Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation

In order to decrease salt crystal formation, reduce the aggregation of viruses in atomic force microscopy (AFM) preparations and obtain an acceptable AFM image, the AFM sampling of viruses must be optimized. In this paper, centrifugal AFM sampling of recombinant adeno-associated virus-2 (rAAV-2) was used. The prepared rAAV-2 virus was imaged by AFM using the tapping mode in air. The results indicate that the rAAV-2 viruses prepared by centrifugal AFM sampling methods could be well imaged by AFM. The rAAV-2 viruses exhibited polymorphous particles in the AFM images. The preliminary off-line section analysis of the individual rAAV-2 virus particle indicated that the half-high widths of the large rAAV-2 particles ranged from 18 to 23 nm, while those of the small rAAV-2 particles ranged from 15 to 17 nm, which is almost in agreement with the results obtained by the off-line particle analysis of the rAAV-2 virus particles. Above all, the aggregation of different-sized rAAV-2 particles was directly imaged by AFM, which verifies the previous speculation that the large number (> ~31 nm) distribution of the mean diameter of rAAV-2 virus particles was probably caused by aggregation of rAAV-2 particles.     

Investigation of foreign substances in food using scanning electron microscopy-x-ray microanalysis

Scanning electron microscopy (SEM) and x-ray microanalysis (EDS) were used to conduct forensic investigations on metal and glass foreign objects. SEM-EDS is an excellent method for identifying metal foreign objects in food, such as wire, dental fillings, bone, and metal packaging, based on their element composition. From a determination of the extent of corrosion of a metal foreign object using SEM-EDS, it is sometimes possible to determine whether the material has been processed with the food product. Case histories of processed aluminum, unprocessed nickel-coated steel, and corrosion resistant stainless steel foreign objects are discussed. A potential product-tampering problem involving a hole defect in a paperboard package was resolved using SEM-EDS. Blue fibers found in the hole were found to contain brass particles from a ball point pen rather than a syringe needle. SEM-EDS has been used to determine the elemental composition of glass foreign objects and is able to distinguish between many types of glass including container, electrical, and bakeware. A case history is presented to show that although container glasses cannot, in general, be distiguished from one another using SEM-ED S, they can be distinguished by trace element semiquantitative spectrograghic analysis. SEM-EDS can be used to distinguish glass-like foreign objects from glass. Case histories of struvite crystals found in salmon and cream of tartar crystals found in grape juice are discussed. Fourier transform infrared (FTIR) identified the cream of tartar crystal as calcium tartrate, and this complemented the calcium, carbon, and oxygen components of the compound found using SEM-EDS.     

Lattice-like array particles on Xenopus oocyte plasma membrane

Plasma membrane from Xenopus laevis oocytes has been used as a model system to study membrane structure and particle components, including native and exogenously expressed proteins. Previous studies by electron microscopy (EM) and atomic force microscopy (AFM) compared intramembrane particles (IMPs) on uninjected oocyte membranes to oocytes expressing proteins of interest. These studies observed randomly distributed IMPs on the surface of the oocyte plasma membrane. In this paper, we introduce a novel technique to isolate oocyte membranes by bursting the oocyte and depositing its membrane on a flat mica substrate. The flat surface membrane preparation allows high-resolution AFM images to beobtained, revealing a novel structure of densely packed particles. These particles exhibit a regular, repeating pattern of a lattice-like array with orderly packing and are thus termed "lattice-like array particles" (LAPs). The LAPs are orderly yet imperfectly packed, are located in depressed pools, occur with a low frequency on the oocyte membrane surface, and have not previously been seen using other isolation and imaging methods. Histogram analysis of the center-to-center distance between LAPs suggest their size to be about 44 nm in diameter, considerably larger than other reported size estimates of IMPs. These results indicate that LAPs represent a novel membrane particle organization, which merits further study.     

Combination of transmission electron and atomic force microscopy techniques to determine volume equivalent diameter of submicrometer particles

Morphological properties of atmospheric particles are directly related to their residence time and transport behaviors, and their deposition patterns in human respiratory systems. The projected properties of particles measured by transmission electron microscopy (TEM) were combined with the particle height measured by atomic force microscopy (AFM) to determine volume equivalent diameter of submicrometer particles. For nonvolatile (refractory) laboratory-generated spherical polystyrene latex and cubic NaCl particles, the measured volume equivalent diameters agreed well with the true values (within 4%). However, for nonrefractory (NH(4))(2)SO(4) particles, the measured volume equivalent diameter was much smaller than the true value due to evaporation of volatile species at low vacuum pressure and high electron-beam intensity conditions in TEM, and deformation of particles in AFM. We observed that the volume equivalent diameter of 100 nm mobility-classified atmospheric particles was 35 ± 5 nm, suggesting that these particles contain nonrefractory species, whereas that of 20 nm mobility-classified atmospheric particles was found to be 19 ± 6 nm, suggesting that these particles were refractory and spherical.     

Investigation of foreign substances in food

Scanning electron microscopy (SEM) together with energy-dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR) were used to investigate foreign substances from seven categories of foreign substances in food. (1) Naturally occurring foreign substances--Using FTIR, a foreign substance was identified as a natural resin probably from the product. (2) Foreign substances introduced during food processing. Scanning electron microscopy-EDS was used to identify a foreign material found on surf clams as calcium phosphate from a product/ingredient interaction. Using SEM-EDS, a crystalline material in a meat product was identified as calcium salts of chloride and phosphate. Fourier transform infrared spectroscopy was used to identify foreign material that clogged an aerosol valve as chipboard. Using SEM-EDS, the metal in the heel of a glass bottle was identified as copper sulfide-containing metal inclusion. (3) Insects, reptiles, and rodents--Scanning electron microscopy was used to determine that a mouse found in food was not processed with the food, but entered the container after it left the factory. (4) Glass fragments--Glass from various sources can be distinguished from one another using SEM-EDS either by the level of the major elements in glass or by the presence of elements in one glass, but not in another. (5) Glass-like particles--Using SEM-EDS, glass-like particles found on beets were determined to be a fatty acid. (6) Metal foreign objects--Using SEM-EDS, metals from a variety of sources can be easily distinguished. For example, a tin-soldered container can be distinguished from a lead-soldered can. Using SEM-EDS, the metal fiber found on the bottom of a two-piece can likely enter the can during the final stage of the manufacture of the drawn and ironed food can. (7) Drug capsule identification--Fourier transform infrared spectroscopy was used to determine that a pill found in food was ibuprofen.     

Use of atomic force microscopy (AFM) for microfabric study of cohesive soils

Microfabric reflects the imprints of the geologic and stress history of the soil deposit, the depositional environment and weathering history. Many investigators have been concerned with the fundamental problem of how the engineering properties of clay depend on the microfabric, which can be defined as geometric arrangement of particles within the soil mass. It is believed that scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the only techniques that can reveal particle arrangements of clayey soils directly; however, current research introduces a novel and more advanced technique, atomic force microscopy, to evaluate the microfabric of cohesive materials. The atomic force microscopy has several advantages over SEM/TEM for characterizing cohesive particles at the sub‐micrometre range by providing 3D images and 2D images with Z‐information used in quantitative measurements of soil microfabric using SPIP software, and having the capability of obtaining images in all environments (ambient air, liquids and vacuums). This paper focuses on the use of atomic force microscopy technique to quantify the microfabric of clayey soils by developing the criteria for average and maximum values of angle of particle orientation within the soil mass using proposed empirical equations for intermediate and extreme microfabrics (dispersed, flocculated).     

Influence of Poisson's ratio variation on lateral spring constant of atomic force microscopy cantilevers

Atomic force microscopy (AFM) can be used to measure the surface morphologies and the mechanical properties of nanostructures. The force acting on the AFM cantilever can be obtained by multiplying the spring constant of AFM cantilever and the corresponding deformation. To improve the accuracy of force experiments, the spring constant of AFM cantilever must be calibrated carefully. Many methods, such as theoretical equations, the finite element method, and the use of reference cantilever, were reported to obtain the spring constant of AFM cantilevers. For the cantilever made of single crystal, the Poisson's ratio varies with different cantilever-crystal angles. In this paper, the influences of Poisson's ratio variation on the lateral spring constant and axial spring constant of rectangular and V-shaped AFM cantilevers, with different tilt angles and normal forces, were investigated by the finite element analysis. When the cantilever's tilt angle is 20 degrees and the Poisson's ratio varies from 0.02 to 0.4, the finite element results show that the lateral spring constants decrease 11.75% for the rectangular cantilever with 1muN landing force and decrease 18.60% for the V-shaped cantilever with 50nN landing force, respectively. The influence of Poisson's ratio variation on axial spring constant is less than 3% for both rectangular and V-shaped cantilevers. As the tilt angle increases, the axial spring constants for rectangular and V-shaped cantilevers decrease substantially. The results obtained can be used to improve the accuracy of the lateral force measurement when using atomic force microscopy.     

Investigation of Wear Particles Generated in Human Knee Joints Using Atomic Force Microscopy

Wear particle analysis can be potentially developed as an effective method for assessment of osteoarthritis (OA). To achieve this goal, the surface morphological and mechanical properties of human wear particles extracted from the osteoarthritic synovial joints with different OA grades need to be studied. Atomic force microscopy (AFM) has been used for cartilage analysis owing to its high resolution and the capability of revealing both mechanical properties and surface topographical data in three-dimensions. Few studies have been conducted on human wear particles due to difficulties in obtaining the samples and technical challenges in preparing wear debris samples for AFM investigations in a hydrated environment. This work aimed to develop a suitable preparation technique to study the mechanical properties and surface morphology of human wear particles using AFM. Wear particles were separated from synovial fluid samples which were collected from OA patients and deposited on an aldehyde functional plasma polymer surface to immobilise wear particles. They were imaged for the first time using AFM. The nanoscaled surface topographies and nanomechanical properties of the particles were obtained in a hydrated mode. The methodology established in this study enables investigations of the surface morphology and mechanical properties of wear particles at the nanoscale for better understanding of OA and the possibility of developing a new diagnostic method based on the wear debris analysis technique.     

Microscopy as a tool to investigate the influence of ammonium polyphosphate particle size on the flame retardant properties of polymer composites

The influence of ammonium polyphosphate (APP) particle size on the performance of an intumescent formulation and on the synergistic action of a series of montmorillonite samples with different d‐spacings for the production of flame retardant composites was investigated. The polymer matrix employed was poly(ethylene‐co‐butyl acrylate), EBA 30, and the intumescent formulation consisted of APP and pentaerythritol (PER). After being processed, the composites were submitted to scanning electron microscopy (SEM), thermogravimetric analysis, heating microscopy, and limiting oxygen index tests. The results indicate that the greater interaction between the APP and PER molecules, caused by the increase of the contact area promoted by the reduction of the APP particle size, could favor the esterification reaction between APP423 and PER, allowing the formation of a greater amount of char precursors in shorter period of time. In addition, the montmorillonite d‐spacings had a more pronounced influence on the clays synergistic action with the intumescent formulation containing the APP with smaller particle size. Microscopy has shown to be an important tool to investigate APP particle size effect on the fire retardancy. AFM results enabled the detection of nanometric particles in the sample containing the smallest particle size of APP. SEM micrographs showed that those nanometric particles were better dispersed in the matrix, interacting more effectively with the other components, a factor probably responsible for the superior fire retardancy results. Heating microscopy revealed that the material with smaller APP particle size did show some remaining structure at the temperature of 850°C.     

AFM investigation of Martian soil simulants on micromachined Si substrates

The micro and nanostructures of Martian soil simulants with particles in the micrometre‐size range have been studied using a combination of optical and atomic force microscopy (AFM) in preparation for the 2007 NASA Phoenix Mars Lander mission. The operation of an atomic force microscope on samples of micrometre‐sized soil particles is a poorly investigated area where the unwanted interaction between the scanning tip and loose particles results in poor image quality and tip contamination by the sample. In order to mitigate these effects, etched silicon substrates with a variety of features have been used to facilitate the sorting and gripping of particles. From these experiments, a number of patterns were identified that were particularly good at isolating and immobilizing particles for AFM imaging. This data was used to guide the design of micromachined substrates for the Phoenix AFM. Both individual particles as well as aggregates were successfully imaged, and information on sizes, shapes and surface morphologies were obtained. This study highlights both the strengths and weaknesses of AFM for the potential in situ investigation of Martian soil and dust. Also presented are more general findings of the limiting operational constraints that exist when attempting the AFM of high aspect ratio particles with current technology. The performance of the final designs of the substrates incorporated on Phoenix will be described in a later paper.     

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.     

Macro and microtribological studies of CrO2 video tapes

Atomic force microscopy (AFM) and its modification-friction force microscopy (FFM) are becoming increasingly important in the understanding of friction, wear, lubrication and nanomechanical property measurements. We describe modified AFM and FFM techniques and present data on microtribological studies of two CrO₂ video tapes. Macro-scale friction measurements were also made on the two tapes. We have observed that macro-scale friction is higher than that on micro-scale. Lower values of micro-scale friction as compared to macro-scale friction may be because of less ploughing contribution in micro-scale friction measurements. The directionality effects observed in micro-scale friction may come from the asymmetrical transfer of wipe material or from the calendering. Differences in the macro-scale friction in the two tapes appear to correlate with the changes in scratch resistance, wear resistance and the hardness of the tape surface on a micro-scale to nano-scales. It is demonstrated that AFM/FFM techniques are valuable in the fundamental understanding of tribology of magnetic tapes.     

Prototype cantilevers for quantitative lateral force microscopy

Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The "hammerhead" cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever "torque sensitivity" to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.     

Measuring absolute nanoparticle number concentrations from particle count time series

Single‐particle microscopy is important for characterization of nanoparticulate matter for which accurate concentration measurements are crucial. We introduce a method for estimating absolute number concentrations in nanoparticle dispersions based on a fluctuating time series of particle counts, known as a Smoluchowski process. Thus, unambiguous tracking of particles is not required and identification of single particles is sufficient. However, the diffusion coefficient of the particles must be estimated separately. The proposed method does not require precalibration of the detection region volume, as this can be estimated directly from the observations. We evaluate the method in a simulation study and on experimental data from a series of dilutions of 0.2‐ and 0.5‐μm polymer nanospheres in water, obtaining very good agreement with reference values.     

Adhesion and wear of colloidal silica probed by force microscopy

The adhesion and wear of colloidal silica nanoparticles (50 nm diameter) dispersed in a film have been directly studied using atomic force microscopy (AFM) under aqueous solution conditions. The adhesion between surface‐bound silica particles and the AFM tip is found to peak in strength between pH 4 and 5. Using the JKR contact mechanics model, the energy for a single Si–OH/Si–OH interaction was estimated to be 0.4 ± 0.1 kcal/mol. Tribochemical wear of the silica particles, and their displacement from the film, is enhanced at high pH due to the increased facility of silica dissolution and the concomitant increase in attendant inter‐particle repulsion. This revised version was published online in September 2006 with corrections to the Cover Date.     

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.     

Morphological characteristics of fulvic acid fractions observed by atomic force microscopy

Structural studies on fulvic acids (FAs) are significantly important since they are believed to be involved in many environmentally important processes, such as adsorption and transportation of organic and inorganic pollutants. In this research, morphology characteristics of FAs were studied by atomic force microscopy (AFM). FAs that were isolated from three soil layers (A1, B and C) of the same vertical profile in a Korean pine forest were divided into four fractions (FA‐1, FA‐2, FA‐3 and FA‐4) by a sequence of successive elution processes. Most of FAs appeared as a platy particle in the AFM topographic and phase images. Among these platy particles, some have a regular shape, such as round flake and oblong flake; others have irregular structures, such as sand heaps. Particle morphologies of different FA fractions, including hydrophilic and hydrophobic FAs fractions, were similar. However, particle sizes and distributions of FA fractions from different soil layers at the same vertical profile did differ. Particle sizes of hydrophobic FAs were relevant with respect to the soil depth. They were increased with the increasing of the soil depth. FAs from C layers were more heterogeneous with respect to the A1 and B. Our results may foster a better understanding for the relevance between the morphology of FA particles with the soil layers and the soil depth.     

Frictional effects in atomic force microscopy of Langmuir-Blodgett films

Frictional effects in atomic force microscopy (AFM) of Langmuir-Blodgett films of 1, 2-dipalmitoyl-snglycero-phosphoglycerol were examined. Height measurements of the Langmuir layers are strongly influenced by the orientation of the cantilevers used in AFM relative to the sample. A simple model is used to describe the frictional effects and to calculate the real height of the monolayers.     

Direct deformation study of AFM probe tips modified by hydrophobic alkylsilane self-assembled monolayers

The in-use wear of atomic force microscopy (AFM) probe tips is crucial for the reliability of AFM measurements. Increase of tip size for several nanometers is difficult to monitor but it can already taint subsequent AFM data. We have developed a method to study the shape evolution of AFM probe tips in nanometer scale. This approach provides direct comparison of probe shape profiles, and thus can help in evaluation of the level of tip damage and quality of acquired AFM data. Consequently, the shape degradation of probes modified by hydrophobic alkylsilane self-assembled monolayers (SAMs) was studied. The tip wear length and wear volume were adopted to quantitatively verify the effectiveness of hydrophobic coatings. When compared with their silicon counterparts, probes modified by SAM materials exhibit superior wear-resistant behavior in tapping mode scans.     

Contact mechanics and tip shape in AFM-based nanomechanical measurements

Stiffness-load curves obtained in quantitative atomic force acoustic microscopy (AFAM) measurements depend on both the elastic properties of the sample and the geometry of the atomic force microscope (AFM) tip. The geometry of silicon AFM tips changes when used in contact mode, affecting measurement accuracy. To study the influence of tip geometry, we subjected ten AFM tips to the same series of AFAM measurements. Changes in tip shape were observed in the scanning electron microscope (SEM) between individual AFAM tests. Because all of the AFAM measurements were performed on the same sample, variations in AFAM stiffness-load curves were attributed to differences in tip geometry. Contact-mechanics models that assumed simple tip geometries were used to analyze the AFAM data, but the calculated values for tip dimensions did not agree with those provided by SEM images. Therefore, we used a power-law approach that allows for a nonspherical tip geometry. We found that after several AFAM measurements, the geometry of the tips at the very end is intermediate between those of a flat punch and a hemisphere. These results indicate that the nanoscale tip-sample contact cannot easily be described in terms of simple, ideal geometries.