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1.
Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti‐RSV drugs/therapeutics, it is indispensable to study the RSV–host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ~150–200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti‐RSV therapies.  相似文献   

2.
Atomic force microscopy enables the simultaneous acquisition of high-resolution topographical and biophysical data allowing integrated analysis of cell surfaces during development and pathogenesis, and, critically, can link molecular and biophysical events. Here we used atomic force microscopy to analyse endometrial epithelial cells and neuronally differentiated P19 cells. Optimized reproducible sample preparation techniques enabled micro- and nanoscale multi-parameter analysis. Comparative analysis using atomic force microscopy and scanning electron microscopy demonstrated the utility of atomic force microscopy for examining tissue morphology, and its ability to generate data allowing differentiation of cells from different origins to be monitored. At low resolution atomic force microscopy produced topographic data complementary to scanning electron microscopy images, whilst at high resolution atomic force microscopy captured novel cell surface structural detail for both epithelial and neuronal cell types. Analysis of surface roughness provided biophysical data which enabled qualitative and quantitative differences between samples to be measured. This study provides an important optimization of sample preparation enabling more generalized atomic force microscopy utilization for cellular analysis required for advanced cell surface morphological studies.  相似文献   

3.
Retroviruses are membrane‐enveloped, RNA‐containing viruses that produce a wide range of threatening diseases in higher animals. Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents. We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces. The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means. The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions. Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLVand HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins. Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold‐labeled antibodies directed against the envelope protein of MuLV. The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population. Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus‐promoted diseases.  相似文献   

4.
One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus—HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine–phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.  相似文献   

5.
Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.  相似文献   

6.
A new male contraceptive given the name RISUG® (an acronym for Reversible Inhibition of Sperm Under Guidance) has been developed by our research group. RISUG® is a bioactive polymer and is injected into the lumen of the vas deferens using a no-scalpel approach. The polyelectrolytic nature of this contraceptive induces a surface charge imbalance on sperm membrane system leading to its destabilization. Complete disintegration of the plasma membrane with subsequent rupture and dispersion of the acrosomal contents is observed on RISUG® treatment. In the present study, micro-structural properties of human spermatozoa exposed to RISUG® in vitro have been quantitatively analysed using atomic force microscopy. The parameters used to quantify these morphological changes include amplitude (peak–valley height difference, arithmetic roughness, root mean square roughness) and spatial roughness. Factor loadings (Varimax rotation) have been used to determine the parameters displaying maximum variation. Further, sperm cells have been classified in various principal-component planes using principal-component analysis. The periodic structural features of the atomic force microscopy images have also been obtained using power spectral analysis.  相似文献   

7.
Semliki Forest virus (SFV), an alphavirus, is a single-stranded positive-sense RNA virus. The RNA genome is surrounded by a protein shell known as the capsid which itself is surrounded by a lipid envelope of host cell origin. In this study, SFV strain L10 enveloped virus and its capsid were immobilised onto silicon wafer supports which had been pre-coated with a monolayer of the relevant anti-viral antibody. After drying, the samples were imaged in air, using non-contact mode atomic force microscopy (AFM). Quantification of the AFM images has revealed that both the strain L10 enveloped virus and capsid collapse when immobilised in this manner. The capsid undergoes more significant collapse compared to the enveloped virus. The dimensions of the immobilised enveloped virus and capsid have been compared to a model where the free spherical particles collapse into ellipsoids during immobilisation. For the immobilised capsid the dimensions are consistent with this model whereas for the enveloped virus the model is less effective. The dimensions of the enveloped virus appear to be affected by the antibody used for immobilisation.  相似文献   

8.
The aim of this study is to evaluate the effect of whitening toothpaste on the surface roughness of resin-based restorative materials by different measurement methods. Twenty four specimens from each of human enamel, a microhybrid composite and two nanohybrid composites discs (8.0 diameter × 4.0 mm thick) were divided into two groups (n = 12) according to toothbrushing solutıon and subjected to simulation toothbrushing (30,000 cycles) with both distilled water and whitening toothpaste containing blue covarine. Surface roughness was examined using atomic force microscopy (AFM), profilometer, and scanning electron microscopy (SEM), and the data obtained were subjected to analysis. Ra values of Tescera (TES) were significantly higher than Sonicfill 2 (SF2) when brushing both toothbrushing solutions for initial or 30,000 cycles. Roughness increased for SF2 and TES when brushed for 30,000 cycles and was higher than enamel and Herculite XRV Ultra (HXU). Human enamel was obtained lower surface roughness values brushed with toothpaste compared with distilled water. Evaluation of the surface roughness of control groups using the AFM revealed no statistically significant difference between the groups, but significant differences were found using a profilometer. The use of abrasive whitening toothpaste containing blue covarine and the number of brushing cycles affect the surface properties of human enamel and the restorative material, and also, the clinical success of the restoration. Toothbrushing for 30,000 cycles increased the surface roughness of all materials. The type of toothbrushing solution partially has affected surface roughness.  相似文献   

9.
Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. The complementarity of the microscopy methods, scanning electron microscopy, electron probe microanalysis and atomic force microscopy assessed and correlated form and function of the surface modifications. Stainless steel disks (1 cm in diameter) were laser‐cut from the same sheets of stainless steel and treated by electropolishing or left untreated for controls. Each treatment was analysed separately using each technique. First, the disks were examined by visual inspection and electron probe microanalysis for surface characteristics and elemental composition, respectively. Aliquots of bacterial suspensions (saline rinses of poultry carcasses from a commercial broiler processing plant) were then diluted in broth and monitored for growth by spectrophotometry. Stainless steel disks (1 cm in diameter) were added and the cultures were grown to sufficient density to allow attachment of bacterial cells to test surfaces. Relative differences in the surface morphology shown by atomic force microscopy, including Z ranges, roughness and other measurements, corresponded by treatment with the differences in reduction of bacterial counts shown by scanning electron microscopy. A model of wet‐processing conditions tested the effects of corrosive treatment of surfaces. Less bacterial attachment occurred after corrosive treatment on controls and electropolished samples. Electropolishing significantly reduced bacterial numbers and the effects of corrosive action compared to the controls. Thus, the multiple imaging techniques showed that engineered changes on stainless steel surfaces improved the resistance of the surface finish to bacterial attachment, biofilm formation, and corrosive action.  相似文献   

10.
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.  相似文献   

11.
Scanning and transmission electron microscopy were used to analyse the ultrastructure of peritoneal mouse macrophage cells infected with Brazilian flavivirus (yellow fever, Rocio, Bussuquara and Saint Louis encephalitis viruses). Macrophage cells collected 3 days after viral infection had a flattened shape, with an increased number of large spikes of cytoplasm prolongations, giving an appearance of hairy cells. Cytopathological changes to the macrophage cells were similar regardless of the infecting flavivirus. Rough and smooth endoplasmic reticulum of the macrophage cells infected with flavivirus were abundant, hypertrophic and enlarged. A large number of free ribosomes were seen in the cytoplasm of these infected cells. Spherical particles approximately 50–70 nm in diameter, some of which were empty, were observed in the cytoplasm, generally inside vesicles. These particles probably correspond to viral particles.  相似文献   

12.
目的:探讨原子力显微镜(AFM)在研究人脐静脉内皮细胞(ECV304)表面形貌、超微结构及纳米机械性质等方面的应用,讨论ECV304超微结构和机械性质与其功能的关系。方法:利用AFM对ECV304细胞的表面形貌及生物机械性质进行表征与测量。结果:在AFM下观察到用普通光学显微镜难以观察到的ECV304细胞的独特的形态结构,如细胞骨架、伪足及细胞边缘微丝等。ECV304细胞呈现长梭形、多角形、圆形等多种形态,细胞表面平均粗糙度为320.52±75.98 nm,表面均匀分布微绒毛,细胞周围有铺展的圆盘状物质。力曲线定量分析得出针尖与细胞表面的非特异性粘附力为75±14 pN。结论:通过AFM成像和力曲线测量表明,ECV304细胞呈圆形,多角形,梭形等多种形态,针尖与细胞膜表面问的粘附力比较小,约75±14pN。  相似文献   

13.
Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules.  相似文献   

14.
Retroviruses are membrane-enveloped, RNA-containing viruses that produce a wide range of threatening diseases in higher animals. Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents. We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces. The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means. The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions. Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLV and HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins. Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold-labeled antibodies directed against the envelope protein of MuLV. The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population. Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus-promoted diseases.  相似文献   

15.
Antitumor immunotherapies, as a prospective approach for local cancer treatment, are attracting increasing interests. To detect the reacting course of immune and tumor cells, we have observed the process of K562 cells (a human erythroleukemic cell line) coculturing with peripheral lymphocytes, and the morphological and ultrastructural alterations of K562 cells and lymphocytes were investigated as well using atomic force microscopy (AFM). AFM morphological imaging revealed that after coculture the apoptosis‐like structures such as blebbing, pores, and apoptotic bodies were observed on the K562 cells. Also, the cell‐surface roughness decreased significantly, which implied the changes in chemical composition of cell membranes. Moreover, the lymphocytes were damaged to some extent induced by the coculture. The data demonstrated that K562 cells could be attacked and induced apoptosis by lymphocytes, and they would make damages to lymphocytes to escape the surveillance of immune system. SCANNING 35:7‐11, 2013. © 2012 Wiley Periodicals, Inc.  相似文献   

16.
ZnO films were deposited onto glass, ITO coated glass, and sapphire substrate by spray pyrolysis, and subsequently annealed at the same temperature of 400°C for 3 h. The role of substrate on the properties of ZnO films was investigated. The structural and optical properties of the films were investigated by X‐ray diffractometer (XRD) and photoluminescence (PL) spectrophotometer, respectively. The surface morphology of the nanostructured ZnO film was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Crystallographic properties revealed that the ZnO films deposited on sapphire and ITO substrates exhibit a strong c‐axis orientation of grains with hexagonal wurtzite structure. Extremely high UV emission intensity was determined in the film on ITO. The different luminescence behaviors was discussed, which would be caused by least value of strain in the film. Films grown on different substrates revealed differences in the morphology. ZnO films on ITO and sapphire substrates revealed better morphology than that of the film on glass. AFM images of the films prepared on ITO show uniform distribution of grains with large surface roughness, suitable for application in dye sensitized solar cells. Microsc. Res. Tech. 77:211–215, 2014. © 2013 Wiley Periodicals, Inc.  相似文献   

17.
The structure and surface chemistry of soil particles has extensive impact on many bulk scale properties and processes of soil systems and consequently the environments that they support. There are a number of physiochemical mechanisms that operate at the nanoscale which affect the soil's capability to maintain native vegetation and crops; this includes soil hydrophobicity and the soil's capacity to hold water and nutrients. The present study used atomic force microscopy in a novel approach to provide unique insight into the nanoscale properties of natural soil particles that control the physiochemical interaction of material within the soil column. There have been few atomic force microscopy studies of soil, perhaps a reflection of the heterogeneous nature of the system. The present study adopted an imaging and force measurement research strategy that accounted for the heterogeneity and used model systems to aid interpretation. The surface roughness of natural soil particles increased with depth in the soil column a consequence of the attachment of organic material within the crevices of the soil particles. The roughness root mean square calculated from ten 25 microm(2) images for five different soil particles from a Netherlands soil was 53.0 nm, 68.0 nm, 92.2 nm and 106.4 nm for the respective soil depths of 0-10 cm, 10-20 cm, 20-30 cm and 30-40 cm. A novel analysis method of atomic force microscopy phase images based on phase angle distribution across a surface was used to interpret the nanoscale distribution of organic material attached to natural and model soil particles. Phase angle distributions obtained from phase images of model surfaces were found to be bimodal, indicating multiple layers of material, which changed with the concentration of adsorbed humic acid. Phase angle distributions obtained from phase images of natural soil particles indicated a trend of decreasing surface coverage with increasing depth in the soil column. This was consistent with previous macroscopic determination of the proportions of organic material chemically extracted from bulk samples of the soils from which specimen particles were drawn. Interaction forces were measured between atomic force microscopy cantilever tips (Si(3)N(4)) and natural soil and model surfaces. Adhesion forces at humic acid free specimen surfaces (Av. 20.0 nN), which are primarily hydrophilic and whose interactions are subject to a significant contribution from the capillary forces, were found to be larger than those of specimen surfaces with adsorbed humic acid (Av. 6.5 nN). This suggests that adsorbed humic acid increased surface hydrophobicity. The magnitude and distribution of adhesion forces between atomic force microscopy tips and the natural particle surfaces was affected by both local surface roughness and the presence of adsorbed organic material. The present study has correlated nanoscale measurements with established macroscale methods of soil study. Thus, the research demonstrates that atomic force microscopy is an important addition to soil science that permits a multiscale analysis of the multifactorial phenomena of soil hydrophobicity and wetting.  相似文献   

18.
本研究利用原子力显微技术(AFM)观察原代培养的神经胶质细胞及其相互间的纳米连接结构。选择生长良好的神经胶质细胞用戊二醛固定30分钟,固定于AFM基底上进行扫描成像,用AFM脱机软件(SPM OFFLINE 2.20)进行检测。观察到胶质细胞平铺于培养皿的底部,胞体形状不规则,表面较扁平。突起丰富,但没有极性,无轴突树突之分,还观察到两胶质细胞间存在长程纤维管状连接结构。  相似文献   

19.
The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.  相似文献   

20.
This study was undertaken to assess in vivo the corrosion in two commercial nickel–titanium (NiTi) orthodontic archwires removed from the oral cavity of patients using fluoride mouthwashes. Five volunteers took part in this study on the corrosion behavior of two brands of NiTi archwires (3M and AO (brand of archwire)) during use of two mouthwashes with neutral sodium fluoride 1.1%, one with acidulated fluoride 1.1%, and one with placebo and a control group. Each patient used one mouthwash in three different periods of time for 1 min a day for 30 days. The archwires were assessed with scanning electron microscopy and atomic force microscopy for qualitative and quantitative analysis. The values obtained with atomic force microscopy (AFM) were submitted to normality test, two‐way analysis of variance, and Tukey's test at a significance level of 5%. The AFM images showed a gradual qualitative increase in the roughness of both types of wire between the treatments: control < placebo < neutral fluoride < acidulated fluoride. The arithmetic average of the roughness and root mean square of the roughness were similar. As for 3M archwires, only the acidulated fluoride group differed statistically from the others. As for AO archwires, the control and placebo groups did not differ from each other, but differed from the other fluoride treatments. The group using neutral fluoride also differed significantly from the acidulated fluoride group. 3M archwires were not affected by daily oral challenges. AO archwires were not affected by daily oral challenges either; their association with fluoride, either neutral or acidulated, increased their roughness.  相似文献   

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