pH-induced changes in IgE molecules measured by atomic force microscopy.

The environment surrounding proteins is tightly linked to its dynamics, which can significantly influence the conformation of proteins. This study focused on the effect of pH conditions on the ultrastructure of Immunoglobulin E (IgE) molecules. Herein, the morphology, height, and area of IgE molecules incubated at different pH were imaged by atomic force microscopy (AFM), and the law of IgE changes induced by pH value was explored. The experiment results indicated that the morphology, height and area of IgE molecules are pH dependent and highly sensitive. In particular, IgE molecules were more likely to present small-sized ellipsoids under acidic conditions, while IgE molecules tend to aggregate into large-sized flower-like structures under alkaline conditions. In addition, it was found that the height of IgE first decreased and then increased with the increase of pH, while the area of IgE increased with the increase of pH. This work provides valuable information for further study of IgE, and the methodological approach used in this study is expected to developed into AFM to investigate the changes of IgE molecules mediated by other physical and chemical factors. RESEARCH HIGHLIGHTS: The ultrastructure of IgE molecules is pH dependent and highly sensitive. IgE molecules were tend to present small-sized ellipsoids under acidic pH. Alkaline pH drives IgE self-assembly into flower-like aggregates.

Repair effect of Centella asiatica (L.) extract on damaged HaCaT cells studied by atomic force microscopy.

People's choice of cosmetics is no longer just 'Follow the trend', but pays more attention to the ingredients of cosmetics, whether the ingredients of cosmetics are beneficial to people's skin health; therefore, more and more skin-healthy ingredients have been discovered and used in cosmetics. In this work, atomic force microscope (AFM) is used to provide physical information about biomolecules and living cells; it brings us a new method of high-precision physical measurement. Centella asiatica (L.) extract has the ability to promote skin wound healing, but its healing effect on damaged HaCaT cells needs to be investigated, which plays a key role in judging the effectiveness of skincare ingredients. The objective of this study was to explore the impact of Centella asiatica (L.) extract on ethanol-damaged human immortalised epidermal HaCaT cells based on AFM. We established a model of cellular damage and evaluated cell viability using the MTT assay. The physical changes of cell height, roughness, adhesion and Young's modulus were measured by AFM. The findings indicated that the Centella asiatica (L.) extract had a good repair effect on injured HaCaT cells, and the optimal concentration was 75 µg/mL.

Responses of INS-1 cells to glucose stimulation patterns.

Diabetes has become a major public health problem in the world for many years, and it is driving us to probe into its complex mechanism of insulin secretion in pancreatic ß cells. The nanoscale resolution characterization of pancreatic ß cells in response to glucose led to insights into diverse mechanical and functional processes at the single cell level. Recent advances allowed the direct observations of cytoskeleton dynamics which were quantitatively determined. Here, we firstly performed the glucose stimulation with multiple physiologically relevant glucose patterns. Atomic force microscopy (AFM) produced high spatial resolution mechanical images together with the insulin secretions linking the physical interactions to the biochemical process of INS-1 cells. Altered material properties of the INS-1 cells revealed the regulation of multiple glucose stimulation patterns. Rapidly responded to high glucose (HG), INS-1 cells presented the unique meshing networks of elasticities. The decreases of Young's modulus (YM) and insulin secretion suggested that mechanical changes affected the insulin release. Furthermore, the frequency and gradient of glucose patterns induced nanomechanical and secreting changes of the INS-1 cells and gained the knowledge on the potential controllability of glucose. The relationships between the cellular mechanics and insulin secretion of INS-1 cells could contribute to establish a mechanical cell model for the study of ß cells in diabetes. The results also indicated the cell mechanics as promising mechanical biomarkers for ß cells, and promoted the understanding of specific mechanical mechanism of glucose regulation, which lighted on the further application of functional glucose regulation in therapy.

Subsurface phase imaging of tapping-mode atomic force microscopy at phase resonance.

The phase image of tapping-mode atomic force microscopy (TM-AFM) contains energy dissipation, which is related to the sample information on the physical properties such as the sample Young's modulus, adhesion, surface morphology and subsurface morphology. When TM-AFM is used for sample measurement, the frequency near the first resonance peak of probe is usually selected to drive the probe vibration. When the probe vibration is driven by the frequency, the probe has a high amplitude sensitivity, but the phase sensitivity is relatively low. In this paper, the frequency at the probe phase resonance peak was selected for driving the probe vibration to measure the sample, which improved the image resolution. Phase imaging was performed on three uniform photoresist samples with different thicknesses and the same structure. When the scanning parameters were fixed and the probe setpoint value was changed alone, it was found that with the decrease of setpoint value the horizontal resolution of the phase subsurface image was decreased, and the depth sensitivity was increased first and then decreased. The result shows that TM-AFM working at the phase resonance peak can better realise the subsurface imaging of samples at different depths. Phase subsurface imaging at the resonance can be used to quantitatively obtain subsurface phase images of different depths.

Nanoscopic characterization of hepatocytes treated with normoxic and hypoxic tumor-derived exosomes.

Hypoxia is a key factor in tumor microenvironments. Tumor-derived exosomes under hypoxia have their functions of communication between local and remote cells, and play an important role in tumor growth and metastasis. However, the effect of tumor-derived exosomes on cell structures and functions under hypoxia is unknown. In this work, the effects of exosomes derived from hepatocellular carcinoma cells (HCC-LM3) under normoxia (N-exos) and hypoxia (H-exos) environments on the biological and physical properties of target cells were stduied. The N-exos promoted the proliferation of hepatocytes (HL-7702) at 1.5 mg/mL, while the H-exos promoted the proliferation of hepatocytes at a lower concentration (1.0 mg/mL). After the cells cultured with the same concentrations of N-exos and H-exos for different time periods, the cell migration was enhanced. The stress fibers of the cells became loose and the cytoskeleton was rearranged, which were time dependent. The changes in morphological and mechanical parameters of the HL-7702 cells were detected by atomic force microscopy (AFM). The results showed that the cell edges became irregular and the filopodia were increased versus the exosomes treatment time. The heights and elastic moduli of cells were reduced. Compared with N-exos, H-exos had a more significant effect on the biological and physical properties of target cells. The results provide a method for studying how tumor-derived exosomes affect the interaction between tumor cells and their hypoxic microenvironment.

Electrical characterization of tumor-derived exosomes by conductive atomic force microscopy.

The physical properties of tumor-derived exosomes have gained much attention because they are helpful to better understand the exosomes in biomedicine. In this study, the conductive atomic force microscopy (C-AFM) was employed to perform the electrical characterizations of exosomes, and it obtained the topography and current images of samples simultaneously. The exosomes were absorbed onto the mica substrates coated with a gold film of 20 nm thick for obtaining the current images of samples by C-AFM in air. The results showed that the single exosomes had the weak conductivity. Furthermore, the currents on exosomes were measured at different bias voltages and pH conditions. It illustrated that the conductivity of exosomes was affected by external factors such as bias voltages and solutions with different pH values. In addition, the electrical responses of low and high metastatic potential cell-derived exosomes were also compared under different voltages and pH conditions. This work is important for better understanding the physical properties of tumor-derived exosomes and promoting the clinical applications of tumor-derived exosomes.

Implementation of flexible virtual microchannels based on optically induced dielectrophoresis.

Micro-nano particle manipulation methods in liquid environments have been widely used in the fields such as medicine, biology and material science. Nevertheless, the methods usually rely on pre-prepared physical microfluidic channels. In this work, virtual electrodes based on the optically induced dielectrophoresis (ODEP) method were used as virtual microchannels instead of traditional physical microfluidic channels. Virtual microchannels with different shapes were implemented by the designs of projected light patterns, which made the virtual microchannels have great flexibility and controllability. The theory of ODEP was verified by simulation and analysis of electric field distributions. The relationship between the manipulation force and the alternating current (AC) voltage or the AC frequency exerted on the cells was assessed. The experimental results indicated that the manipulation force was increased with the increase of the AC voltage, and it was reduced with the increase of the AC frequency. Moreover, different virtual microchannels were designed to carry out the transportation, aggregation and sorting of yeast cells and rat basophilic leukemia cells (RBL-2H3 cells) and the survival rate of the cells was evaluated. This work shows that the virtual microchannels can be flexibly realized by ODEP in liquid environments.

Effect of trypsin concentration on living SMCC-7721 cells studied by atomic force microscopy.

Trypsin is playing an important role in the processes of cancer proliferation, invasion and metastasis which require the precise information of morphology and mechanical properties on the nano-scale for the related research. In this work, living human hepatoma (SMCC-7721) cells were treated with different concentrations of trypsin solution. The morphology and mechanical properties of the cells were measured via atomic force microscope (AFM). Statistical analyses of measurement data indicated that with the increase of trypsin concentration, the average cell height and the surface roughness were both increased, but the cell viability, the cell surface adhesion and the elasticity modulus were decreased significantly. The force required to puncture the cells was also gradually reduced. It indicates that trypsin not only hydrolyses the proteins between the cell and the substrate but also the membrane proteins. The results offer valuable clues for the cancerous process study, pathological analysis and trypsin inhibitor drug development. And this work provides an effective way for overcoming the cell membrane in drug injection for cell-targeted therapy.

High-reliability graphene-wrapped nanoprobes for scanning probe microscopy.

The nanoprobe is a powerful tool in scanning probe microscopy (SPM) that is used to explore various fields of nanoscience. However, the tips can wear out very fast due to the low stability of conventional probes, especially after the measurement of high currents or lateral friction, which results in image distortion and test imprecision. Herein, a novel functional nanoprobe is presented using graphene sheets in a high-quality graphene solution wrapped round a plasma-treated conventional Pt-Ir coated nanoprobe, which shows highly stability and resistance to degradation, leading to a significantly increased lifetime. Furthermore, we show that the graphene-wrapped nanoprobes have the advantages of enhanced electrical conductivity and reduced tip-sample friction, compared with Pt-Ir coated nanoprobes. The simplicity and low cost of this method make it valuable to various functional graphene-wrapped nanoprobes and applications.

Self-Assembly of DNA molecules in magnetic Fields.

In this work, a rich variety of self-assembled DNA patterns were obtained in the magnetic field. Herein, atomic force microscopy (AFM) was utilized to investigate the effects of the concentration of DNA solution, intensity and direction of magnetic field and modification of mica surface by different cations on the self-assembly of DNA molecules. It was found that owning to the change of the DNA concentration, even under the same magnetic field, the DNA self-assembly results were different. Thein situtest results showed that the DNA self-assembly in an magnetic field was more likely to occur in liquid phase than in gas phase. In addition, whether in a horizontal or vertical magnetic field, a single stretched dsDNA was obtained in a certain DNA concentration and magnetic field intensity. Besides, the modification of cations on the mica surface significantly increased the force between the DNA molecules and mica surface, and further changed the self-assembly of DNA molecules under the action of magnetic field.

Electrical conductivity measurement ofλDNA molecules by conductive atomic force microscopy.

Conductive atomic force microscopy (C-AFM) is a powerful tool used in the microelectronics analysis by applying a certain bias voltage between the conducting probe and the sample and obtaining the electrical information of sample. In this work, the surface morphological information and current images of the lambda DNA (λDNA) molecules with different distributions were obtained by C-AFM. The 1 and 10 ngµl-1DNA solutions were dripped onto mica sheets for making randomly distributed DNA and DNA network samples, and another 1 ngµl-1DNA sample was placed in a DC electric field with a voltage of 2 V before being dried for stretching the DNA sample. The results show that the current flowing through DNA networks was significantly higher than the stretched and random distribution of DNA in the experiment. TheI-Vcurve of DNA networks was obtained by changing the bias voltage of C-AFM from -9 to 9 V. The currents flowing through stretched DNA at different pH values were studied. When the pH was 7, the current was the smallest, and the current was gradually increased as the solution became acidic or alkaline.

The influence of different liquid environments on the atomic force microscopy detection of living bEnd.3 cells.

Atomic force microscopy (AFM) is one of the most important tools in the field of biomedical science, and it can be used to perform the high-resolution three-dimensional imaging of samples in liquid environments to obtain their physical properties (such as surface potentials and mechanical properties). The influence of the liquid environment on the image quality of the sample and the detection results cannot be ignored. In this work, quantitative imaging (QI) mode AFM imaging and mechanical detection were performed on mouse brain microvascular endothelial (bEnd.3) cells in different liquid environments. The gray-level variance product (SMD2) function was used to evaluate the imaging quality of the cells in liquids with different physical properties, and the variations in cell mechanical properties were quantitatively analyzed. An AFM detection liquid containing less ions and organics compared with the traditional culture medium, which is beneficial for improving the imaging quality, is introduced, and it shows similar mechanical detection results within 3 h. This can greatly reduce the detection costs and could have positive significance in the field of AFM living-cell detection.

Direct imaging of antigen-antibody binding by atomic force microscopy.

Direct observation of antigen-antibody binding at the nanoscale has always been a considerable challenging problem, and researchers have made tremendous efforts on it. In this study, the morphology of biotinylated antibody-specific Immunoglobulin E (IgE) immune complexes has been successfully imaged by atomic force microscopy (AFM) in the tapping-mode. The AFM images indicated that the individual immune complex was composed of an IgE and a biotinylated antibody. Excitingly, it is the first time that we have actually seen the IgE binding to biotinylated antibody. Alternatively, information on the length of IgE, biotinylated antibodies and biotinylated antibody-specific IgE immune complexes were also obtained, respectively. These results indicate the versatility of AFM technology in the identification of antigen-antibody binding. This work not only lays the basis for the direct imaging of the biotinylated antibody-IgE by AFM, but also offers valuable information for studying the targeted therapy and vaccine development in the future.

A study on the effects of tumor-derived exosomes on hepatoma cells and hepatocytes by atomic force microscopy.

Tumor-derived exosomes (exos) are closely related to the occurrence, development and treatment of tumors. However, it is not clear how the exosomes affect the physical properties, which lead to the deterioration of the target cells. In this paper, atomic force microscopy (AFM) was used to study the effects of exosomes in HCC-LM3 cells and other cells (SMMC-7721 and HL-7702). The results showed that the HCC-LM3-exos (the exosomes secreted by HCC-LM3 cells, 50 µg mL-1) significantly promoted the proliferation and migration of HCC-LM3 cells. HCC-LM3-exos also promoted the proliferation and migration of SMMC-7721 and HL-7702 cells at 1000 and 1500 µg mL-1, respectively. With an increase in time and concentration, the proliferation effect was more significant. On comparing the mechanical properties of the three types of cells (HCC-LM3, SMMC-7721 and HL-7702 cells), the degradation degree and migration ability of the cells were from high to low in the above order. In turn, the surface roughness of the cells decreased, and adhesion and elastic modulus increased. With an increase in treatment time, surface roughness increased, while adhesion and elastic modulus decreased. These suggested that the HCC-LM3-exos could change the mechanical properties of cells, leading to their deterioration, and enhance their migration and invasion ability. In this paper, the effects of exosomes were analyzed from the perspective of the physical parameters of cells, which provide a new idea to study cancer metastasis and prognosis.

Dynamic mechanics of HK-2 cell reaction to HG stimulation studied by atomic force microscopy.

Renal tubular cell injury by exposure to high glucose (HG) stimulation mainly accounts for diabetic nephropathy (DN). To understand the mechanism of injury by HG, quantitative characterization has commonly focused on the cells that are already impaired, which ignores the signals for the process of being injured. In this study, the architecture and morphology of HK-2 cells were observed dynamically by multiple imaging methods. AFM (atomic force microscopy)-based single-cell force spectroscopy was employed to investigate the dynamic mechanics quantitatively. The results showed that the Young's modulus increased continuously from 2.44 kPa up to 4.15 kPa for the whole period of injury by HG, while the surface adhesion decreased from 2.43 nN to 1.63 nN between 12 h and 72 h. In addition, the actin filaments of HK-2 cells exposed to HG depolymerized and then nucleated with increasing Young's modulus. The absence of cell pseudopodia coincided with the reduced cell adhesion, strongly suggesting close relationships between the cell architecture, morphology and mechanical properties. Furthermore, the stages of cell reactions were identified and assessed. Overall, the dynamic mechanics of the cells facilitate the identification of injured cells and the assessment of the degree of injury for accurate diagnoses and treatments.

Modeling and correction of image pixel hysteresis in atomic force microscopy.

In an atomic force microscope (AFM) system, the measurement accuracy in the scan images is determined by the displacement accuracy of piezo scanner. The hysteresis model of piezo scanner displacement is complex and difficult to correct, which is the main reason why the output displacement of the piezo scanner does not have high precision. In this study, an image pixel hysteresis model of the piezo scanner displacement in the AFM system was established. An AFM was used to scan a two-dimensional (2D) grating in the 0 ° and 90 ° directions and a polynomial fitting method was employed to obtain the image pixel hysteresis model parameters of the piezo scanner displacement in the X-direction and Y-direction. The image pixel hysteresis model was applied to correct the AFM scan image of regular octagons. The results showed that the relative measurement error in the X-direction was decreased from 12.47% to 0.52% after the correction and that in the Y-direction decreased from 28.57% to 0.35%. The image pixel hysteresis model can be applied in the post-processing software of a commercial AFM system. The model solves the hysteresis problem of the AFM system and improves the measuring accuracy of AFM in 2 degrees of freedom (2 DOF).

Linewidth study of pixelated aluminum nanowire gratings on polarization performance.

Nowadays, nanowire gratings are widely used in various applications such as imaging sensors and high-resolution microscopes. Structure parameters are the main factors that affect the optical performance of the gratings. This work aims to present the influence of the linewidth of pixelated aluminum nanowire gratings with a fixed period on the transmittance and extinction ratio in the visible region. By controlling the exposure doses of electron beam lithography (EBL), different linewidths of pixelated aluminum nanowire gratings with a period of 170 nm were fabricated. The significant effects of linewidth difference on the polarization performance were verified by the simulations of finite-difference time-domain (FDTD) software. The simulations were divided into two parts: the discussion of the pure aluminum without considering oxidation and the discussion of the surface aluminum being oxidized into the aluminum oxide. An optical system was built to evaluate the performance of the fabricated structures. The results show that the trends of the measurement results are consistent with that of simulation. This work will give a guide to the fabrication and evaluation of the nanowire gratings.

Anti-degranulation response of herbal formula in RBL-2H3 cells.

Allergic diseases not only bring serious economic burden to the patients, but also consume a lot of substantial resources of social medical systems. Thus, the prevention and treatment of allergic diseases are imperative. In this study, the anti-degranulation activity of herbal formula was evaluated using the rat basophil leukemia cells (RBL-2H3) as in vitro model. The morphological and biophysical properties of RBL-2H3 cells before and after treatment with herbal formula were also determined. Notably, the herbal formula exhibits clearly inhibited degranulation by RBL-2H3 cells in a concentration-dependent manner without cytotoxic effect. Therefore, this herbal formula can be used as an alternative and promising therapeutic agent to ameliorate allergic diseases.

Self-repair behaviour of the neuronal cell membrane by conductive atomic force indentation.

Conductive atomic force indentation (CAFI) was proposed to study the self-repair behaviour of the neuronal cell membrane here. CAFI was used to detect the changes of membrane potentials by performing the mechanical indentation on neurons with a conductive atomic force microscope. In the experiment, a special insulation treatment was made on the conductive probe, which turned out to be a conductive nanoelectrode, to implement the CAFI function. The mechanical properties of the neuronal cell membrane surface were tested and the membrane potential changes of neurons cultured in vitro were detected. The self-repair behaviour of the neuronal cell membrane after being punctured was investigated. The experiment results show that CAFI provides a new way for the study of self-repair behaviours of neuronal cell membranes and mechanical and electrical properties of living cells.

Controlled Self-Assembly of λ-DNA Networks with the Synergistic Effect of a DC Electric Field.

Large-scale and morphologically controlled self-assembled λ-DNA networks were successfully constructed by the synergistic effect of a DC electric field. The effect of DNA concentration, direction, and intensity of the electric field, even the modification of the mica surface using Mg2+ on the characteristics of the as-prepared DNA networks, were investigated in detail by atomic force microscopy (AFM). It was found that the horizontal electric field was more advantageous to the formation of DNA networks with more regular structures. At the same concentration, the height of DNA network was not affected significantly by the intensity change of the horizontal electric field. The modification of Mg2+ on mica surface increased the aggregation of DNA molecules, which contributed to the morphological change of the DNA networks. Furthermore, DNA molecules were obviously stretched in both horizontal and vertical electric fields at low DNA concentrations.