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1.
The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.  相似文献   

2.
A combined optical and atomic force microscope for live cell investigations   总被引:6,自引:0,他引:6  
We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.  相似文献   

3.
Adapting atomic force microscopy for cell biology   总被引:11,自引:0,他引:11  
We present details of our AFM modifications to produce an adaptable imaging system for the cell biologist. We have designed and validated a new inverted microscope interface and a scan head with increased Z-range, based upon the TopoMetrix Explorer AFM. We have utilised these changes, together with home-made glass ball cantilevers, to obtain topographical information over cells with large Z-dimension (over 15 microm high), and mapped calcitonin-calcitonin receptor binding forces in living bone cells. We conclude that modified AFM can be used to evaluate intermolecular events in living cells and that this approach will ensure general application to the study of receptor-ligand interactions under truly physiological conditions.  相似文献   

4.
Uehara H  Osada T  Ikai A 《Ultramicroscopy》2004,100(3-4):197-201
Asymmetric localizations of cellular proteins and mRNAs are important for cell functions such as division, differentiation and development. The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins and thereby restricting their locations to appropriate cellular regions. We have previously reported a novel method based on atomic force microscopy (AFM) for examining gene expression in a single living cell without killing or destroying it. An AFM tip was inserted into a living cell to extract mRNAs, which were analyzed after multiplication by RT-PCR and quantitative PCR. By applying this method, in this study we performed quantitative measurement of mRNA at different loci within individual living cells.  相似文献   

5.
Kim H  Tsuruta S  Arakawa H  Osada T  Ikai A 《Ultramicroscopy》2004,100(3-4):203-210
To develop force measurements using an atomic force microscope (AFM) in a quantitative manner, it is necessary to estimate the number density of target molecules on a sample surface, and for this, the sensitivity of detection should be known. In this study, the AFM was used as a mechanical detector and an antigen and its antibody were used as a model to evaluate the sensitivity of detection. Antigens were immobilized on a glass surface and number density was estimated by monitoring optical absorbance due to product formation by the reaction of crosslinkers. The concentration of antigen was controlled by mixing control peptides. A microbead was used as a probe and antibodies were immobilized on the bead. AFM force measurements were then made for a range of number densities in the order of 10–106 antigen molecules per square micrometer of surface and were compared to evaluate the sensitivity of detection. Our result establishes the reliability of estimating a number of molecules like receptors on the cell surface, and indicates that the AFM is useful as a mechanical detector with high sensitivity.  相似文献   

6.
Osada T  Itoh A  Ikai A 《Ultramicroscopy》2003,97(1-4):353-357
The distribution of the receptor-associated protein (RAP) binding protein and the adhesion forces between RAP and its binding protein on living fibroblast cells were examined using an atomic force microscope (AFM). The distribution of RAP binding protein was obtained on 256 (16x16) locations in 2x2 micro m sections over the surface of living cells. The adhesion forces between RAP and the binding protein were measured with an AFM tip functionalized with RAP. In the presence of RAP in the scanning solution, the number of force curves with large adhesion force decreased. These results indicate that the adhesive forces observed here represent specific binding between RAP and the binding protein. This method will be a useful application of AFM to examine receptors on cell surfaces in high resolution.  相似文献   

7.
The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.  相似文献   

8.
Chen Y  Cai J  Zhao T  Wang C  Dong S  Luo S  Chen ZW 《Ultramicroscopy》2005,103(3):173-182
The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.  相似文献   

9.
Chen B  Wang Q  Han L 《Scanning》2004,26(4):162-166
In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.  相似文献   

10.
The viscoelastic properties of a large number of mouse fibroblast NIH3T3 cells (n?130) were investigated by combining atomic force microscopy (AFM) with a microarray technique. In the experiments, the cells were arranged and cultured in the wells of a microarray substrate, and a force modulation mode experiment was used to measure the complex shear modulus, G*, of individual cells in a frequency range 0.5–200 Hz. The frequency dependence of G* of the cells exhibited a power-law behavior and similar frequency dependencies have been observed in several cell types cultured on flat substrates. This indicated that the NIH3T3 cells cultured in the wells of a microarray have analogous structural organization to those cells cultured on flat substrates. The number distribution of both the storage and loss moduli of G* fitted well to a log-normal distribution function, whereas the power-law exponent estimated by a power-law structural damping model showed a normal distribution function. These results showed that combining AFM with a microarray technique was a suitable approach for investigating the statistics of rheological properties of living cells without the requirement of cell surface modification.  相似文献   

11.
Atomic force microscopy (AFM) holds great potential for studying the nanoscale surface structures of living cells, and to measure their interactions with abiotic surfaces, other cells, or specific biomolecules. However, the application of AFM in microbiology is challenging due to the difficulty of immobilising bacterial cells to a flat surface without changing the cell surface properties or cell viability. We have performed an extensive and thorough study of how to functionalise surfaces in order to immobilise living bacteria for AFM studies in liquid environments. Our aim was to develop a scheme which allows bacterial cells to be immobilised to a flat surface with sufficient strength to avoid detachment during the AFM scanning, and without affecting cell surface chemistry, structure, and viability. We compare and evaluate published methods, and present a new, reproducible, and generally applicable scheme for immobilising bacteria cells for an AFM imaging.  相似文献   

12.
We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.  相似文献   

13.
Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.  相似文献   

14.
Membrane deformation of living glial cells using atomic force microscopy   总被引:3,自引:0,他引:3  
Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi-fluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper SupertipsTM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.  相似文献   

15.
Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity.  相似文献   

16.
Ge G  Han D  Lin D  Chu W  Sun Y  Jiang L  Ma W  Wang C 《Ultramicroscopy》2007,107(4-5):299-307
Magnetic AC mode (MAC mode) atomic force microscopy (AFM), a novel type of tapping mode AFM in which the cantilever is driven directly by a magnetic field, is a powerful tool for imaging with high spatial resolution and better signal-to-noise in liquid environment. It may largely extend the application of AFM to living samples, especially those are sensitive to cantilever forces, even to multilayer tissue samples. However, there are few reports on the imaging of living cells by MAC mode AFM previously. In our present study, we explore the optimal imaging conditions of MAC mode AFM on living astrocytes and fresh arterial intima surface. We also used nude tips for PicoTREC panel (i.e., Aux in BNC, a new data collecting channel) to image living samples and discussed its difference with phase imaging. We show that living biological samples can be imaged by MAC mode AFM at details of comparable resolution as those by high resolution scanning electron microscopy. Furthermore, the combination of height, amplitude, phase and TREC panel signals provide abundant informations for the characteristics of living samples, such as topography, profile, stiffness and adhesion.  相似文献   

17.
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.  相似文献   

18.
Temperature-dependent imaging of living cells by AFM   总被引:1,自引:0,他引:1  
Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.  相似文献   

19.
Li X  Ji T  Hu J  Sun J 《Ultramicroscopy》2008,108(9):826-831
High resolution imaging of intracellular structures of ultrathin cell section samples is critical to the performance of precise manipulation by atomic force microscopy (AFM). Here, we test the effect of multiple factors during section sample preparation on the quality of the AFM image. These factors include the embedding materials, the annealing process of the specimen block, section thickness, and section side. We found that neither the embedding materials nor the temperature and speed of the annealing process has any effect on AFM image resolution. However, the section thickness and section side significantly affect the surface topography and AFM image resolution. By systematically testing the image quality of both sides of cell sections over a wide range of thickness (40-1000 nm), we found that the best resolution was obtained with upper-side sections approximately 50-100 nm thick. With these samples, we could observe precise structure details of the cell, including its membrane, nucleoli, and other organelles. Similar results were obtained for other cell types, including Tca8113, C6, and ECV-304. In brief, by optimizing the condition of ultrathin cell section preparation, we were able to obtain high resolution intracellular AFM images, which provide an essential basis for further AFM manipulation.  相似文献   

20.
Atomic Force Microscopy (AFM) has proven itself over recent years as an essential tool for the analysis of microbial systems. This article will review how AFM has been used to study microbial systems to provide unique insight into their behavior and relationship with their environment. Immobilization of live cells has enabled AFM imaging and force measurement to provide understanding of the structure and function of numerous microbial cells. At the macromolecular level AFM investigation into the properties of surface macromolecules and the energies associated with their mechanical conformation and functionality has helped unravel the complex interactions of microbial cells. At the level of the whole cell AFM has provided an integrated analysis of how the microbial cell exploits its environment through its selective, adaptable interface, the cell surface. In addition to these areas of study the AFM investigation of microbial biofilms has been vital for industrial and medical process analysis. There exists a tremendous potential for the future application of AFM to microbial systems and this has been strengthened by the trend to use AFM in combination with other characterization methods, such as confocal microscopy and Raman spectroscopy, to elucidate dynamic cellular processes. SCANNING 32: 134–149, 2010. © 2010 Wiley Periodicals, Inc.  相似文献   

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