Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes.Furthermore,the principle of protein assembly gives guidelines for new biomimetic materials with potential appli-cations in medicine,energy,and nanotechnology.Atomic force microscopy(AFM)is a powerful tool for investigating protein assembly and interactions across spatial scales(single molecules to cells)and temporal scales(milliseconds to days).It has significantly contributed to understanding nanoscale architectures,inter-and intra-molecular interactions,and regulatory elements that determine protein structures,assemblies,and functions.This review describes recent advancements in elucidating protein assemblies with in situ AFM.We discuss the structures,diffusions,interac-tions,and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging,bimodal imaging,live cell imaging,and machine-learning-enhanced data analysis.These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.

Observation and characterization of asphalt microstructure by atomic force microscopy

The microstructure of asphalt is investigated by atomic force microscopy （AFM）. In order to analyze the impacts of asphalt types on microstructures, two neat asphalts with different penetration grades （50# and 70#） and one styrene-butadiene-styrene （SBS） modified asphalt are chosen. The influence of short-term aging is also studied. Based on the knowledge of asphalt＇s microproperties, the relationship between microstructures and healing property is analyzed. The results indicate that the microstructures of three asphalts are quite different and the effects of aging on the surface characteristics for different asphalts are also different. It is proposed that the bee structure is a type of wax crystal and it has a close relationship with the ＂bridge-healing＂ mechanism. The findings may reveal the formation mechanism of microstructure and the healing property for asphalts.

Interaction Study between DNA and Histone Proteins on Single-molecule Level using Atomic Force Microscopy

DNA and histone protein are important in the formation of nucleosomal arrays, which are the first packaging level of DNA into a more compact chromatin structure. To characterize the interactions of DNA and histone proteins, we reconstitute nucleosomes using lambda DNA and whole histone proteins by dialysis and perform direct atomic force microscopy （AFM） imaging. Compared with non-specific DNA and histone binding, nucleosomes are formed within the assembled “beads-on-a-string” nucleosomal array by dialysis. These observations facilitate the establishment of the molecular mechanisms of nucleosome and demonstrate the capability of AFM for protein-DNA interaction analysis.

AN ATOMIC FORCE MICROSCOPY STUDY ON THE AGGREGATION OF ISOTACTIC POLY(METHYL METHACRYLATE)

Aggregation process of isotactic poly（methyl methacrylate） （i-PMMA） has been studied extensively for many years, and considerable progress has been made in both experimental and theoretical studies. They are, however, seldom sustained by real-space observations of the underlying morphology. In this paper, the aggregation process of i-PMMA in concentrated acetone solutions and the fractal structure of the resulting three-dimensional clusters were characterized on the basis of real-space AFM observations of their two-dimensional projection. It was found that spherical multiple-chain particles formed upon collapse and aggregation of the involving chains as a whole during quenching the solution to room temperature. By keeping the solution at room temperature, the initially formed particles stick together upon contact to form larger particles through reassembling very slowly. The succeeding collision of the enlarged spherical particles leads to the formation of small clusters. These newly formed small clusters grow when they meet with other clusters or single Brownian particles. This leads to the formation of large clusters with fractal dimension of 1.95 ± 0.05, which suggest a reaction-limited cluster aggregation of i-PMMA in a concentrated acetone solution. This is in accordance with the conclusion obtained by light scattering measurements.

Analysis of the injection layer of PTCDA in OLEDs using x-ray photoemission spectroscopy and atomic force microscopy

Through the investigation of the sample surface and interface of 3, 4, 9, 10-perylenetetracarboxylic dianhydride （PTCDA）/indium-tin-oxide （ITO） thin films using atomic force microscopy, it has been found that the surface is complanate, the growth is uniform and the defects cover basically the surface of ITO. Furthermore, the number of pinholes is small. The analysis of the sample surface and interface further verifies this result by using x-ray photoemission spectroscopy. At the same time, PTCDA is found to have the ability of restraining the diffusion of chemical constituents from ITO to the hole transport layer, which is beneficial to the improvement of the performance and the useful lifetime of the organic light emitting diodes （OLEDs）.

Effect of Rho-kinase pathway on neurite outgrowth of rat hippocampal neurons under atomic force microscopy

Hippocampal neurons of neonatal rats were cultured in serum-free culture medium for 5 days in vitro, and treated with the Rho-kinase inducer lysophosphatidic acid. Atomic force microscopy revealed that the numbers of level-1, -2 and -3 neurites protruding from rat hippocampal neurons was significantly reduced. After treatment with the Rho kinase inhibitor Y27632, a significant increase in the numbers of these neurites was observed. Our experimental findings indicate that the Rho-kinase pathway is closely associated with the neurites of hippocampal neurons.

Effects of caveolin-1 on bone marrow mesenchymal stem cells differentiating into neuron-like cells An atomic force microscopy observation

Bone marrow mesenchymal stem cells （MSCs） from rats were transfected with Rn-siRNA-caveolin-1 and differentiated into neuron-like cells using fasudil hydrochloride. Membrane ultrastructural changes in MSCs were observed under atomic force microscopy. Caveolin-l-transfected rat MSCs exhibited weak nuclear refraction, dense caveolae and long finger-like cellular processes prior to fasudil hydrochloride treatment. MSCs differentiating into neuron-like cells exhibited weak nuclear refraction and large cellular processes without caveolae. We hypothesize that caveolin-1 plays an important role in the regulation of bone marrow MSC differentiating into neuron-like cells.

Characterizing silicon intercalated graphene grown epitaxially on Ir films by atomic force microscopy

An efficient method based on atomic force microscopy（AFM） has been developed to characterize silicon intercalated graphene grown on single crystalline Ir（111） thin films. By combining analyses of the phase image, force curves,and friction–force mapping, acquired by AFM, the locations and coverages of graphene and silicon oxide can be well distinguished. We can also demonstrate that silicon atoms have been successfully intercalated between graphene and the substrate. Our method gives an efficient and simple way to characterize graphene samples with interacted atoms and is very helpful for future applications of graphene-based devices in the modern microelectronic industry, where AFM is already widely used.

Application of atomic force microscopy in blood research

AIM: To find suitable solutions having lesser granules and keeping erythrocytes in normal shapes under atomic force microscopy (AFM). METHODS: Eight kinds of solutions, 1% formaldehyde, PBS buffer (pH7.2), citrate buffer (pH6.0), 0.9% NaCl, 5% dextrose, TAE, 1640 medium and 5% EDTA-K2, were selected from commonly used laboratory solutions, and venous blood from a healthy human volunteer was drawn and anticoagulated with EDTA-K2. Before scanned by AFM (NanoScopeIIIa SPM, Digital Instruments, Santa Barbara, CA), a kind of intermixture was deposited on freshly cleaved mica and then dried in the constant temperature cabinet (37 ℃). RESULTS: One percent formaldehyde, citrate buffer, 5% dextrose, TAE, were found to keep human erythrocytes in normal shape with few particles. Processed by these solutions, fine structures of human erythrocyte membrane were obtained. CONCLUSION: One percent formaldehyde, citrate buffer, 5% dextrose and TAE may be applied to dispose erythrocytes in AFM. The results may offer meaningful data for clinical diagnosis of blood by AFM.

Environmental temperature effect on dimensional measurements of atomic force microscopy

Atomic force microscopy(AFM)is increasingly being used as a fundamental tool for dimensional measurements at the nanoscale in the laboratory and in industry.Since the environmental temperature is not controlled in many measurements,or is even varied on purpose,quantification of its effects on AFM dimensional measurements is needed.In this paper,the influences of the temperature in the entire environment of the AFM(excluding only the controller and computer)and that in the local environment around the tip–sample are investigated.The results show that lateral dimensional measurements are affected mainly by the entire environmental temperature.However,vertical measurements are influenced by the temperature of both the entire environment and the local environment.The effects become significant for temperatures higher than some threshold,here between 35 and 40 XC.

Investigation of Adsorption of Xanthan Gum on Enamel by Tapping Mode Atomic Force Microscopy

By using tapping mode atomic force microscopy(TMAFM), a polymer layer was found on the enamel surface after the exposure to xanthan gum solutions. The layer thickness is closely related to the exposure time and the concentration of xanthan gum solution. The thickness data were evaluated by a Kruskal-Wallis test and Box-Whisker Plot at a 95% confidence level(p<0.05), and a statistically significant difference among the thickness data groups was demonstrated. After the exposure to 1000, 400, 100 mg/L xanthan gum solutions, the mean of layer thickness at the adsorption equilibrium is in the ranges of 103.5_122.06, 82.4_88.94 and 45.27_55.55 nm, respectively. This phenomenon suggests that the viscosity modifying agents in the beverage might be adsorbed on the enamel surface during consumption, which may form a barrier that can protect the enamel from being attacked by acid and therefore reduce dental erosion.

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM)

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined withtapping-mode scanning force microscopy under conditionsclose to their native environment. Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of ～ 15-20nm, height of ～ 2-3nm for eachindividual nucleosome) can be consistently observed. Furthermore, superbeads (width of ～ 40nm, height of ～ 7nm)are demonstrated. Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained eitherby using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (～ 50-60nm and ～90-110nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning thefolding pattern of interphase chromatin fibers.

A Study of the Probe Effect on the Apparent Image of Biological Atomic Force Microscopy

The probe effect on the apparent image of biological atomic force microscopy was explored in this study, and the potential of AFM in conformational study of gene related biological processes was illustrated by the specific nanostructural information of a new antitumor drug binding to DNA.

Effects of Environments on the Noise of Atomic Force Microscopy Cantilevers

Thermal vibration of the cantilevers is the main source of noise in atomic force microscopy(AFM).When immersed in liquids,the dynamic behavior of the cantilevers will be significantly affected by the environment.In this study,the effects of environments on the noise of AFM were investigated.The results show that the V⁃shaped cantilever exhibited very low noise in high vacuum.The noise in atmospheric environment was also satisfactory.In liquid environments,the noise dramatically increased.The systematic noise was low and dependent on the distance between the tip and substrate.As the velocity increased,the noise of V⁃shaped cantilever was increased in a similar trend.It is also found that the variation trend was more pronounced as the liquid viscosity increased.The noise of rectangular cantilever was lower than that of V⁃shaped cantilever in the same environment.The present study indicates that high vacuum is an ideal environment for single molecule force spectroscopy(SMFS)due to the lower noise and higher resolution.For a better signal⁃to⁃noise ratio,both the cantilever velocity and liquid viscosity should be as low as possible.These results will be helpful when a lower noise is desired in weak force detection.

Microstructure Morphologies of Asphalt Binders using Atomic Force Microscopy

We investigated microstructure morphologies of three asphalts（SK, Karamay, and Esso） used in China using atomic force microscopy（AFM）. The topography and phase contrast images were obtained. Topographic profile and three dimensional images were described. Roughnesses of microstructure were calculated. And the chemical compositions of asphalt were tested to explain the microstructural mechanism of the asphalt. The results show that the topography and phase image in atomic force microscopy are appropriate to evaluate the microstructure of the asphalt binder. There are significant differences in microstructural morphologies including bee-like structure, topographic profile, 3D image, and roughness for three asphalts in this study. There are three different phases in microstructure of asphalt binder. The oil source and chemical composition of asphalt, especially asphaltenes content have a great influence on the microstructure.

Theory of higher harmonics imaging in tapping-mode atomic force microscopy

The periodic impact force induced by tip-sample contact in a tapping mode atomic force microscope （AFM） gives rise to the non-harmonic response of a micro-cantilever. These non-harmonic signals contain the full characteristics of tip-sample interaction. A complete theoretical model describing the dynamical behaviour of tip-sample system was developed in this paper. An analytic formula was introduced to describe the relationship between time-varying tip-sample impact force and tip motion. The theoretical analysis and numerical results both show that the timevarying tip-sample impact force can be reconstructed by recording tip motion. This allows for the reconstruction of the characteristics of the tip-sample force, like contact time and maximum contact force. It can also explain the ability of AFM higher harmonics imaging in mapping stiffness and surface energy variations.

Morphologies of Eumelanins from the Ink of Six Cephalopods Species Measured by Atomic Force Microscopy

The morphologies of eumelanin, isolated from the six cephalopods species Sepia esculenta, Sepia lycidas, Sepia pharaonis, Sepiella japonica, Euprymna berryi, and Uroteuthis(Photololigo) edulis, were investigated using atomic force microscopy(AFM). The results showed that the hierarchical aggregate structures of irregular spherical particles with different diameters are the common characteristics of these eumelanins. Furthermore, the diameters of these spherical particles present an uneven distribution in a wide range and mainly concentrate in the range of about 20-150 nm. In addition, the eumelanin from different cephalopods species show obvious differences in the morphologies, which is illustrated by different assembly forms of diverse aggregate units and the quantitative features of eumelanin particles derived from the images.

Improvement of the high-k/Ge interface thermal stability using an in-situ ozone treatment characterized by conductive atomic force microscopy

In this work, an in-situ ozone treatment is carried out to improve the interface thermal stability of HfO_2/Al_2O_3 gate stack on germanium（Ge） substrate. The micrometer scale level of HfO_2/Al_2O_3 gate stack on Ge is studied using conductive atomic force microscopy（AFM） with a conductive tip. The initial results indicate that comparing with a non insitu ozone treated sample, the interface thermal stability of the sample with an in-situ ozone treatment can be substantially improved after annealing. As a result, void-free surface, low conductive spots, low leakage current density, and relative high breakdown voltage high-κ/Ge are obtained. A detailed analysis is performed to confirm the origins of the changes.All results indicate that in-situ ozone treatment is a promising method to improve the interface properties of Ge-based three-dimensional（3D） devices in future technology nodes.

Atomic Force Microscopy Measurement in the Lignosulfonate/Inorganic Silica System:From Dispersion Mechanism Study to Product Design

Designing and preparing high-performance lignin-based dispersants are crucial steps in realizing the value-added utilization of lignin on an industrial scale.Such process depends heavily on an understanding of the dispersion mechanism of lignin-based dispersants.Here,atomic force microscopy(AFM)is employed to quantitatively investigate the dispersion mechanism of a lignosulfonate/silica(LS/SiO_(2))system under different pH conditions.The results show that the repulsive force between SiO_(2)particles in LS solution is stronger than it is in water,resulting in better dispersion stability.The Derjaguin–Landau–Verwey–Overbeek(DLVO)formula as well as the DLVO formula combined with steric repulsion is utilized for the fitting of the AFM force/distance(F/D)curves between the SiO_(2)probe and substrate in water and in LS solution.Based on these fitting results,electrostatic and steric repulsive forces are respectively calculated,yielding further evidence that LS provides strong steric repulsion between SiO_(2)particles.Further studies indicate that the adsorbance of LS on SiO_(2)(Q),the normalized interaction constant(A),and the characteristic length(L)are the three critical factors affecting steric repulsion in the LS/SiO_(2)system.Based on the above conclusions,a novel quaternized grafted-sulfonation lignin(QAGSL)dispersant is designed and prepared.The QAGSL dispersant exhibits good dispersing performance for SiO_(2)and real cement particles.This work provides a fundamental and quantitative understanding of the dispersion mechanism in the LS/inorganic particle system and provides important guidance for the development of high-performance lignin-based dispersants.

Adaptive semi-empirical model for non-contact atomic force microscopy

Non-contact atomic force microscope is a powerful tool to investigate the surface topography with atomic resolution.Here we propose a new approach to estimate the interaction between its tips and samples,which combines a semi-empirical model with density functional theory(DFT)calculations.The generated frequency shift images are consistent with the experiment for mapping organic molecules using CuCO,Cu,CuCl,and CuO_(x)tips.This approach achieves accuracy close to DFT calculation with much lower computational cost.