane (molecular weight cutoff rated at 10 kg/mol) was imaged by AFM. Sample mounting methods tried include cyanoacrylate, double-sided tape, and paraffin. Wax and tape bonding did not lead to usable images. Cyanoacrylate bonding resulted in images that appear to show 2.8° 10⁹ pores/m² approximately 3 nm in diameter, creating a porosity of 2%. This is consistent with estimates of molecular sizes for 10 kg/mol proteins, but not with the results of other AFM studies of similar membranes. The discrepancies can be explained largely by differences in sample preparation techniques.     

Atomic and magnetic force microscopy imaging of thin-film recording heads

Investigations on the use of the scanning probe microscope (SPM) in the atomic force microscopy (AFM) mode for topography imaging and the magnetic force microscopy (MFM) mode for magnetic imaging are presented for a thin-film recording head. Results showed that the SPM is suitable for imaging the surface profile of the recording head, determining the width of the pole gap region, and mapping the magnetic field patterns of the recording head excited under current bias conditions of different polarity. For the cobalt sputter-coated tips used in MFM imaging, it was found that the magnetic field patterns obtained under different polarities of the current bias to the recording head were similar. This can be explained by the nature of the thin-film MFM tip, in which the direction of the tip magnetic moment can follow the stray magnetic field of the sample as the current bias to the recording head reverses in direction.     

Atomic force microscopic investigation of respiratory syncytial virus infection in HEp‐2 cells

Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti‐RSV drugs/therapeutics, it is indispensable to study the RSV–host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ～150–200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti‐RSV therapies.     

Adhesion artefacts in atomic force microscopy imaging

Artefacts that affect contrast and arise from adhesion forces in atomic force microscopy images of aramid fibres (both fresh and plasma-treated) are investigated. It is demonstrated that these stem not only from variations in the chemical composition of the surface but also from certain topographical features (which may appear hidden or enhanced in the images), resulting in changes in the lateral forces that are detected by the cantilever and are comparable to the vertical forces. It is also shown that both types of contribution to the forces can be uncoupled to yield images free from these artefacts, thus allowing more accurate quantitative measurements. These artefactual effects are also generally applicable to many other materials.     

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

Atomic force microscopy of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.     

Trypsin-Banding Induced Volume Changes of Human Metaphase Chromosomes Analyzed by Scanning Force Microscopy

A procedure for volume estimation based on scanning force microscopy images is applied to the study of banding-induced structural changes of chromosomes. Therefore, metaphase chromosomes were imaged before and after trypsin digestion, and the resulting three-dimensional data sets were used for a determination of the volumes of the imaged structures. The procedure is based on a histogram-based thresholding. The estimated volume is corrected for the background signal using the average background value from the histogram, so that an automated analysis of the images is possible. A first set of experimental data processed according to this approach is presented.     

Atomic force and scanning electron microscopy of atmospheric particles

Atomic force microscopy (AFM) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) have been used for both morphological and elemental mass analysis study of atmospheric particles. As part of the geometrical particle analysis, and in addition to the traditional height profile measurement of individual particles, AFM was used to measure the volume relative to the projection area for each particle separately, providing a particle shape model. The element identification was done by the EDS analysis, and the element mass content was calculated based on laboratory calibration with particles of known composition. The SEM-EDS mass measurements from two samples collected at 150 and 500 m above the surface of the Mediterranean Sea were found to be similar to mass calculations derived from the AFM volume measurements. The AFM results show that the volume of most of the aerosols that were identified as soluble marine sulfate and nitrate aerosol particles can be better estimated using cylindrical shapes than spherical or conical geometry.     

Atomic force microscopy of purple membranes

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.     

Atomic vacancy-induced friction on the graphite surface: observation by lateral force microscopy

Lateral force microscopy has been employed to investigate the frictional behaviour of atomic vacancies on the graphite surface. Such a study was only made possible by the controlled expansion of originally single‐atom vacancies into multiatom vacancies, employing oxygen plasma etching for this purpose. Enhanced friction was observed on the vacancy regions compared with pristine areas of graphite, the origin of which is examined and discussed.     

Scanning force microscopy of chromatin fibers in air and in liquid

We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin structures in the scanning force microscope (SFM) in air and in liquid. The beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying, whereas supranucleosomal structures were preserved after treatment of cell nuclei with detergent. In the latter case, the nucleosomes were still distinct but appeared more condensed. A modified droplet diffusion-spreading technique of chromatin from Namalwa cells (a human B-lymphoid line) yielded a uniform filamentous morphology and similar fiber appearance. A reversible swelling of spread chromatin was observed upon exposure of air-dried samples to solutions differing in salt concentrations.     

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al . (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science , 1996, pp. 924–930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.     

Atomic force microscopic analysis of hydrogen peroxide bleached kraft northern black spruce fibres

Hydrogen peroxide is a potent, relatively inexpensive oxidant that chemically degrades chromophoric components in pulps and textiles. Oxidation of cellulose is a byproduct of this process step that decreases the tensile strength of individual fibres. The residence time of pulp in the bleaching reactor must be optimized to achieve the desired brightness and minimizing fibre degradation. To evaluate the impact of peroxide bleaching at the microfibrillar level, a single black spruce tree was chosen and kraft pulped. Peroxide bleaching was conducted via benchtop polyethylene bag bleaching in a temperature-controlled waterbath. Atomic force microscopy (AFM) topographical images acquired before and after the bleaching step show dramatic changes in fibre structure consistent with delignification and defects in the surface topography. This was further verified by X-ray work at Brookhaven National Laboratory, NY, U.S.A.     

Atomic force microscopy as a tool for study of human hair

Atomic force microscopy (AFM) is a newly developed microscopic technique that offers high-resolution power, less intrusive measurement, and requires little sample pretreatment for elucidating structures of biological materials in three dimensions and in their natural environment. In this study, AFM has been used not only as an imaging technique for examining human hair structure at high resolution, but also as a tool for quantitative assessment of the effect of treatment in 10 mM phosphate buffered saline of pHs 3.0, 7.0, and 11.0 and heating on human hair structure. It is observed that the hair cuticle is a sensitive indicator of external influences on hair structure, and that its height can be used as a parameter for quantitative assessment. The experimental results obtained show that the swelling of hair caused by the incubation in the buffer decreases with the increase of the pH values and that, depending on the duration of heating, the hair undergoes structural expansion and shrinkage. This study demonstrates that AFM can be used as a valuable alternative to conventional microscopic techniques for hair research.     

Karyotyping of barley chromosomes by a new fluorescence banding technique combined with scanning probe microscopy

Fluorescence banding has been used to classify chromosomes, except those of barley. Four of the seven barley chromosomes are indistinguishable by length or arm ratio. C-banding has been used for classification; however, it requires a long aging period. Here, we describe a new fluorescence banding method for barley. The chromosomes are treated with warm acetate followed by staining with a fluorescent dye, YOYO-1. Using this method, all seven barley chromosomes can be clearly distinguished. Atomic force microscopy and scanning near-field microscopy analyses revealed that the surfaces of the banded chromosomes were flat, indicating that the fluorescence intensity reflected the internal DNA density or condensation of chromatin.     

Atomic force microscopy of silica nanoparticles and carbon nanohorns in macrophages and red blood cells

The emerging interest in understanding the interactions of nanomaterial with biological systems necessitates imaging tools that capture the spatial and temporal distributions and attributes of the resulting nano–bio amalgam. Studies targeting organ specific response and/or nanoparticle-specific system toxicity would be profoundly benefited from tools that would allow imaging and tracking of in-vivo or in-vitro processes and particle-fate studies. Recently we demonstrated that mode synthesizing atomic force microscopy (MSAFM) can provide subsurface nanoscale information on the mechanical properties of materials at the nanoscale. However, the underlying mechanism of this imaging methodology is currently subject to theoretical and experimental investigation. In this paper we present further analysis by investigating tip-sample excitation forces associated with nanomechanical image formation. Images and force curves acquired under various operational frequencies and amplitudes are presented. We examine samples of mouse cells, where buried distributions of single-walled carbon nanohorns and silica nanoparticles are visualized.     

Investigation of human hair fibers using lateral force microscopy

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used to investigate the morphologic and surface changes associated with various surface modifications to human hair. These included extraction with a series of solvents, bleaching, and treatment with a cationic copolymer. The study assessed the ability of these techniques to distinguish the changes in surface properties, including morphology and friction coefficient, as manifested in changes brought about by the indicated surface modifications. While topographic morphology can easily be investigated with contact AFM. LFM offers an additional tool for probing the surface distribution of oils and waxes. The removal of surface lipids from the fiber surface was accomplished using soxhlet extraction with t-butanol and n-hexane, while the free internal lipids (within the fiber structure) were removed by extraction with a mixture of chloroform and methanol (70:30, v/v). In addition, the surface of hair was modified with the cationic polymer, co(vinyl pyrrolidone-methacrylamidopropyl trimethylammonium chloride [PVP/MAPTAC]), and its distribution on the surface was monitored. Ambient AFM and LFM studies of surface modified and native fibers clearly indicate that when investigated as a function of tip loading force, the different modifications result in changes of the friction coefficient, which increase in this order: native, bleached, solvent extracted, and polymer-treated hair. Friction images show surface variations that are interpreted as areas of varying lipid film coverage. In addition, topographic images of the fibers show the presence of small pores, which become increasingly prevalent upon solvent extraction.