Characterization of human ovarian teratoma hair by using AFM, FT-IR, and Raman spectroscopy

The structural, physical, and chemical properties of hair taken from an ovarian teratoma (teratoma hair) was first examined by atomic force microscopy (AFM), Fourier transform infrared (FT-IR), and Raman spectroscopy. The similarities and differences between the teratoma hair and scalp hair were also investigated. Teratoma hair showed a similar morphology and chemical composition to scalp hair. Teratoma hair was covered with a cuticle in the same manner as scalp hair and showed the same amide bonding modes as scalp hair according to FT-IR and Raman spectroscopy. On the other hand, teratoma hair showed different physical properties and cysteic acid bands from scalp hair: the surface was rougher and the adhesive force was lower than the scalp hair. The cystine oxides modes did not change with the position unlike scalp hair. These differences can be understood by environmental effects not by the intrinsic properties of the teratoma hair.     

Morphological changes induced in erythrocyte membrane by the antiepileptic treatment: An atomic force microscopy study

Atomic force microscopy (AFM), a powerful characterization tool widely applied in problems in a large range of disciplines of the natural sciences, including cellular biology, was used to obtain information about the morphological changes induced in the erythrocyte membrane at the patients with epilepsy that undergo a long time treatment that operates upon one or several neuronal ionic channels, comparative with a healthy donor. This technique allowed non‐invasive imaging of erythrocyte membrane, revealing details and specific characteristics down to the nanometer level with characterization of surface texture parameters, such as average height, average roughness and coefficient of kurtosis at micrometer/nanometer resolution. For the healthy donor the AFM morphology appears to have all the characteristics of a normal red blood cell membrane. Instead, the closer examination of the erythrocytes membrane surface morphology for the samples collected from the patients diagnosed with epilepsy and treated with specific drugs did not reveal similar structures with those obtained for the healthy donor. The nanostructure of the membrane was drastically damaged, depending on the administrated treatment, and probably in time will affect their functionality. Therefore, the anticomital drugs have influence not only at the neuronal level, but also at the red blood cell level.     

Molecular ultrastructure of the urothelial surface: Insights from a combination of various microscopic techniques

The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno‐TEM), and freeze‐fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno‐FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896–901, 2014. © 2014 Wiley Periodicals, Inc.     

Visualization of the nanoscale assembly of type I collagen on PLA by AFM

Collagen adsorption and the morphology of its assemblies at polymer surface play an important role in improving the biocompatibility of materials. In this study, the nanoscale organization of type I collagen on Polylactide (PLA) was observed directly by high‐resolution atomic force microscopy. The results show that the supramolecular structure of adsorbed collagen was affected by the concentration of collagen solution, appropriate pH and electrolyte composition of the buffer. On PLA substrate, network structures formed in high humidity atmosphere. In addition, collagen formed well‐oriented nano‐patterns at nearly neutral pH and appropriate electrolyte composition. Particularly, the typical 65 nm D‐periodicity of collagen fibers was observed in the presence of potassium ions. Our investigation provides useful insights into the regulation of collagen assembly by substrates and environmental conditions, which is important for understanding the mechanism of collagen adsorption and assembly on polymer surfaces. It also offers a potential way to create surfaces of bio‐functioned and nano‐patterned materials for biotechnological and biomedical applications. SCANNING 32: 104–111, 2010. © 2010 Wiley Periodicals, Inc.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.     

Platelet‐rich fibrin and collagen membrane in the preservation of the alveolar bone: Feasibility of the elemental inorganic composition and scanning electron microscopy analysis

The success of dental implants is related to the amount, quality, and composition of the alveolar bone. The placement of platelet‐rich fibrin (PRF) clot associated with a resorbable collagen membrane (RCM) in a postextraction alveolus is a technique used for ridge preservation. This case report study analyzed the ultrastructural characteristics of cross‐sectioned alveolar bone that received PRF and RCM using scanning electron microscopy and the inorganic composition using “energy dispersive X‐ray spectrometry,” in order to explore the feasibility of this method to clinical studies. Three alveolar bone samples from two male patients (37 and 58 years old), obtained in the procedure of placing the dental implant, were analyzed. Two bone samples previously received PRF and RCM (M37 and M58), the third sample represented a physiological bone formation without treatment (M37‐control). The bone sample M37 showed irregularly shaped islets of calcified material intermingled with connective tissue. The other samples, from the 58‐year‐old patient with PRF and RCM (M58); and the other untreated bone sample from the same 37‐year‐old patient (M37‐control) showed similar ultrastructural morphology with trabecular conformation without islets agglomerations. The inorganic composition analysis showed higher concentrations of calcium and phosphorus in both samples treated with PRF and RCM in comparison to the untreated bone sample. The Ca/P ratio was higher in the M37 sample compared to the others samples. The results showed morphology and inorganic composition differences among the treatments used, suggesting that this method is feasible to analyze parameters of the alveolar bone tissue.     

Mechanical properties of L929 cells measured by atomic force microscopy: Effects of anticytoskeletal drugs and membrane crosslinking

To shed light on the architecture of the cytoskeleton, we used the atomic force microscope (AFM) to measure the elasticity, viscoelasticity, and plasticity of L929 cells. The initial elastic response (Young's modulus ～ 4,000 Pa) of the cells to an applied force was followed by a slow compression of the cytoskeleton (τ_1/2 ≈ 10 s). When force application was terminated, the cytoskeleton underwent a sudden partial decompression and a subsequent slow, incomplete recovery. The role of the cytoskeletal elements in cell mechanics was accessed in AFM measurements carried out on cells treated with cytochalasin D, nocodazole, or col-cemid. Cytochalasin D treatment reduced both elasticity (～45%) and cytoplasmic viscosity (～65%), whereas cells treated with nocodazole or colcemid exhibited a marked increase in elasticity (～100%) and a slight increase in viscosity (～15%). The AFM force measurements also provided evidence that the cell membrane and the cytoskeleton are mechanically coupled. Tightly adherent cells were stiffer than cells that were loosely attached. Moreover, cells crosslinked with either glutaraldehyde, 3,3 ‘-dithiobis’sul-fosuccinimidylpropionate] (DTSSP), or Concanavalin A were more rigid than untreated cells. It is of interest that cells crosslinked with Concanavalin A, but not DTSSP, displayed plastic behaviors that may reflect the induction of cytoskeletal reorganization by Concanavalin A.     

Fractal compression and adaptive sampling: reducing the image acquisition time in scanning probe microscopy

In typical scanning probe microscope experiment a three‐dimensional image of a substrate is obtained. For a given scanning mechanism, the time needed to image an area depends mainly on the number of samples and the size of the image. The imaging speed is further compromised by drifts associated with the substrate and the piezoscanner. It is therefore desirable to improve the imaging speed with limited impact to the effective resolution of the resulting image. By utilizing an adaptive sampling scheme with fractal compression technique, we have demonstrated that the number of the required samples can be significantly reduced with minimal impact to the image quality. SCANNING 30: 463–473, 2008. © 2008 Wiley Periodicals, Inc.     

Effects of probe pollutants on morphological and mechanical measurements of muscle and collagen fibers using atomic force microscopy

Because of the interaction between probes and samples, pollutants in buffer solution or in the air would easily bind to probes and make the probe polluted, which might influence the morphological and mechanical measurements with atomic force microscopy. The polluted probes might transfer the pollutants onto the samples and thus change the surface ultrastructure of samples, or collect the deviated feedback signals to make the phantasm images. The former process is irreversible even if a new probe is employed, and the latter one is a reversible process as long as changing the used/polluted probe. Effects of polluted probes on morphological and mechanical characteristics of insect flight muscle and rat tail tendon collagen I fibers had been discussed in this study, in which, we constructed a series of methods to avoid/reduce the collecting of phantasm images and deviated mechanical information, such as changing the scanning direction and scanning force, replacing the new probes, or cleaning the polluted probes. SCANNING 32:113–121, 2010. © 2010 Wiley Periodicals, Inc.     

Comparison of Two Methods of Methyl Group Grafting to the Silica Thin Film Surface and Its Tribological Properties Measured by Atomic Force Microscopy

Two different methods are used to modify silica surfaces and the results of the different modification methods on the frictional properties are presented in this paper. Methyl groups were introduced to the silica film directly during the synthesis step with the use of triethoxymethylsilane or by post-synthesis grafting from solution using trimethylchlorosilane. Atomic Force Microscopy (AFM) was used to compare the frictional behaviour of samples after modifiying the silica surface using the two methods. It was found, that depending on the presence of methyl groups on the surface or in bulk, the frictional properties are strongly influenced not only by methyl groups but also by elasticity of final material.     

Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo‐scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin–eosin, Masson's trichrome (for total collagen), and picrosirius‐red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively. Microsc. Res. Tech. 77:183–188, 2014. © 2013 Wiley Periodicals, Inc.     

Effects of atopic dermatitis on the morphology and water content of scalp hair

The effects of atopic dermatitis (AD) on scalp hair properties, such as morphology and water content, were investigated using atomic force microscopy (AFM) and thermogravimetric analyzer. Hairs from lesional and nonlesional scalp regions of eight patients with AD were investigated. The severity of the disease, which was evaluated using the SCORing Atopic Dermatitis index, was 48.75 (range, 40-80). Hairs from 15 normal adults were also examined as controls. The surface images were taken in an area of 20 × 20 μm(2) with 512 × 512 pixels and a scan speed of 0.8 line/s. AD affected the cuticle structures and scales of scalp hair. The edges of cuticles were torn and collapsed, and the scales were very thick. The water contents of both types of AD hair were less than the control: 12% ± 0.7%, 11.7% ± 0.4%, and 13% ± 0.8% for lesional AD hair, nonlesional AD hair, and control hair, respectively. The scalp hair of patients with AD can be characterized by thick and globular scale patterns. The hair of patients with AD has less water content than normal hair showing a good agreement with the property of skin having AD.     

Characterization of the tip-loading force-dependent tunneling behavior in alkanethiol metal–molecule–metal junctions by conducting atomic force microscopy

We report on a tip-loading force-dependent tunneling behavior through alkanethiol self-assembled monolayers formed in metal–molecule–metal junctions, using conducting atomic force microscopy. The metal–molecule contacts were formed by placing a conductive tip in a stationary point contact on alkanethiol self-assembled monolayers under a controlled tip-loading force. Current–voltage characteristics in the alkanethiol junctions are simultaneously measured, while varying the loading forces. Tunneling current through the alkanethiol junctions increases and decay coefficient β_N decreases, respectively, with increasing tip-loading force, which results from enhanced intermolecular charge transfer in a tilted molecular configuration under the tip-loading effect.     

Error analysis and regression mode of the V‐grooved sample in the atomic force microscope simulation measurement mode by the molecular mechanics

Based on the molecular mechanics, this study uses the two‐body potential energy function to construct a trapezoidal cantilever nano‐scale simulation measurement model of contact mode atomic force microscopy (AFM) under the constant force mode to simulate the measurement the nano‐scale V‐grooved standard sample. We investigate the error of offset distance of the cross‐section profile when using the probes with different trapezoidal cantilever probe tip radii (9.5, 8.5, and 7.5 Å) to scan the peak of the V‐grooved standard sample being reduced to one‐tenth (1/10) of its size, and use the offset error to inversely find out the regression equation. We analyze how the tip apex as well as the profile of the tip edge oblique angle and the oblique edge angle affects the offset distance. Furthermore, a probe with a larger radius of 9.5 nm is used to simulate and measure the offset error of scan curve, and acquire the regression equation. By the conversion proportion coefficient of size (ω), and revising the size‐reduced regression equation during the small size scale, a revised regression equation of a larger size scale can be acquired. The error is then reduced, further enhancing the accuracy of the AFM scanning and measurement. SCANNING 31: 147–159, 2009. © 2009 Wiley Periodicals, Inc.     

Returning to the same area of hair surfaces before and after treatment: a longitudinal AFM technique

We report the use of longitudinal (aspect ratio > 1 : 1) scanning atomic force microscopy as an aid in returning to the same area of hair fibres after bleaching, treatment with a commercial shampoo or the application of a ‘leave‐on’ conditioner product. The bleaching treatment used in this study was not found to affect the cuticular architecture and lateral force microscopy (LFM) also showed little difference after treatment, reflecting the homogeneity of the newly revealed surfaces. After treatment with a commercial shampoo, the hair sample again showed very little difference in topography or lateral force characteristics. Hair treated with the leave‐on conditioner product also showed no major topographical changes. LFM traces, however, showed regions between the ghost edge, marking the original position of the scale edge before cuticular erosion, and the existing scale edge, to have higher frictional properties than distal regions of the cuticle. A thin film of the leave‐on product thus seems to form in this region and extends from the foot of the scale edge.     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.