Immobilizing live bacteria for AFM imaging of cellular processes

Coccoid cells of the bacterial species Staphylococcus aureus have been mechanically trapped in lithographically patterned substrates and imaged under growth media using atomic force microscopy (AFM) in order to follow cellular processes. The cells are not perturbed as there is no chemical linkage to the surface. Confinement effects are minimized compared to trapping the cells in porous membranes or soft gels. S. aureus cells have been imaged undergoing cell division whilst trapped in the patterned substrates. Entrapment in lithographically patterned substrates provides a novel way for anchoring bacterial cells so that the AFM tip will not push the cells off during imaging, whilst allowing the bacteria to continue with cellular processes.     

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.     

Effects of substrates on biofilm formation observed by atomic force microscopy

Formation of biofilm is known to be strongly dependent on substrates including topography, materials, and chemical treatment. In this study, a variety of substrates are tested for understanding biofilm formation. Sheets of aluminum, steel, rubber, and polypropylene have been used to examine their effects on formation of Pseudomonas aeruginosa biofilm. In particular, the morphological variation, transition, and adhesiveness of biofilm were investigated through local measurement by atomic force microscopy (AFM). Mechanism of removing biofilm from adhering to substrate is also analyzed, thus the understanding of the mechanism can be potentially useful to prevent the biofilm formation. The results reveal that formation of biofilm can remain on rough surface regardless of substrates in hot water, which may easily induce extra-polymeric substances detachment from bacterial surface. By probing using AFM, local force–distance characterization of extra-cellular materials extracted from the bacteria can exhibit the progress of the biofilm formation and functional complexities.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

The role of substrates on the structural,optical, and morphological properties of zno nanotubes prepared by spray pyrolysis

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.     

Study of the antibacterial effects of chitosans on Bacillus cereus (and its spores) by atomic force microscopy imaging and nanoindentation

Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely distributed in nature. Its intrinsic thermal resistance coupled with the extraordinary resistance against common food preservation techniques makes it one of the most frequent food-poisoning microorganisms causing both intoxications and infections. In order to control B. cereus growth/sporulation, and hence minimize the aforementioned hazards, several antimicrobial compounds have been tested. The aim of this work was to assess by atomic force microscopy (AFM) the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon both vegetative and resistance forms of B. cereus. The use of AFM imaging studies helped us to understand how chitosans with different MW act differently upon B. cereus. Higher MW chitosans (628 and 100 kDa) surrounded both forms of B. cereus cells by forming a polymer layer—which eventually led to the death of the vegetative form by preventing the uptake of nutrients yet did not affect the spores since these can survive for extended periods without nutrients. Chitooligosaccharides (COS) (<3 kDa), on the other hand, provoked more visible damages in the B. cereus vegetative form—most probably due to the penetration of the cells by the COS. The use of COS by itself on B. cereus spores was not enough for the destruction of a large number of cells, but it may well weaken the spore structure and its ability to contaminate, by inducing exosporium loss.     

利用原子力显微技术对神经胶质细胞超微结构及其相互间的纳米连接结构的观察

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。     

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This "fix-and-look" approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the "fix-and-look" approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.     

Tribological Properties of Self-Assembled Monolayers and Their Substrates Under Various Humid Environments

Using friction force microscopy (FFM) under controlled environments, we have systematically investigated the humidity effect on the frictional properties of two important classes of self-assembled monolayers (SAMs), i.e., N-octadecyltrimethoxysilane (OTE, CH₃(CH₂)₁₇Si(OCH₃)₃) on SiO₂(OTE/SiO₂), and N-alkanethiols on Au(111), together with their respective substrates. Experimental results show that both OTE and alkylthiol SAMs can decrease the friction force between a Si₃N₄ atomic force microscope (AFM) tip and substrates. The nearly humidity-independent friction of the two kinds of SAMs indicates that these SAMs are ideal lubricants in applications of micro-electro-mechanical systems (MEMS) under different environments. The humidity dependence—as the humidity increases, the friction first increases and then decreases—of the two substrates, SiO₂ and Au(111), can be explained by the adsorption of water. The decrease in the friction at high humidity is attributed to the low viscosity in the multilayers of water, while the increase in the friction at low humidity can be explained by the high viscosity between the water monolayer and the surfaces (AFM tip and sample), possibly due to the confinement effects. The effect of modification of the AFM tip with alkanethiol molecules on the humidity dependence of Au(111) friction has also been investigated.     

Substrate influence on cell shape and cell mechanics: HepG2 cells spread on positively charged surfaces

A human hepatoma cell line (HepG2) was cultured on positively and negatively charged polyelectrolytes. Cell/surface adhesion and cell shape evolution were followed with quartz microbalance with dissipation (QCM‐D) and optical microscopy as a function of time, respectively. In particular, substrates coated with poly(ethyleneimine) (PEI) led to fast cell attachment and further spreading, with average maximum frequency Δf = 79 Hz and dissipation ΔD = 40 × 10^?6. On the contrary, no cell spreading was observed on poly(sodium‐4‐styrenesulfonate) (PSS), with Δf = 33 Hz and ΔD = 4.5 × 10^?6. Atomic force microscopy (AFM) was used to investigate the influence of cell shape on its mechanical properties. Considering the cells as an homogenous solid material, the corresponding elastic modulus was estimated using the Hertz model. The elastic modulus was calculated at the central part of the cell, and the average values obtained were 191 ± 14 Pa and 941 ± 58 Pa for cells adsorbed on PSS and PEI, respectively. Thus, different cell–substrate interaction implied different cell mechanical properties reflected in a higher elastic modulus for stronger cell/substrate interaction. The combination of QCM‐D, AFM, and optical microscopy allowed the online study of the cell adhesion process, and the mechanical properties of the adhered cells. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Further results on non-Newtonian power-law flows past a two-dimensional flat plate with finite length

The flow of a non-Newtonian, power-law fluid directed either tangentially or normally to a flat plate of finite length and infinite width (two-dimensional flow) is considered. The problem is investigated numerically using the code ANSYS FLUENT. This problem has been investigated in the past but only for shear-thinning fluids (n < 1). We extend the investigation for the case of shear-thinning, Newtonian and shear-thickening fluids, covering a wide range of Reynolds numbers (from very low to very high). For low Reynolds numbers and low power-law index (n < 0.6) the drag coefficient obeys the relationship c _D = A/Re, both for tangential and normal flow. Equations for the quantity A have been derived as functions of the power-law index. For normal flow, the drag coefficient tends to become independent of the power-law index, both for shear-thinning and shear-thickening fluids at high Reynolds numbers.     

Spectroscopy and atomic force microscopy of biomass

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.     

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.     

Effect of capillary formation on friction and pull-off forces measured on submicron-size asperities

Asperities with hemispherical peaks were fabricated on a silicon substrate using a focused ion beam. Pull-off and friction forces were measured on each asperity using atomic force microscopy (AFM) in high vacuum (HV) of 2 × 10^–5 Pa. The probe of the AFM cantilever had a flat square tip, approximately 1 × 1 m² in area. The radius of curvature of the asperity peaks ranged from 70 to 610 nm. The results showed that the pull-off force was roughly proportional to this radius. The friction force was proportional to the pull-off force. Effects of the substrate temperature on pull-off force on a plane (the flat substrate) and friction force on an asperity were also examined. The pull-off force on the flat substrate increased with increasing contact time at a substrate temperature of 100 °C or lower, but was independent of contact time at 190 °C or higher. This suggests that the capillary cannot form at a substrate temperature of 190 °C or higher. The friction force increased with lower sliding velocities at 100 °C or lower, suggesting the capillary has a lubricating effect that prevents direct solid contact.     

Rippling on Wear Scar Surfaces of Nanocrystalline Diamond Films After Reciprocating Sliding Against Ceramic Balls

The formation of nanoscopic ripple patterns on top of material surfaces has been reported for different materials and processes, such as sliding against polymers, high-force scanning in atomic force microscopy (AFM), and surface treatment by ion beam sputtering. In this work, we show that such periodic ripples can also be obtained in prolonged reciprocating sliding against nanocrystalline diamond (NCD) films. NCD films with a thickness of 0.8 µm were grown on top of silicon wafer substrates by hot-filament chemical vapor deposition using a mixture of methane and hydrogen. The chemical structure, surface morphology, and surface wear were characterized by Raman spectroscopy, scanning electron microscopy (SEM), and AFM. The tribological properties of the NCD films were evaluated by reciprocating sliding tests against Al₂O₃, Si₃N₄, and ZrO₂ counter balls. Independent of the counter body material, clear ripple patterns with typical heights of about 30 nm induced during the sliding test are observed by means of AFM and SEM on the NCD wear scar surfaces. Although the underlying mechanisms of ripple formation are not yet fully understood, these surface corrugations could be attributed to the different wear phenomena, including a stress-induced micro-fracture and plastic deformation, a surface smoothening, and a surface rehybridization from diamond bonding to an sp ² configuration. The similarity between ripples observed in the present study and ripples reported after repeated AFM tip scanning indicates that ripple formation is a rather universal phenomenon occurring in moving tribological contacts of different materials.     

Structural properties of 0.65Pb(Mg1/3Nb2/3)O3–0.35PbTiO3 relaxor ferroelectric thin films on SrRuO3 conducting oxides

Coating of 0.65Pb(Mg_1/3Nb_2/3)O₃–0.35PbTiO₃ (PMN–PT) relaxor ferroelectrics by a sol–gel method is followed by growth of epitaxial SrRuO₃ (SRO) metallic oxide electrodes on SrTiO₃ (STO) single-crystal substrate by pulsed laser deposition. High-quality PMN–PT films on SRO with preferred growth orientation were successfully fabricated by controlling the operation parameters. Structural properties of relaxor ferroelectric PMN–PT thin films on SRO/STO substrates have been studied by X-ray diffraction (XRD), transmission electron microscopy (TEM) and atomic force microscopy (AFM). In-plane and out-of-plane alignments of the heterostructure are confirmed and the structural twinning of the materials are also revealed.     

Structural properties of 0.65Pb(Mg1/3Nb2/3)O3-0.35PbTiO3 relaxor ferroelectric thin films on SrRuO3 conducting oxides

Coating of 0.65Pb(Mg(1/3)Nb(2/3))O(3)-0.35PbTiO(3) (PMN-PT) relaxor ferroelectrics by a sol-gel method is followed by growth of epitaxial SrRuO(3) (SRO) metallic oxide electrodes on SrTiO(3) (STO) single-crystal substrate by pulsed laser deposition. High-quality PMN-PT films on SRO with preferred growth orientation were successfully fabricated by controlling the operation parameters. Structural properties of relaxor ferroelectric PMN-PT thin films on SRO/STO substrates have been studied by X-ray diffraction (XRD), transmission electron microscopy (TEM) and atomic force microscopy (AFM). In-plane and out-of-plane alignments of the heterostructure are confirmed and the structural twinning of the materials are also revealed.     

Immobilisation of living bacteria for AFM imaging under physiological conditions

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.     

Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells

The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the ‘porosome’,—the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.     

Characterization of MEMS piezoresistive pressure sensors using AFM

Atomic force microscope (AFM) is adapted to characterize an ultrasensitive piezoresistive pressure sensor based on microelectromechanical system (MEMS) technology. AFM is utilized in contact mode to exert force on several different micromachined diaphragm structures using a modified silicon cantilever with a particle attached to its end. The applied force is adjusted by changing the trigger voltage during each engage step of the probe-tip on the diaphragm surface. The contact force is determined from the force plots obtained for each trigger voltage in advanced force mode. Low force values in the range of 0.3–5 μN have been obtained with this method. This force induces strain on the bridge-arm of the diaphragm where the polysilicon resistor is located. The resultant change in the resistance produced due to varying force/pressure is measured using a delta mode current–voltage (I–V) measurement set-up. The contact mode AFM in conjunction with a nanovoltmeter enables the calibration of very sensitive force sensors down to 0.3 μN.