Application Of Atomic Force Microscopy Research Articles

Brief reports on the use of atomic force microscopy in visualization of Mycobacterium tuberculosis

Background: By invention of the atomic force microscope (AFM) in 1986, the imaging of surfaces objects at nanometer-scale resolutions becomes possible. Although, in the beginning, AFM was applied almost exclusively to characterize the surfaces of nonbiological materials, at present the application of the AFM to biological and biomedical research has increased exponentially. Methods: In this study, we tried to investigate and visualize Mycobacterium tuberculosis under AFM. The transformation of bacterial shape and the formation of “hard” shell in resistant or dormant conditions were characterized and identified. Results: Application of AFM for the study of antibiotic-resistant forms of M. tuberculosis revealed the presence of round-shaped bacteria along with conventional rod-shaped ones. There has also been concluded changing of the surface charge of the cell membrane for mutated forms since round-shaped bacteria fixed on the charged surface less strongly and can be moved along the surface by a microscope tip with easily. Conclusions: In brief, this article highlights the optimal operation modes and base principal to study dangerous bacilli under AFM.

Atomic force microscopy study of the biofouling and mechanical properties of virgin and industrially fouled reverse osmosis membranes

The mechanical properties of virgin and industrially fouled reverse osmosis membranes (composite polyamide) used for the purification and desalination of seawater in desalination processes were characterised using novel atomic force microscopy (AFM) methods. Polymeric surface elasticity has previously been demonstrated to strongly affect the adhesion of bacteria; hence the study examined membrane surface elasticity to demonstrate how AFM can be used to assess the bio-fouling potential of membranes. An AFM colloid probe technique was used to determine the mechanical properties of the membrane, the adhesion forces and the work of adhesion at the membrane surfaces. The mean values of Young's modulus for the virgin membrane decreased in magnitude with increasing pH values, where these values were significantly different (p<0.017) between both pH3 (1450kPa), pH7 (1327kPa) and pH9 (788kPa). These differences were attributed to differences in membrane swelling and indicate possible control parameters that could be exploited to improve membrane cleaning regimes. A membrane with a higher modulus will be stronger and potentially more resistant to chemical and physical processes during operation and cleaning. Significant differences (p<0.017) in force measurements were also found between different electrolytic conditions for each of the membranes, where for the virgin membrane the adhesion force values were 6.00nN at pH3, 1.77nN at pH7 and 0.98N at pH9, and also the work of adhesion were 153.6nJ at pH3, 22.8nJ at pH7 and 9.9nJ at pH9 in 0.6M NaCl. These observations further confirm the importance of the electrolytic environment on the nanoscale interactions of the membrane which should be considered to control fouling during operation and cleaning cycles. AFM images and streaming potential measurements of virgin and fouled membranes were also obtained to aid analysis of the industrial membrane system. The novel application of AFM to membranes to measure Young's moduli and work of adhesion is a new addition to the AFM tools that can be used to unravel separation processes at the membrane surface. In addition, this study further demonstrates that AFM force spectroscopy can be used as part of a sophisticated membrane autopsy procedure for the elucidation of the mechanisms involved in membrane fouling.

Development of Nano-Biology with Atomic Force Microscopy

The first observation of double-stranded DNA by atomic force microscopy in the late 1980’s greatly encouraged many biological researchers to jump into the nano-world. Here we briefly review the history of how AFM has been utilized to reveal nanometer-scale structures of DNA-protein complexes, and we highlight key technical developments that have accelerated applications of AFM to molecular biology, physiology, biophysics, and cell biology. Biology is a visual science. Understanding the ‘biological events’ around us through visualization and observation has always been a fundamental part of biological scientific inquiry. Since the early days in the 17th century, biological investigations of the fundamental components of biological systems have relied on microscopes, the resolution of which is limited to one half the wavelength of light. Electron microscopy (EM), invented in the 1920-1930’s, brought a hundred times greater resolution than the light microscope, and it continues to enable us to visualize biological materials in the nanometer range [1]. However, EM requires special specimen preparation and operational constraints, e.g., coating the sample with a fine layer of gold and observing it under vacuum. These limitations were in large part circumvented in the 1980’s by an altogether new concept. Shortly after Binnig invented atomic force microscopy (AFM) [2], Hansma [3] proposed many possible uses for AFM in biology [3]. However it took almost 20 years for it to become an indispensable technique in biological research that allows observations in solution without fixation of the specimen. Commercially available instruments equipped with a scanning method known as the tapping mode [4,5], have yielded unprecedented views of biological materials such as DNA and proteins in their native state. The subsequent invention of high-speed AFM [6], which can scan biological samples in solution with sub-second temporal resolution, was a landmark accomplishment that has contributed greatly to the establishment of nanobiology as a major field in bioscience [7].

AFM visualization of sub-50 nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope

It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50nm particles resembling polyplexes in morphology compared with control preparations.

Controlled tip wear on high roughness surfaces yields gradual broadening and rounding of cantilever tips.

Tip size in atomic force microscopy (AFM) has a major impact on the resolution of images and on the results of nanoindentation experiments. Tip wear is therefore a key limitation in the application of AFM. Here we show, however, how wear can be turned into an advantage as it allows for directed tip shaping. We studied tip wear on high roughness polycrystalline titanium and diamond surfaces and show that tip wear on these surfaces leads to an increased tip size with a rounded shape of the apex. Next, we fitted single peaks from AFM images in order to track the changes in tip radius over time. This method is in excellent agreement with the conventional blind tip reconstruction method with the additional advantage that we could use it to demonstrate that the increase in tip size is gradual. Moreover, with our approach we can shape and control the tip size, while retaining identical chemical and cantilever properties. This significantly expands the reproducibility of AFM force spectroscopy data and is therefore expected to find a wide applicability.

Structural transformations in femtosecond laser-processed n-type 4H-SiC

We present a comprehensive study of morphological modification induced on and below the surface of n-type 4H-SiC by irradiation with femtosecond laser pulses under tight focusing condition. Spectroscopic investigation of local electronic and structural transformations in SiC-micro/nanostructures suggested bond breaking i.e. transformation of crystalline SiC to amorphous silicon (a-Si) and amorphous carbon (a-C). These observations were augmented by investigations applying atomic force microscopy (AFM), micro-photoluminescence (PL) spectroscopy, and high resolution X-ray diffraction (HRXRD). A high-resolution cross-sectional study of laser-modified region with transmission electron microscope (TEM) showed a thin amorphous layer in the vicinity of the geometrical focus with deformations and stacking faults in the sub-surface area. Having considered the existing ablation theories, a complex interplay of fast laser heating followed by melting and rapid re-solidification as well as dynamic relaxation of the laser-induced stresses seems to be responsible for formation of the observed structural changes.

Automated force controller for amplitude modulation atomic force microscopy.

Atomic Force Microscopy (AFM) is widely used in physics, chemistry, and biology to analyze the topography of a sample at nanometer resolution. Controlling precisely the force applied by the AFM tip to the sample is a prerequisite for faithful and reproducible imaging. In amplitude modulation (oscillating) mode AFM, the applied force depends on the free and the setpoint amplitudes of the cantilever oscillation. Therefore, for keeping the applied force constant, not only the setpoint amplitude but also the free amplitude must be kept constant. While the AFM user defines the setpoint amplitude, the free amplitude is typically subject to uncontrollable drift, and hence, unfortunately, the real applied force is permanently drifting during an experiment. This is particularly harmful in biological sciences where increased force destroys the soft biological matter. Here, we have developed a strategy and an electronic circuit that analyzes permanently the free amplitude of oscillation and readjusts the excitation to maintain the free amplitude constant. As a consequence, the real applied force is permanently and automatically controlled with picoNewton precision. With this circuit associated to a high-speed AFM, we illustrate the power of the development through imaging over long-duration and at various forces. The development is applicable for all AFMs and will widen the applicability of AFM to a larger range of samples and to a larger range of (non-specialist) users. Furthermore, from controlled force imaging experiments, the interaction strength between biomolecules can be analyzed.

Vesicle Size Regulates Nanotube Formation in the Cell.

Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.

AFM Applications to the Analysis of Plasma-Treated Surface Growth and Nanocomposite Materials

Background: The atomic force microscope (AFM) technique has proven to be a useful and versatile tool for the surface characterization of various materials. AFM is capable of providing three dimensional representations of surfaces down to the sub-nanometer scale resolution, with even atomic resolution. The aims of this mini review are to briefly illustrate our personal experience in AFM application for characterizing plasma-treated surface growth on different substrates, such as fluorocarbon (CFx) nano-structured films, polyethylene oxide (PEO) substrates and plasma deposited acrilic acid (pdAA) coatings, and nanocomposite materials, such as polydimethylsiloxane-gold (PDMS-Au) and chitosan-Au (CTO-Au), including the characterization of nanoparticle powder. Methods: The CFx films were obtained by plasma enhanced-chemical vapor deposition in the non-contact AFM mode and pdAA coatings by radiofrequency glow discharges fed with AA vapors and analyzed in the contact AFM mode. Coating morphology was analized by X-Ray photoelectron microscopy (XPS) and water contact angle (WCA). The AFM images of PDMS-Au and CTO-Au nanocomposites was also acquired and analyzed for their topography. Results: The surface topography, the root-mean square (RMS) surface roughness and the mean surface height of CFx coatings plasma-polymerised on polyethyleneterephthalate (PET) substrates were evaluated by AFM as a function of the deposition time, and AFM images obtained were used to gain detailed topographical information of the single nanostructure. By comparing the AFM images of pure PDMS with those of PDMS-Au it was possible to observe the topography of nanofillers embedded in a polymeric matrix or generated on a polymeric surface and also other main differences between the two materials. Conclusion: The AFM technique was shown to be a versatile and promising tool for the morphological characterization of growth of plasma-treated surfaces, such as CFx nano-structured films, PEO substrates and pdAA coatings, and for the topographical characterization of nanocomposite materials such as PDMs-Au and CTO-Au. Finally, AFM can be used as a simple method able to characterize the topography of as-received nanofillers, based on the attachment of the nanopowders on a bi-adhesive tape and on 3D image processing. Keywords: AFM, CFx nano-structured films, nanocomposite materials, nanofillers topography, plasma deposited acrylic acid coatings.

A monolithic MEMS position sensor for closed-loop high-speed atomic force microscopy

The accuracy and repeatability of atomic force microscopy (AFM) imaging significantly depend on the accuracy of the piezoactuator. However, nonlinear properties of piezoactuators can distort the image, necessitating sensor-based closed-loop actuators to achieve high accuracy AFM imaging. The advent of high-speed AFM has made the requirements on the position sensors in such a system even more stringent, requiring higher bandwidths and lower sensor mass than traditional sensors can provide. In this paper, we demonstrate a way for high-speed, high-precision closed-loop AFM nanopositioning using a novel, miniaturized micro-electro-mechanical system position sensor in conjunction with a simple PID controller. The sensor was developed to respond to the need for small, lightweight, high-bandwidth, long-range and sub-nm-resolution position measurements in high-speed AFM applications. We demonstrate the use of this sensor for closed-loop operation of conventional as well as high-speed AFM operation to provide distortion-free images. The presented implementation of this closed-loop approach allows for positioning precision down to 2.1 Å, reduces the integral nonlinearity to below 0.2%, and allows for accurate closed loop imaging at line rates up to 300 Hz.

Atomic force microscopy study of ionomycin-induced degranulation in RBL-2H3 cells.

Mast cell degranulation is the typical anaphylaxis process of mast cells associated with the release of cytokines, eicosanoids and their secretory granules, which play very important roles in the allergic inflammatory response of the human body upon anaphylactogen stimulation. The calcium ionophore ionomycin is widely used as a degranulation induction agent for mast cell degranulation studies. In the present work, ionomycin-induced degranulation of RBL-2H3 basophilic leukemia cell line cells was investigated in vitro by high resolution atomic force microscopy (AFM). Ionomycin, which could increase the intracellular free Ca2+ level and β-Hexosaminidase release, was found to induce the formation of a kind of peculiar vesicles in the cytoplasm area of RBL-2H3 cells. Those vesicles induced by ionomycin would desintegrate to release a larger amount of granules surrounding RBL-2H3 cells by the controlling of F-actin. These results provide the precise morphological information of ionomycin-induced mast cell degranulation at nanoscale, which could benefit our understanding of ionomycin-induced mast cell anaphylaxis model and also validate the applicability of AFM for the detection of allergic inflammatory response in mast cells. SCANNING 38:525-534, 2016. © 2016 Wiley Periodicals, Inc.

Optimizing force spectroscopy by modifying commercial cantilevers: Improved stability, precision, and temporal resolution.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100μm) cantilever's gold coating. Next, this sub-pN bandwidth is extended by a factor of ∼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40μm) cantilever. Finally, FIB-modifying an ultrashort (L=9μm) cantilever improves its force stability and precision while maintaining 1-μs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.

Atomic force microscopy and graph analysis to study the P-cadherin/SFK mechanotransduction signalling in breast cancer cells.

Physical forces mediated by cell-cell adhesion molecules, as cadherins, play a crucial role in preserving normal tissue architecture. Accordingly, altered cadherins' expression has been documented as a common event during cancer progression. However, in most studies, no data exist linking pro-tumorigenic signaling and variations in the mechanical balance mediated by adhesive forces. In breast cancer, P-cadherin overexpression increases in vivo tumorigenic ability, as well as in vitro cell invasion, by activating Src family kinase (SFK) signalling. However, it is not known how P-cadherin and SFK activation impact cell-cell biomechanical properties. In the present work, using atomic force microscopy (AFM) images, cell stiffness and cell-cell adhesion measurements, and undirected graph analysis based on microscopic images, we have demonstrated that P-cadherin overexpression promotes significant alterations in cell's morphology, by decreasing cellular height and increasing its area. It also affects biomechanical properties, by decreasing cell-cell adhesion and cell stiffness. Furthermore, cellular network analysis showed alterations in intercellular organization, which is associated with cell-cell adhesion dysfunction, destabilization of an E-cadherin/p120ctn membrane complex and increased cell invasion. Remarkably, inhibition of SFK signaling, using dasatinib, reverted the pathogenic P-cadherin induced effects by increasing cell's height, cell-cell adhesion and cell stiffness, and generating more compact epithelial aggregates, as quantified by intercellular network analysis. In conclusion, P-cadherin/SFK signalling induces topological, morphological and biomechanical cell-cell alterations, which are associated with more invasive breast cancer cells. These effects could be further reverted by dasatinib treatment, demonstrating the applicability of AFM and cell network diagrams for measuring the epithelial biomechanical properties and structural organization.

Imaging DNA Structure by Atomic Force Microscopy.

Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine superstructure and secondary structure of surface-bound DNA. The method is also readily applicable to probe DNA-DNA interactions and DNA-protein complexes.

DETERMINATION OF FILLER STRUCTURE IN SILICA-FILLED SBR COMPOUNDS BY MEANS OF SAXS AND AFM

ABSTRACT The structure of the silica particles network in two different solution styrene–butadiene rubbers (S-SBRs) was studied by means of small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). S-SBR compounds with different silica contents were analyzed in comparison with their oil extended counterparts. A study into the application of SAXS experiments was defined to quantify the structures of silica primary particles and clusters in filled rubber compounds up to very high levels of filler content. We propose a modified structure model that is physically more sound than the widely used Beaucage model and that leads to more robust quantification of the silica structures. In addition, an independent characterization of the filler structure was performed by means of AFM. The cluster and particle sizes deduced from both techniques are in close agreement, supporting the proposed approach. The synergetic application of SAXS and AFM allows a consistent and robust characterization of primary particles and clusters in terms of size and structure. These results were compared and discussed in the framework of previously published works.

Atomic Force Microscopy for Solar Fuels Research: An Introductory Review

Although research on solar fuel production via water oxidation, hydrogen evolution, and carbon dioxide reduction (i.e., artificial photosynthesis) has grown tremendously in recent years, practical solar fuel devices remain elusive. For these multi-electron transfer processes to take place efficiently, integrated systems constructed from materials with very different chemical, mechanical, and electrical properties are required. This complicated integration presents numerous challenges, including the need for increased characterization of material surfaces and interfaces for both fundamental studies and device optimization. Atomic force microscopy (AFM) is a powerful tool for surface and interface analysis. Although solar fuel research has frequently utilized the basic AFM function of topographic mapping for routine surface analysis, some researchers have avoided more advanced AFM methodologies due to the complexity of these integrated solar fuel generating systems. This article provides researchers in this area with an introduction to various advanced AFM techniques for mechanical, electrical, and chemical analysis on the nanoscale. It also discusses the possibility of in situ study while providing an outline of the working principles of different AFM application modes.

Atomic force microscopy to investigate asphalt binders: a state-of-the-art review

Atomic force microscopy (AFM) is a non-destructive imaging tool, which is capable of qualitative and quantitative surface analysis with sub-nanometer resolution. Simultaneously with the topology at the micro-scale, AFM is capable of acquiring micro-mechanical information such as relative stiffness/Young's modulus, stickiness/adhesion, hardness, energy loss and sample deformation quantitatively. This paper presents an extensive review on the applications of AFM to investigate different physiochemical properties and performances of asphalt binder. AFM techniques and principles, different sample preparation techniques and its effect on observed micro-structures, chemical origin, surface or bulk phenomenon and temperature sensitivity of these micro-structures are also discussed in this paper. All of the studies conducted on this topic clearly indicated that AFM can successfully be utilised as a tool to better understand how the surface morphology and its physicochemical properties are interlinked and related to the binder performances.

Direct Measurement of Three-Dimensional Forces in Atomic Force Microscopy

Direct measurement of three-dimensional (3-D) forces between an atomic force microscope (AFM) probe and the sample benefits diverse applications of AFM, including force spectroscopy, nanometrology, and manipulation. This paper presents the design and evaluation of a measurement system, wherein the deflection of the AFM probe is obtained at two points to enable direct measurement of all the three components of 3-D tip-sample forces in real time. The optimal locations for measurement of deflection on the probe are derived for a conventional AFM probe. Further, a new optimal geometry is proposed for the probe that enables measurement of 3-D forces with identical sensitivity and nearly identical resolution along all three axes. Subsequently, the designed measurement system and the optimized AFM probe are both fabricated and evaluated. The evaluation demonstrates accurate measurement of tip-sample forces with minimal cross-sensitivities. Finally, the real-time measurement system is employed as part of a feedback control system to regulate the normal component of the interaction force, and to perform force-controlled scribing of a groove on the surface of polymethyl methacrylate.

Applications of Atomic Force Microscopy in Exploring Drug Actions in Lymphoma-Targeted Therapy at the Nanoscale

Rituximab, a monoclonal antibody against the CD20 molecule expressed on B cells, has revolutionized the treatment of B cell lymphomas. The use of rituximab significantly improves the long-term survival rates of lymphoma patients. However, there are still many patients who develop resistance to rituximab. Hence, the urgent need is enhancing the potency of anti-CD20 antibodies beyond that achieved with rituximab to provide effective therapies for more patients. Traditional studies about the killing mechanisms of rituximab are commonly based on optical microscopy, which cannot reveal the detailed situations due to the limit of 200-nm resolution. Quantitatively investigating the actions of rituximab at the nanoscale is still scarce. The advent of atomic force microscopy (AFM) provides a powerful and versatile platform for in situ investigating the nanoscale behaviors of individual live cells. The applications of AFM in life sciences in the past decade have yielded a lot of novel insights into cell biology and related diseases. In this article, the typical applications (e.g., ultra-microstructures, mechanical properties, and molecular recognition) of AFM in investigating the three killing mechanisms of rituximab at the nanoscale are summarized; the challenges facing AFM single-cell assay and its future directions are also discussed.

Investigation of quercetin-induced HepG2 cell apoptosis-associated cellular biophysical alterations by atomic force microscopy.

Quercetin, a wildly distributed bioflavonoid, has been proved to possess excellent antitumor activity on hepatocellular carcinoma (HCC). In the present study, the biophysical properties of HepG2 cells were qualitatively and quantitatively determined using high resolution atomic force microscopy (AFM) to understand the anticancer effects of quercetin on HCC cells at nanoscale. The results showed that quercetin could induce severe apoptosis in HepG2 cells through arrest of cell cycle and disruption of mitochondria membrane potential. Additionally, the nuclei and F-actin structures of HepG2 cells were destroyed by quercetin treatment as well. AFM morphological data showed some typical apoptotic characterization of HepG2 cells with increased particle size and roughness in the ultrastructure of cell surface upon quercetin treatment. As an important biophysical property of cells, the membrane stiffness of HepG2 cells was further quantified by AFM force measurements, which indicated that HepG2 cells became much stiffer after quercetin treatment. These results collectively suggest that quercetin can be served as a potential therapeutic agent for HCC, which not only extends our understanding of the anticancer effects of quercetin against HCC cells into nanoscale, but also highlights the applications of AFM for the investigation of anticancer drugs.