Intercellular interaction observed by atomic force microscopy

The cultured myoblasts, focusing on the microprocesses related to the intercellular interaction, were observed by taking topological images. For atomic force microscopy (AFM), cells were fixed and either dried as in usual scanning electron microscopy or kept in the buffer. The dried cells were used for observing intercellular interactions related to the fusion. The prefusing myoblasts aligned in a chain were mostly spindle in shape and were characterized by the presence of many microprocesses along the facing edges of adjacent aligned myoblasts. The space between fusing myoblasts and between myotubes and myoblasts were often traversed by filopodia and cellular bridges formed by the connection of microvilli. These results suggest that microprocesses may be involved in the fusion of myoblasts. The best images of the fixed cell in liquid were obtained using the contact mode of AFM. AFM observation is an efficient tool in the study on the interaction between cells, and the fixation, imaging in liquid is a good approach to understand the cellular dynamics.     

Engineering a multi-nucleated myotube, the role of the actin cytoskeleton

Myoblasts in vitro form characteristic arrays of bipolar-shaped cells prior to fusion. We have shown that the actin cytoskeleton re-organizes in these fusing cells and that the interaction of non-muscle myosin 2A with actin at the plasma membrane helps to generate the bipolar shape of myoblasts, which is key for fusion. Here we discuss how fusion occurs, and in particular how the actin cytoskeleton is involved. Myoblast fusion is essential to form the multi-nucleated muscle fibres that make up the skeletal muscle. Skeletal muscle fibres contain many nuclei, roughly one nucleus to every 15 sarcomeres (35 microm) in adult muscle, although this varies with muscle type (Bruusgaard et al., 2006). Thus a muscle fibre 30 cm long contains about 8000 nuclei and is formed by the fusion of about 8000 cells during development. The formation of multi-nucleated myotubes has been intensively studied for many years using a number of different systems. Many different proteins have been identified using Drosophila as a model system (e.g. see reviews by Taylor, 2000, 2002) that have given an insight into what happens in mammals. However, the process of fusion of mammalian cells is less well understood, and this paper will cover some of the aspects of mammalian myoblast fusion, with a particular focus on the role of the actin cytoskeleton.     

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.     

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.     

Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton

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.     

Membrane deformation of living glial cells using atomic force microscopy

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 Supertips^TM 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.     

Using carbon nanotube probes for high-resolution three-dimensional imaging of cells

While atomic force microscopy (AFM) has become a promising tool for visualizing membrane morphology of cells, many studies have reported the presence of artifacts such as cliffs on the edges of cells. These artifacts shield important structural features such as lamellopodia, filopodia, microvilli and membrane ridges, which represent characteristic status in signaling processes such as spreading and activation. These cliff-like edges arise from a premature contact of the probe side contact with the cell prior to the probe top apex-cell contact. Carbon nanotube (CNT) modified AFM probes were utilized to address this drawback. Using rat basophilic leukemia (RBL) cells, this work revealed that CNT probes diminish cliff-like artifacts and enabled visualization of entire membrane morphology and structural features in three dimensions. The high aspect ratio of CNT probes provides a very effective remedy to the cliff-like artifacts as well as tip convolution of conventional probes, which shall enhance the validity and application of AFM in cellular biology research.     

The distribution of caveolin-3 immunofluorescence in skeletal muscle fibre membrane defined by dual channel confocal laser scanning microscopy, fast Fourier transform and image modelling

Membrane domains rich in caveolin‐3 overlie sarcomeric actin in skeletal muscle. The membrane exhibits a regular array of caveolin‐3 immunofluorescence using confocal laser scanning microscopy (CLSM). Fourier analysis of tissue imaged by CLSM accurately defines a repeating intensity with a long‐axis spacing of 1.48 µm confirmed by measurement of direct images. Reverse fast Fourier transform (FFT) and image‐modelling allow reconstruction of the pattern. Mathematical modelling has allowed replication of several features of the FFT, including the second order maxima that confirm the relatively high information content of the original images. Measurements of membrane‐pattern primary long‐axis spacings are consistent with our measurements of the I‐band sarcomere repeat in similarly prepared specimens labelled with fluorescent phalloidin or imaged using differential interference contrast microscopy. Dual‐channel CLSM analysis of the sarcomeric banding pattern of actin and the repeating pattern of muscle fibre membrane caveolin showed that caveolae overlie the I‐band. The anti‐caveolin immunofluorescence is deficient over the Z‐disc and maximal toward each of the I‐band extremities. A mechanism of membrane shape change in which membrane–lipid molecules are interposed between more stable anchored rafts associated with caveolae can be envisaged. Thus, increasing girth and reducing length of the sarcolemma in rapid contraction may be explained.     

QD as a bifunctional cell-surface marker for both fluorescence and atomic force microscopy

Fluorescent quantum dots (QDs) are a new class of fluorescent label and have been extensively used in cell imaging. Streptavidin-conjugated QDs have a diameter of ca. 10–15 nm; therefore when used as probes to label cell-surface biomolecules, they can provide contrast enhancement under atomic force microscopy (AFM) and allow specific proteins to be distinguished from the background. In addition, the size and fluorescent properties potentially make them as probes in correlative fluorescence microscopy (FM) and AFM. In this study, we tested the feasibility of using QD-streptavidin conjugates as probes to label wheat germ agglutinin (WGA) receptors on the membrane of human red blood cells (RBCs) and simultaneously obtain fluorescence and AFM images. The results show that the distribution of QDs labeled on human RBCs was non-uniform and that the number of labeled QDs on different erythrocytes varied significantly, which perhaps indicates different ages of the erythrocytes. Thus, QDs may be employed as bifunctional cell-surface markers for both FM and AFM to quantitatively investigate the distribution and expression of membrane proteins or receptors on cell surface.     

Velocity dependent friction laws in contact mode atomic force microscopy

Friction forces in the tip–sample contact govern the dynamics of contact mode atomic force microscopy. In ambient conditions typical contact radii between tip and sample are in the order of a few nanometers. In order to account for the large interaction area the dynamics of contact mode atomic force microscope (AFM) is investigated under the assumption of a multi-asperity contact interface between tip and sample. Thus, the kinetic friction force between tip and sample is the product of the real contact area between both solids and the interfacial shear strength. The velocity strengthening of the lateral force is modeled assuming a logarithmic relationship between shear-strength and velocity. Numerical simulations of the system dynamics with this empirical model show the existence of two different regimes in contact mode AFM: steady sliding and stick–slip where the tip undergoes periodically stiction and kinetic friction. The state of the system depends on the scan velocity as well as on the velocity dependence of the interfacial friction force between tip and sample. Already small viscous damping contributions in the tip–sample contact are sufficient to suppress stick–slip oscillations.     

Temperature-dependent imaging of living cells by AFM

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.     

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.     

Penetrability of AH plus and MTA fillapex after endodontic treatment and retreatment: A confocal laser scanning microscopy study

The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta‐percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re‐prepared up to F5 instruments and filled with gutta‐percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups (P > 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment (P > 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. Microsc. Res. Tech. 77:467–471, 2014. © 2014 Wiley Periodicals, Inc.     

Novel operation mode for eliminating influence of inclination angle and friction in atomic force microscopy

The accuracy of topography imaging in contact force mode of atomic force microscopy (AFM) depends on the one-to-one corresponding relationship between the cantilever deflection and the tip–sample distance, whereas such a relationship cannot be always achieved in the presence of friction and incline angle of sample surface. Recently, we have developed a novel operation mode in which we keep the van der Waals force as constant instead of the applied normal force, to eliminate the effect of inclination angle and friction on topography imaging in the contact force mode. We have improved our AFM to enable the new operation mode for validation. Comparative experiments have been performed and the results have shown that the effect of friction and inclination angle on topography imaging in contact mode of AFM can be eliminated or at least decreased effectively by working in the new operation mode we present.     

AFM/CLSM data visualization and comparison using an open-source toolkit

There is a vast difference in the traditional presentation of AFM data and confocal data. AFM data are presented as surface contours while confocal data are usually visualized using either surface- or volume-rendering techniques. Finding a common meaningful visualization platform is not an easy task. AFM and CLSM technologies are complementary and are more frequently being used to image common biological systems. In order to provide a presentation method that would assist us in evaluating cellular morphology, we propose a simple visualization strategy that is comparative, intuitive, and operates within an open-source environment of ImageJ, SurfaceJ, and VolumeJ applications. In order to find some common ground for AFM-CLSM image comparison, we have developed a plug-in for ImageJ, which allows us to import proprietary image data sets into this application. We propose to represent both AFM and CLSM image data sets as shaded elevation maps with color-coded height. This simple technique utilizes the open source VolumeJ and SurfaceJ plug-ins. To provide an example of this visualization technique, we evaluated the three-dimensional architecture of living chick dorsal root ganglia and sympathetic ganglia measured independently with AFM and CLSM.     

Multifractal characterization of morphology of human red blood cells membrane skeleton

The purpose of this paper is to show applicability of multifractal analysis in investigations of the morphological changes of ultra‐structures of red blood cells (RBCs) membrane skeleton measured using atomic force microscopy (AFM). Human RBCs obtained from healthy and hypertensive donors as well as healthy erythrocytes irradiated with neutrons (45 μGy) were studied. The membrane skeleton of the cells was imaged using AFM in a contact mode. Morphological characterization of the three‐dimensional RBC surfaces was realized by a multifractal method. The nanometre scale study of human RBCs surface morphology revealed a multifractal geometry. The generalized dimensions D_q and the singularity spectrum f(α) provided quantitative values that characterize the local scale properties of their membrane skeleton organization. Surface characterization was made using areal ISO 25178‐2: 2012 topography parameters in combination with AFM topography measurement. The surface structure of human RBCs is complex with hierarchical substructures resulting from the organization of the erythrocyte membrane skeleton. The analysed AFM images confirm a multifractal nature of the surface that could be useful in histology to quantify human RBC architectural changes associated with different disease states. In case of very precise measurements when the red cell surface is not wrinkled even very fine differences can be uncovered as was shown for the erythrocytes treated with a very low dose of ionizing radiation.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

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.     

Microscopic analysis of the quality of obturation and physical properties of MTA Fillapex

This study analyzed the quality of obturation and physical properties of MTA Fillapex and AH Plus sealer. A sample of 30 human maxillary central incisors were instrumented with Protaper until a F5 (50/05) file. Both sealers were mixed with Rhodamine‐B dye to allow visualization on a confocal laser‐scanning microscope (CLSM). Next, the canals were filled using the single cone technique. After setting, all samples were sectioned at 2, 4, and 6 mm from the apex. CLSM was used to analyze the gaps and sealer penetration into the dentinal tubules. All samples were scanned 10 µm below the dentin surface and images were recorded at 100× magnification using the fluorescent mode. Additionally, the solubility, flowability and setting time of the sealers were evaluated. All the measured quantities of the examined materials were evaluated for significant differences by means of statistical analysis. The CLSM analysis of the MTA Fillapex showed the highest percentage of gaps at all sections (P = 0.0001). Physical tests revealed adequate properties for both sealers except for a higher solubility of the MTA Fillapex (P = 0.0001). The MTA Fillapex presented flowability and intratubular penetration similar to the AH Plus. Nevertheless, the MTA Fillapex sealer presented a higher solubility and considerable quantity of gaps between the sealer/dentin interface in relation to the AH Plus sealer. Clinicians must take into consideration, the quality of endodontic sealers as it is essential in the outcome of the root canal filling. Microsc. Res. Tech. 77:1031–1036, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of Zn doping on the sublimation rate of pentaerythritol tetranitrate using atomic force microscopy

A series of Zn ion‐doped pentaerythritol tetranitrate (PETN) nanoislands in the form of thin films were prepared on Si substrates using spin coating. The effect of Zn concentrations on the sublimation energy was investigated by atomic force microscopy (AFM). The pure and Zn‐doped nanoislands are imaged by AFM in contact mode at room temperature after annealing isothermally for a given time. The volume of the islands starts to decrease after annealing at 45°C for pure PETN, whereas Zn‐doped nanoislands start to decrease in height and volume after annealing at 55–58°C. The minimum activation energy is found to be 29.7 Kcal/mol for 1,000 ppm Zn concentration. These studies are important for the long‐term stabilization of PETN. SCANNING 31: 181–187, 2009. © 2009 Wiley Periodicals, Inc.     

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.