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.

Progress on mechanical and tribological characterization of 2D materials by AFM force spectroscopy

Two-dimensional(2D)materials are potential candidates for electronic devices due to their unique structures and exceptional physical properties,making them a focal point in nanotechnology research.Accurate assessment of the mechanical and tribological properties of 2D materials is imperative to fully exploit their potential across diverse applications.However,their nanoscale thickness and planar nature pose significant challenges in testing and characterizing their mechanical properties.Among the in situ characterization techniques,atomic force microscopy(AFM)has gained widespread applications in exploring the mechanical behaviour of nanomaterials,because of the easy measurement capability of nano force and displacement from the AFM tips.Specifically,AFM-based force spectroscopy is a common approach for studying the mechanical and tribological properties of 2D materials.This review comprehensively details the methods based on normal force spectroscopy,which are utilized to test and characterize the elastic and fracture properties,adhesion,and fatigue of 2D materials.Additionally,the methods using lateral force spectroscopy can characterize the interfacial properties of 2D materials,including surface friction of 2D materials,shear behaviour of interlayers as well as nanoflake-substrate interfaces.The influence of various factors,such as testing methods,external environments,and the properties of test samples,on the measured mechanical properties is also addressed.In the end,the current challenges and issues in AFM-based measurements of mechanical and tribological properties of 2D materials are discussed,which identifies the trend in the combination of multiple methods concerning the future development of the in situ testing techniques.

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.

IMPROVED FABRICATION METHOD FOR CARBON NANOTUBE PROBE OF ATOMIC FORCE MICROSCOPY(AFM)

An improved arc discharge method is developed to fabricate carbon nanotube probe of atomic force microscopy （AFM） here. First, silicon probe and carbon nanotube are manipulated under an optical microscope by two high precision microtranslators. When silicon probe and carbon nanotube are very close, several tens voltage is applied between them. And carbon nanotube is divided and attached to the end of silicon probe, which mainly due to the arc welding function. Comparing with the arc discharge method before, the new method here needs no coat silicon probe with metal film in advance, which can greatly reduce the fabrication＇s difficulty. The fabricated carbon nanotube probe shows good property of higher aspect ratio and can more accurately reflect the true topography of silicon grating than silicon probe. Under the same image drive force, carbon nanotube probe had less indentation depth on soft triblock copolymer sample than silicon probe. This showed that carbon nanotube probe has lower spring constant and less damage to the scan sample than silicon probe.

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.

基于光偏转原理的AFM光电检测系统设计

利用光偏转原理设计并实现了原子力显微镜(AFM)的光电检测系统,研究了影响光偏转噪声的影响因素。设计了新型螺旋式嵌套结构调节准直光斑,使激光发散角降低到0.053°,利用压电陶瓷特性设计了高精度三维调节结构,可以在α,β,γ方向精准调节偏转角度,36mm的聚焦焦距满足了小型化、集成化的设计目标。基于该高灵敏度光电检测系统的设计,AFM系统得到了清晰的硅材料表面台阶结构,系统分辨率达到了0.1nm,为今后的亚原子测量奠定了实验基础。

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.

基于大高宽比CD-AFM探针的设计与制备

针对传统原子力显微镜(AFM)探针和关键尺寸原子力显微镜(CD-AFM)探针受限于针尖有效扫描高度较低,无法对深沟槽和大悬垂侧壁结构进行精准扫描成像的问题,提出了一种大高宽比针尖结构的新型CDAFM探针设计与制备方案。开发的新型CD-AFM探针针尖有效高度为5.1~5.8μm,高宽比达到14,相较于传统硅基CD-AFM探针,其有效高度提升了约4倍。利用开发的探针完成了标称深度为2.3μm、深宽比为4.6的深沟槽样品测试。

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.

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.

Quantitative measurement of local elasticity of SiO_x film by atomic force acoustic microscopy

In this paper the elastic properties of SiOx film are investigated quantitatively for local fixed point and qualitatively for overall area by atomic force acoustic microscopy （AFAM） in which the sample is vibrated at the ultrasonic frequency while the sample surface is touched and scanned with the tip contacting the sample respectively for fixed point and continuous measurements. The SiOx films on the silicon wafers are prepared by the plasma enhanced chemical vapour deposition （PECVD）, The local contact stiffness of the tip-SiOx film is calculated from the contact resonance spectrum measured with the atomic force acoustic microscopy. Using the reference approach, indentation modulus of SiOx film for fixed point is obtained. The images of cantilever amplitude are also visualized and analysed when the SiOx surface is excited at a fixed frequency. The results show that the acoustic amplitude images can reflect the elastic properties of the sample.

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.

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）.

Single DNA molecular manipulation with atomic force microscopy

Nanomanipulation of DNA molecules or other biomolecules to form artificial patterns or structures at nanometer scale has potential applications in the construction of molecular devices in future industries. It may also lead to new insights into the interesting properties and behavior of this fantastic nature-selected molecule at the sin- gle-molecular level. Here we present a special method based on the combination of macroscopic “molecular comb- ing” and microscopic “molecular cutting” to manipulate DNA molecules and form complex patterns at nanometer scale on solid surfaces. A possible strategy for ordered DNA sequencing based on this nanomanipulation technique has also been proposed.

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.

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.

The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomicforce microscopy

The structure of the nuclosome core particle of chromatin in chicken erythrocytes has been examined by usingAFM. The 146 hp of DNA wrapped twice around the corehistone octamer are clearly visualized. Both the ends ofentry/exit of linker DNA are also demonstrated. The dimension of the nucleosome core particles is- 1-4 um inheight and ～13-22 um in width. In addition, superbeads(width of - 48-57 urn, beight of-2-3 nm) are occasionallyrevealed, two turns of DNA around the core particles arealso detected.

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.

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.

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.