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.     

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.     

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.     

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.     

Characterization of ultrastructure and collagen composition of the teratoma membrane: Comparison to the amniotic membrane

The structural and morphological properties of the teratoma membrane were investigated to better understand the pathogenesis of ovarian teratomas. A mature cystic teratoma and amnion were obtained from patients who underwent laparoscopic cystectomy and uncomplicated delivery, respectively. The teratoma membrane was divided into three layers according to the results of the histological analysis. Each layer showed distinct morphological properties, including an outer layer that was uniformly arranged, a middle layer with an irregular pattern of fibers, and an inner layer that was structurally dense with a wavy pattern of fibers. The morphology of the layers of the amniotic membrane was the reverse that of the teratoma membrane. In the teratoma membrane, the outer layer was primarily composed of type III collagen and the inner layer had a large amount of type III and IV collagen. The amniotic membrane showed a small amount of type III collagen in the outer layer, whereas the inner layer had large amounts of type I, III, and IV collagen. In the teratoma membrane, the collagen fibrils were arranged regularly in the outer layer, but irregularly in the inner layer. In the amniotic membrane, the arrangement of collagen fibrils was the reverse that of the teratoma membrane. Additionally, the collagen fibrils in the teratoma membrane were thinner than those of the amniotic membrane and had slightly shorter d‐spacing. Two membranes showed the differences in collagen fibril arrangement, which may caused by the different functional roles. Microsc. Res. Tech. 76:432–441, 2013. © 2013 Wiley Periodicals, Inc.     

A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides

We develop a method, which utilizes a combined atomic force microscope (AFM)/fluorescence microscope and small copy number polymerase chain reaction (PCR), to affinity-select individual aptamer species in a single cycle from a small pool of random-sequence oligonucleotides (oligos). In this method, a library of small beads, each of which is functionalized with fluorescent oligos of different sequences, is created. This library of oligo-functionalized beads is flowed over immobilized target molecules on a glass cover slip. High-affinity target-specific aptamers bind tightly to the target for prolonged periods and resist subsequent washes, resulting in a strong fluorescence signal on the substrate surface. This signal is observed from underneath the sample via fluorescence microscopy. The AFM tip, situated above the sample, is then directed to the coordinates of the fluorescence signal and is used to capture a three-dimensional high-resolution image of the surface-bound bead and to extract the bead (plus attached oligo). The extracted oligo is PCR-amplified, sequenced, and may then be subjected to further biochemical analysis. Here, we describe the underlying principles of this method, the required microscopy instrumentation, and the results of proof-of-principle experiments. In these experiments, we selected aptamers in eight trials from a binary pool containing a 1:1 mixture of thrombin aptamer oligo and a nonsense oligo. In each of the eight trials, the positive control aptamer was successfully detected, imaged, extracted, and characterized by PCR amplification and sequencing. In no case was the nonsense oligo selected, indicating good selectivity at this early stage of technology development.     

Evaluation of surface alterations in different retreatment nickel‐titanium files: AFM and SEM study

The aim of this study was to evaluate the surface changes of nickel titanium (Ni‐Ti) rotary retreatment files after three and five uses. Furthermore, the effects of 2% sodium hypochlorite and chloroform solutions and sterilization procedures on the NiTi rotary retreatment surfaces were investigated. ProTaper Retreatment files, R‐endo files, and Mtwo retreatment files were used for this study. The palatinal roots of maxillary molar teeth were obturated with gutta percha and Ah26. Retreatment procedures were performed with these retreatment file systems. The surface changes of untreated NiTi rotary files that were used three and five times, immersed in NaOCl and chloroform and subjected to sterilization procedures were investigated with atomic force microscopy (AFM) and scanning electron microscopy (SEM). The RMS values, three dimensional images and SEM images at various magnifications were obtained. RMS values showed that all three NiTi rotary retreatment file systems showed significant deteriorations after three and five uses. Cracks, damages and spiral construction deteriorations were detected in the SEM images after three and five uses. Furthermore, the Mtwo 15 file was broken off after five uses. AFM data indicated that 2% NaOCl caused significant surface deteriorations on NiTi rotary files and both AFM and SEM evaluation showed that chloroform solution and sterilization procedures did not cause significant surface deteriorations. In conclusion, ProTaper retreatment, R‐endo, and Mtwo retreatment files showed surface damages depending on retreatment procedures. Clinicians have to consider that retreatment files always have a tendency to break off after the third time they have been used. Microsc. Res. Tech. 78:356–362, 2015. © 2015 Wiley Periodicals, Inc.     

Imaging and Measuring the Molecular Force of Lymphoma Pathological Cells Using Atomic Force Microscopy

Atomic force microscopy (AFM) provides a new technology to visualize the cellular topography and quantify the molecular interactions at nanometer spatial resolution. In this work, AFM was used to image the cellular topography and measure the molecular force of pathological cells from B‐cell lymphoma patients. After the fluorescence staining, cancer cells were recognized by their special morphological features and then the detailed topography was visualized by AFM imaging. The AFM images showed that cancer cells were much rougher than healthy cells. CD20 is a surface marker of B cells and rituximab is a monoclonal antibody against CD20. To measure the CD20‐rituximab interaction forces, the polyethylene glycol (PEG) linker was used to link rituximab onto the AFM tip and the verification experiments of the functionalized probe indicated that rituximab molecules were successfully linked onto the AFM tip. The CD20‐rituximab interaction forces were measured on about 20 pathological cells and the force measurement results indicated the CD20‐rituximab binding forces were mainly in the range of 110–120 pN and 130–140 pN. These results can improve our understanding of the topography and molecular force of lymphoma pathological cells. SCANNING 35:40‐46, 2013. © 2012 Wiley Periodicals, Inc.     

Fabricating nanostructures through a combination of nano-oxidation and wet etching on silicon wafers with different surface conditions

This study investigates the surface conditions of silicon wafers with native oxide layers (NOL) or hydrogen passivated layers (HPL) and how they influence the processes of nano-oxidation and wet etching. We also explore the combination of nano-oxidation and wet etching processes to produce nanostructures. Experimental results reveal that the surface conditions of silicon wafers have a considerable impact on the results of nano-oxidation when combined with wet etching. The height and width of oxides on NOL samples exceeded the dimensions of oxides on HPL samples, and this difference became increasingly evident with an increase in applied bias voltage. The height of oxidized nanolines on the HPL sample increased after wet etching; however, the width of the lines increased only marginally. After wet etching, the height and width of oxides on the NOL were more than two times greater than those on the HPL. Increasing the applied bias voltage during nano-oxidation on NOL samples increased both the height and width of the oxides. After wet etching however, the increase in bias voltage appeared to have little effect on the height of oxidized nanolines, but the width of oxidized lines increased. This study also discovered that the use of higher applied bias voltages on NOL samples followed by wet etching results in nanostructures with a section profile closely resembling a curved surface. The use of this technique enabled researchers to create molds in the shape of a silicon nanolens array and an elegantly shaped nanoscale complex structures mold.     

Effect of the metallic modifier is nature on the surface microstructure of the phenolic carboplastic-steel interface

Atomic force microscopy and scanning electron microscopy are used to explore the effect of the nature of fine-grained metallic modifiers (babbit B-83, nickel, copper) on the friction-surface morphology and triboengineering characteristics of phenolic carboplastics. It is demonstrated that introduction of a modifier reduces the wear rate of the composite by 2–10 times, while the mechanism of wear of carboplastics is determined by the modifier is nature.     

Imaging of various surface properties of fluorescently labelled phospholipid Langmuir–Blodgett films with a combined scanning probe microscope

We present results of phase separation of a single-component system of 1,2-dihexadecanoyl- sn -glycero-3-phospho-[ N -(4-nitrobenz)-2-oxa-1,3-diazolyl]ethanolamine in which a liquid-condensed (LC) phase co-exists with a liquid-expanded (LE) phase. Domain formation in the co-existence region was studied using a newly developed combined scanning near-field optical microscope–atomic force microscope (SNOM–AFM). We demonstrate for the first time that the topographic, friction, fluorescence and surface potential distributions for a phase-separated single-component Langmuir–Blodgett film between the LE and LC phases can be simultaneously observed using the SNOM–AFM with a thin-step etched optical fibre probe.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

Effect of in‐office bleaching agents on the surface roughness and morphology of different dental composites: An AFM study

The aim of this study was to evaluate, using atomic force microscopy, the effect of two different bleaching agents on the modification of dental composites materials. This modification will be judged by analyzing the variation of surface roughness and surface morphology of two different composites: one containing nanoparticles and other consisting of microhybrid resin. The bleaching was performed by using two different concentrations of hydrogen peroxide: HP Blue (20% hydrogen peroxide) and Whiteness HP Maxx (35% hydrogen peroxide). Disks of Esthet X and Filtek? Z350 composites were used. Atomic force microscopy was used for analyses of the same place of the sample before and after treatment. A total of 12 analyses were performed per group (n = 12). The samples were analyzed qualitatively by evaluating morphological changes in the images and quantitatively by using roughness parameters (Ra). Data were analyzed statistically using Kruskal–Wallis, Mann–Whitney, and Friedman tests (P < 0.05). Changes were observed both qualitatively and quantitatively only in the groups where Esthet X resin was used. The use of hydrogen peroxide bleaching agents caused changes only in the surface of microhybrid composites, with no changes being observed in the composite containing nanoparticles. Despite being even significant, these alterations are clinically slight and can be eliminated by polishing them. Microsc. Res. Tech. 76:481–485, 2013. © 2013 Wiley Periodicals, Inc.     

Influence of scanning rate on quality of AFM image: Study of surface statistical metrics

The purpose of this work is to study the dependence of AFM‐data reliability on scanning rate. The three‐dimensional (3D) surface topography of the samples with different micro‐motifs is investigated. The analysis of surface metrics for estimation of artifacts from inappropriate scanning rate is presented. Fractal analysis was done by cube counting method and evaluation of statistical metrics was carrying out on the basis of AFM‐data. Combination of quantitate parameters is also presented in graphs for every measurement. The results indicate that the sensitivity to scanning rate growths with fractal dimension of the sample. This approach allows describing the distortion of the images against scanning rate and could be applied for dependences on the other measurement parameters. The article explains the relevance and comparison of fractal and statistical surface parameters for characterization of data distortion caused by inappropriate choice of scanning rate.