The morphological and biomechanical changes of keratocytes cultured on modified p (HEMA‐MMA) hydrogel studied by AFM

The poor integration with host cornea tissue and the low mechanical properties of pHEMA hydrogel for artificial cornea remains a difficult problem to solve. A modified pHEMA hydrogel, MMA copolymerized and type‐I collagen and bFGF immobilized, was previously prepared in an attempt to solve the problems. In this study, the cytotoxicity of Col/bFGF‐p (HEMA‐MMA) and p (HEMA‐MMA) was studied by cell adhesion assay and atomic force microscopy (AFM). The results of cell adhesion assay show that the attachment of keratocytes on the modified membrane is much higher than that of the unmodified membrane. This indicates that the material after modification have better cell–material interaction. The AFM images reveal that the morphology of keratocytes cultured on different substrate is obviously different. The cell cultured on modified membrane presented a completely elongated and spindle‐shape morphology. The force?distance indicates that the biomechanical of keratocytes changes significantly after culturing on different substrates. The adhesion force (2328±523 pN) and Young's modulus (0.51±0.125 kPa) of the cell cultured on modified membrane are much higher, and the stiffness (0.08±0.022 mN/m) is lower than those of the cell cultured on unmodified membrane. These results show that the cytotoxicity of Col/bFGF‐p (HEMA‐MMA) for keratocytes is much improved. SCANNING 31: 246–252, 2009. © 2010 Wiley Periodicals, Inc.     

Time‐series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy

Vesicles or caveolae within endothelial cells, fusing together to form vacuolar organelles, are implicated in macromolecular transport and cellular element transmigration across the blood–brain barrier (BBB) during inflammation and ischemia. Vacuolar organelles have been described by transmission electron microscopy and immunofluorescence, but the details of their dynamics have not been well addressed yet. Herein, by using tapping mode atomic force microscopy (AFM), we observed the time‐series changes of fused vesicles within the serum‐free cultured rat cerebral microvessel endothelial cells. The fused vesicles were certainly proved by fluorescent staining of Fm4‐64 combining simultaneous AFM imaging, as well as the field emission scanning electron microscopy technique. And energy dispersive spectrum results additionally implied that there may be specific structure and compositions around the vesicle region. These results indicate that increased vesicles in BBB may contribute to the formation of fused vesicles and a higher probability to construct the trans‐endothelial channel across endothelium layer. Furthermore, the AFM application may open up a new approach to investigate the details of transcellular process by fused vesicles. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Friction and wear effects on a micro/nano‐scale

In this paper are described tribological effects which can be found in micro‐tribological systems, and in those macro‐systems which can be analysed by micro‐methods, e.g., by atomic force microscopy (AFM) or related methods. Micro‐tribology systems have friction contacts with loads in the micro/nano‐newton range and/or dimensions in the micro/nanometre range. Experiments on the micro/nano‐scale should be easier to explain by theoretical modelling due to their simpler system structure. An example is discussed of adhesion and friction measurements between AFM tips and clean, flat, solid surfaces in ultra‐high vacuum, which shows some of the special aspects of micro/nano‐tribology. Surprising friction characteristics on surfaces with an artificial micro‐structure can be explained by skilled and careful topographical analysis of the friction path with an AFM. In micro‐sensor contacts, ‘single wear events’ can be detected using AFM analysis of the contact region. For ceramic compounds, different friction levels for the components of the material can be found. The problems, difficulties, and dangers of misinterpretation are also discussed.     

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit‐8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM‐based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8±37.9 pN, whereas control group had a higher adhesion force of 1064.2±97.0 pN. The Young's modulus decreased to nearly one‐third, from control group of 0.648±0.037 kPa to treated group of 0.254±0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231±0.084 mN/m to treated group of 1.917±0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti‐carcinogen efficacy against tumor cell. SCANNING 31: 83–89, 2009. © 2009 Wiley Periodicals, Inc.     

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐dielectric barrier discharge plasma

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro‐dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro‐DBD plasma for various exposure times. The elasticity of a single cell was determined by force–distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma‐treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro‐DBD plasma were related to cell mechanical changes.     

AFM studied the effect of celastrol on β1 integrin‐mediated HUVEC adhesion and migration

Integrin‐mediated human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix plays a fundamental role in tumor‐induced angiogenesis. Celastrol, a traditional Chinese medicine plant, has possessed anticancer and suppressed angiogenesis activities. Here, the mechanism underling the antiangiogenesis capacity of celastrol was investigated by exploring the effect of celastrol on β1(CD29) integrin‐mediated cell adhesion and migration. Flow cytometry results showed that the HUVECs highly expressed CD29 and cell adhesion assay indicated that celastrol specifically inhibited the adhesion of HUVECs to fibronectin (FN) without affecting nonspecific adhesion to poly‐L‐lysine (PLL). After cell FN adhesion being inhibited, the cell surface nanoscale structure and adhesion force were detected by atomic force microscope (AFM). High‐resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with celastrol. The membrane average roughness (Ra) and the major forces were decreased from 31.34 ± 4.56 nm, 519.60 ± 82.86 pN of 0 μg/ml celastrol to 18.47 ± 6.53 nm, 417.79 ± 53.35 pN of 4.0 μg/ml celastrol, 10.54 ± 2.85 nm, 258.95 ± 38.98 pN of 8.0 μg/ml celastrol, respectively. Accompanying with the decrease of adhesion force, the actin cytoskeleton in the cells was obviously disturbed by the celastrol. All of these changes influenced the migration of HUVECs from the wound‐healing migration assay. Taken together, our results suggest that celastrol can be as an inhibitor of HUVEC adhesion to FN. This work provides a novel approach to inhibition of tumor angiogenesis and tumor growth. SCANNING 35:316‐326, 2013. © 2012 Wiley Periodicals, Inc.     

Polysaccharide properties probed with atomic force microscopy

In recent years, polysaccharides have been extensively studied using atomic force microscopy (AFM). Owing to its high lateral and vertical resolutions and ability to measure interaction forces in liquids at pico‐ or nano‐Newton level, the AFM is an excellent tool for characterizing biopolymers. The first imaging studies showed the morphology of polysaccharides, but gradually more quantitative image analysis techniques were developed as the AFM grew easier to use in aqueous liquids and in non‐contact modes. Recently, AFM has been used to stretch polysaccharides and characterize their physicochemical properties by application of appropriate polymer stretching models, using a technique called single‐molecule force spectroscopy. From application of such models as the wormlike chain, freely jointed chain, extensible‐freely jointed chain, etc., properties such as the contour length, persistence length and segment elasticity or spring constant can be calculated for polysaccharides. The adhesion between polysaccharides and surfaces has been quantified with AFM, and this application is particularly useful for studying polysaccharides on microbial and other types of cells, because their adhesion is controlled by biopolymer characteristics. This review presents a synthesis of the theory and techniques currently in use to probe the physicochemical properties of polysaccharides with AFM.     

The comparison between force volume and peakforce quantitative nanomechanical mode of atomic force microscope in detecting cell's mechanical properties

Atomic force microscope (AFM) has been widely used in the biological field owing to its high sensitivity (subnanonewton), high spatial resolution (nanometer), and adaptability to physiological environments. Nowadays, force volume (FV) and peakforce quantitative nanomechanical (QNM) are two distinct modes of AFM used in biomechanical research. However, numerous studies have revealed an extremely confusing phenomenon that FV mode has a significant difference with QNM in determining the mechanical properties of the same samples. In this article, for the case of human benign prostatic hyperplasia cells (BPH) and two cancerous prostate cells with different grades of malignancy (PC3 and DU145), the differences were compared between FV and QNM modes in detecting mechanical properties. The results show measured Young's modulus of the same cells in FV mode was much lower than that obtained by QNM mode. Combining experimental results with working principles of two modes, it is indicated that surface adhesion is highly suspected to be a critical factor resulting in the measurement difference between two modes. To further confirm this conjecture, various weight ratios of polydimethylsiloxane (PDMS) were assessed by two modes, respectively. The results show that the difference of Young's modulus measured by two modes increases with the surface adhesion of PDMS, confirming that adhesion is one of the significant elements that lead to the measurement difference between FV and QNM modes.     

Comparisons of the topographic characteristics and electrical charge distributions among Babesia‐infected erythrocytes and extraerythrocytic merozoites using AFM

Tick‐borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite–host relationships. We hereby used atomic force microscopy (AFM) and frequency‐modulated Kelvin probe potential microscopy (FM‐KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM‐KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis‐infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi‐infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium‐infected erythrocytes, FM‐KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis‐infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.     

Nanoscale distribution of CD20 on B‐cell lymphoma tumour cells and its potential role in the clinical efficacy of rituximab

Rituximab is an exciting monoclonal antibody drug approved for treating B‐cell lymphomas and its target is the CD20 antigen which is expressed on the surface of B cells. In recent years, the variable efficacies of rituximab among different lymphoma patients have become an important clinical issue and urgently need to be solved for further development of antibodies with enhanced efficacies. In this work, atomic force microscopy (AFM) was used to investigate the nanoscale distribution of CD20 on the surface of tumour B cells from lymphoma patients to examine its potential role in the clinical therapeutic effects of rituximab. By performing ROR1 fluorescence labelling (ROR1 is a specific tumour cell surface marker) on the bone marrow cells prepared from B‐cell lymphoma patients, the tumour B cells were recognized, and then AFM tips carrying rituximabs via polyethylene glycol crosslinkers were moved to the tumour cells to probe the specific CD20‐rituximab interactions. By applying AFM single‐molecule force spectroscopy (SMFS) at the local areas (500×500 nm²) on the surface of tumour B cells, the nanoscale distributions of CD20 on the surface of tumour B cells were mapped, visually showing that CD20 distributed heterogeneously on the cell surface. Bone marrow cell samples from three clinical B‐cell lymphoma cases were collected to analyze the binding affinity and nanoscale distribution of CD20 on tumour cells. The experimental results showed that CD20 distribution on tumour cells were to some extent related to the clinical therapeutic outcomes while the CD20‐rituximab binding forces did not have distinct effects to the clinical outcomes. These results can provide novel insights in understanding the rituximab's clinical efficacies from the nanoscale distribution of CD20 on the tumour cells at single‐cell and single‐molecule levels.     

Measuring biomaterials mechanics with atomic force microscopy. 1. Influence of the loading rate and applied force (pyramidal tips)

Atomic force microscopy (AFM) is today an established tool in imaging and determination of mechanical properties of biomaterials. Due to their complex organization, those materials show intricate properties such as viscoelasticity. Therefore, one has to consider that the loading rate at which the sample is probed will lead to different mechanical response (properties). In this work, we studied the dependence of the mechanical properties of endothelial cells on the loading rate using AFM in force spectroscopy mode. We employed a sharp, four‐sided pyramidal indenter and loading rates ranging from 0.5 to 20 μm/s. In addition, by variation of the load (applied forces from 100 to 10,000 pN), the dependence of the cell properties on indentation depth could be determined. We then showed that the mechanical response of endothelial cells depends nonlinearly on the loading rate and follows a weak power‐law. In addition, regions of different viscous response at varying indentation depth could be determined. Based on the results we obtained, a general route map for AFM users for design of cell mechanics experiments was described.     

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro‐ and nanosized features of Ti surfaces in the short‐term response of primary human osteoblast‐like cells (HOC). Surfaces were prepared as ground (G‐Ti), hydrofluoric acid etched (HF‐Ti), and sandblasted/HF‐etched (SLA‐Ti), and analyzed using both three‐dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF‐Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA‐Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short‐term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short‐term response. SCANNING 34: 378‐386, 2012. © 2012 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.     

Study on water‐dispersible colloids in saline–alkali soils by atomic force microscopy and spectrometric methods

Recent studies have revealed that water‐dispersible colloids play an important role in the transport of nutrients and contaminants in soils. In this study, water‐dispersible colloids extracted from saline–alkali soils have been characterized by atomic force microscopy (AFM), X‐ray diffraction (XRD), X‐ray photoelectron spectroscopy (XPS), and UV absorption spectra. AFM observation indicated that the water‐dispersible colloids contain some large plates and many small spherical particles. XRD, XPS, and UV absorption measurement revealed that the water‐dispersible colloids are composed of kaolinite, illite, calcite, quartz and humic acid. In addition, UV absorption measurement demonstrated that the humic acids are associated with clay minerals. Water‐dispersible colloids in the saline–alkali soils after hydrolyzed polymaleic anhydride treatment and an agricultural soil (nonsaline–alkali soil) were also investigated for comparison. The obtained results implied that the saline–alkali condition facilitates the formation of a large quantity of colloids. The use of AFM combined with spectrometric methods in the present study provides new knowledge on the colloid characteristics of saline–alkali soils. Microsc. Res. Tech. 79:525–531, 2016. © 2016 Wiley Periodicals, Inc.     

Application of atomic force microscopy in bacterial research

The atomic force microscope (AFM) has evolved from an imaging device into a multifunctional and powerful toolkit for probing the nanostructures and surface components on the exterior of bacterial cells. Currently, the area of application spans a broad range of interesting fields from materials sciences, in which AFM has been used to deposit patterns of thiol‐functionalized molecules onto gold substrates, to biological sciences, in which AFM has been employed to study the undesirable bacterial adhesion to implants and catheters or the essential bacterial adhesion to contaminated soil or aquifers. The unique attribute of AFM is the ability to image bacterial surface features, to measure interaction forces of functionalized probes with these features, and to manipulate these features, for example, by measuring elongation forces under physiological conditions and at high lateral resolution (<1 Å). The first imaging studies showed the morphology of various biomolecules followed by rapid progress in visualizing whole bacterial cells. The AFM technique gradually developed into a lab‐on‐a‐tip allowing more quantitative analysis of bacterial samples in aqueous liquids and non‐contact modes. Recently, force spectroscopy modes, such as chemical force microscopy, single‐cell force spectroscopy, and single‐molecule force spectroscopy, have been used to map the spatial arrangement of chemical groups and electrical charges on bacterial surfaces, to measure cell–cell interactions, and to stretch biomolecules. In this review, we present the fascinating options offered by the rapid advances in AFM with emphasizes on bacterial research and provide a background for the exciting research articles to follow. SCANNING 32: 74–96, 2010. © 2010 Wiley Periodicals, Inc.     

Liposome impaired the adhesion and spreading of HEK293 cells: an AFM study

Gene transfer has been proven to be a promising approach for treatment of several diseases. The cytotoxicity of transfection reagents is one of the key factors for clinical applications. The cytotoxicity of liposome has been extensively studied. However, its effects on the adhesion and spreading of transformed cells are still unclear. In this study, the cytotoxic effects of liposome on cell viability and mitochondrial membrane potential of HEK293 cells were first evaluated. Then, an atomic force microscope (AFM) was recruited to investigate the effects of liposome on the adhesion and spreading of HEK293 cells. AFM data indicated that liposome induced a significant decrease in number of cellular pseudopodia and cell-surface particles, in cell-surface roughness, and in average adhesion force of cell membranes. The AFM data implied that liposome impaired the adhesion and spreading of HEK293 cells.     

The evaluation of surface topography changes in nanoscaled 2,6‐diphenyl anthracene thin films by atomic force microscopy

The physical properties of electronic devices made by 2,6‐diphenyl anthracene (DPA) are influenced by the microtexture of DPA surfaces. This work focused on the experimental investigation of the 3‐D surface microtexture of DPA thin films deposited on OTS (octadecyltrichlorosilane), HMDS (Hexamethyldisilasane), OTMS (octadecyltrimethoxysilane), and Si/SiO₂ (300 nm SiO₂ thickness) substrates with 5 and 50 nm thicknesses and 5 and 10 μm scan size. The thin film surfaces were recorded using atomic force microscopy (AFM) and their images were stereometrically analyzed to obtain statistical parameters, in accordance with ASME B46.1‐2009 and ISO 25178‐2: 2012. The results showed the effect of different manufacturing parameters on microtexture values where the granular structure is confirmed in all films. In addition, root mean square is increased by increasing the thickness from 5 to 50 nm for all types of substrates.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

Mechanical Property Investigation of Soft Materials by Cantilever‐Based Optical Interfacial Force Microscopy

Cantilever‐based optical interfacial force microscopy (COIFM) was applied to the investigation of the mechanical properties of soft materials to avoid the double‐spring effect and snap‐to‐contact problem associated with atomic force microscopy (AFM). When a force was measured as a function of distance between an oxidized silicon probe and the surface of a soft hydrocarbon film, it increases nonlinearly in the lower force region below ∼10 nN, following the Herzian model with the elastic modulus of ∼50 MPa. Above ∼10 nN, it increases linearly with a small oscillatory sawtooth pattern with amplitude 1–2 nN. The pattern suggests the possible existence of the layered structure within the film. When its internal part of the film was exposed to the probe, the force depends on the distance linearly with an adhesive force of −20 nN. This linear dependence suggests that the adhesive internal material behaved like a linear spring with a spring constant of ∼1 N/m. Constant‐force images taken in the repulsive and attractive contact regimes revealed additional features that were not observed in the images taken in the noncontact regime. At some locations, however, contrast inversions were observed between the two contact regimes while the average roughness remained constant. The result suggests that some embedded materials had spring constants different from those of the surrounding material. This study demonstrated that the COIFM is capable of imaging mechanical properties of local structures such as small impurities and domains at the nanometer scale, which is a formidable challenge with conventional AFM methods. SCANNING 35:59‐67, 2013. © 2012 Wiley Periodicals, Inc.     

A comparative study on the self‐assembly of an amyloid‐like peptide at water–solid interfaces and in bulk solutions

In the past years the self‐assembly of amyloid‐like peptides has attracted increasing attentions, because it is highly related to neurodegenerative diseases and has a potential for serving as nanomaterial to fabricate novel and useful nanostructures. In this paper, we focused on the role of interfacial conditions in the self‐assembly of an amyloid‐like peptide, termed Pep11. It was found that, when dissolved in bulk solutions, Pep11 formed into β‐sheet structures and assembled into long filaments in several hours, as revealed by Thioflavin T fluorescence and transmission electron microscopy (TEM) morphology characterization, respectively. When the peptide solution was added onto a mica/HOPG substrate, peptide filaments with three preferred orientations with an angle of 60° to each other were formed immediately, as imaged in situ by atomic force microscopy (AFM). However, the kinetics in filament formation and the morphologies of the formed beta sheet either on HOPG and mica or in bulk solutions were quite different. These results indicate that the interfacial properties dramatically affect the peptide self‐assembly process. Microsc. Res. Tech. 78:375–381, 2015. © 2015 Wiley Periodicals, Inc.