Fabrication of carbon nanotube AFM probes using the Langmuir–Blodgett technique

Carbon nanotube (CNT)-tipped atomic force microscopy (AFM) probes have shown a significant potential for obtaining high-resolution imaging of nanostructure and biological materials. In this paper, we report a simple method to fabricate single-walled carbon nanotube (SWNT) nanoprobes for AFM using the Langmuir–Blodgett (LB) technique. Thiophenyl-modified SWNTs (SWNT-SHs) through amidation of SWNTs in chloroform allowed to be spread and form a stable Langmuir monolayer at the water/air interface. A simple two-step transfer process was used: (1) dipping conventional AFM probes into the Langmuir monolayer and (2) lifting the probes from the water surface. This results in the attachment of SWNTs onto the tips of AFM nanoprobes. We found that the SWNTs assembled on the nanoprobes were well-oriented and robust enough to maintain their shape and direction even after successive scans. AFM measurements of a nano-porous alumina substrate and deoxyribonucleic acid using SWNT-modified nanoprobes revealed that the curvature diameter of the nanoprobes was less than 3 nm and a fine resolution was obtained than that from conventional AFM probes. We also demonstrate that the LB method is a scalable process capable of simultaneously fabricating a large number of SWNT-modified nanoprobes.     

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.     

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip–sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase–distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever–sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.     

原子力显微镜原理与应用技术

本文简述原子力显微镜的工作原理,对比说明敲击模式的优越性,指出针尖-样品卷积效应和假象产生的原因,并例证其应用领域及其测试效果。     

Atomic force microscopy of silica nanoparticles and carbon nanohorns in macrophages and red blood cells

The emerging interest in understanding the interactions of nanomaterial with biological systems necessitates imaging tools that capture the spatial and temporal distributions and attributes of the resulting nano–bio amalgam. Studies targeting organ specific response and/or nanoparticle-specific system toxicity would be profoundly benefited from tools that would allow imaging and tracking of in-vivo or in-vitro processes and particle-fate studies. Recently we demonstrated that mode synthesizing atomic force microscopy (MSAFM) can provide subsurface nanoscale information on the mechanical properties of materials at the nanoscale. However, the underlying mechanism of this imaging methodology is currently subject to theoretical and experimental investigation. In this paper we present further analysis by investigating tip-sample excitation forces associated with nanomechanical image formation. Images and force curves acquired under various operational frequencies and amplitudes are presented. We examine samples of mouse cells, where buried distributions of single-walled carbon nanohorns and silica nanoparticles are visualized.     

AFM imaging of functionalized double-walled carbon nanotubes

We present a comparative study of several non-covalent approaches to disperse, debundle and non-covalently functionalize double-walled carbon nanotubes (DWNTs). We investigated the ability of bovine serum albumin (BSA), phospholipids grafted onto amine-terminated polyethylene glycol (PL-PEG₂₀₀₀-NH₂), as well as a combination thereof, to coat purified DWNTs. Topographical imaging with the atomic force microscope (AFM) was used to assess the coating of individual DWNTs and the degree of debundling and dispersion. Topographical images showed that functionalized DWNTs are better separated and less aggregated than pristine DWNTs and that the different coating methods differ in their abilities to successfully debundle and disperse DWNTs. Height profiles indicated an increase in the diameter of DWNTs depending on the functionalization method and revealed adsorption of single molecules onto the nanotubes. Biofunctionalization of the DWNT surface was achieved by coating DWNTs with biotinylated BSA, providing for biospecific binding of streptavidin in a simple incubation step. Finally, biotin-BSA-functionalized DWNTs were immobilized on an avidin layer via the specific avidin–biotin interaction.     

High-resolution constant-height imaging with apertured silicon cantilever probes

We present high-resolution aperture probes based on non-contact silicon atomic force microscopy (AFM) cantilevers for simultaneous AFM and near-infrared scanning near-field optical microscopy (SNOM). For use in near-field optical microscopy, conventional AFM cantilevers are modified by covering their tip side with an opaque aluminium layer. To fabricate an aperture, this metal layer is opened at the end of the polyhedral probe using focused ion beams (FIB). Here we show that apertures of less than 50 nm can be obtained using this technique, which actually yield a resolution of about 50 nm, corresponding to λ/20 at the wavelength used. To exclude artefacts induced by distance control, we work in constant-height mode. Our attention is particularly focused on the distance dependence of resolution and to the influence of slight cantilever bending on the optical images when scanning at such low scan heights, where first small attractive forces exerted on the cantilever become detectable.     

Phase contrast in Simultaneous Topography and Recognition imaging

The operation of a force microscope in Simultaneous Topography and Recognition (TREC) imaging mode is analyzed by means of numerical simulations. Both topography and recognition signals are analyzed by using a worm-like chain force as the specific interaction between the functionalized tip probe and the sample. The special feedback mechanism in this mode is shown to couple the phase signal to the presence of molecular recognition interactions even in absence of dissipation.     

Analysis of force curves obtained on the live cell membrane using chemically modified AFM probes

Force curves were obtained on the live cell surface using an atomic force microscope mounted with a modified tip with the bifunctional covalent crosslinker, disuccinimidyl suberate, which forms a covalent bond with amino-bearing molecules on the cell surface. A ramp delay time of 1.0 s was introduced before the start of the retraction regime of the force curve to increase the stationary reaction time between the crosslinkers on the tip and the amino groups on the cell surface. While live cell surface responses to forced contact with a non-functionalized tip rarely showed evidence of tip–cell interaction, those obtained with modified tips gave clear indication of prolonged adhesion which was terminated by a single step release of the tip to its neutral position. Under the given experimental conditions of this work, 58% of a total of 198 force curves gave only one jump and 70% of those with one jump gave the final rupture force of 4.5±0.22 nN. The result emphasized the uniqueness of the observed mechanical response of the cell surface when probed with chemically modified tips.     

Nano-slit probes for near-field optical microscopy fabricated by focused ion beams

The near-field probes described in this paper are based on metallized non-contact atomic force microscope cantilevers made of silicon. For application in high-resolution near-field optical/infrared microscopy, we use aperture probes with the aperture being fabricated by focused ion beams. This technique allows us to create apertures of sub-wavelength dimensions with different geometries. In this paper we present the use of slit-shaped apertures which show a polarization-dependent transmission efficiency and a lateral resolution of < 100 nm at a wavelength of 1064 nm. As a test sample to characterize the near-field probes we investigated gold/palladium structures, deposited on an ultrathin chromium sublayer on a silicon wafer, in constant-height mode.     

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 μm within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of ≤10–15 μm into the sample, further compounding the ability to image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology.     

Nanotribology of cluster assembled carbon films

The frictional properties of cluster assembled carbon films have been investigated on nanometric scale by friction force microscopy. The experiment was performed at low loads to avoid plastic deformation and wear of materials. We found that load-dependent measurements acquired on samples with different composition present excellent agreement with the Hertzian-plus-offset model. A quantitative comparison among these films and atom-assembled carbon compounds is finally presented.     

Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM-AFM probes

With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with AFM has recently been introduced. Simultaneous contact mode imaging of a micropatterned sample with immobilized enzyme spots and imaging of enzyme activity is shown. In contrast to force spectroscopy the conversion of an enzymatic byproduct is directly detected during AFM imaging and correlated to the activity of the enzyme.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

Coaxial probes for scanning near-field microscopy

This paper deals with the development of coaxial aperture tips integrated in a cantilever probe for combined scanning near-field infrared microscopy and scanning force microscopy. A fabrication process is introduced that allows the batch fabrication of hollow metal aperture tips integrated on a silicon cantilever. To achieve the coaxial tip arrangement a metal rod is deposited inside the hollow tip using the focused ion beam technique. Theoretical calculations with a finite integration code were performed to study the transmission characteristics of coaxial tips in comparison with conventional aperture probes. In addition, the influence of the geometrical design parameters of the coaxial probe on its optical behaviour is investigated.     

A symmetrical stretching stage for electrical atomic force microscopy

We present a remotely-controlled device for sample stretching, designed for use with atomic force microscopy (AFM) and providing electrical connection to the sample. Such a device enables nanoscale investigation of electrical properties of thin gold films deposited on polydimethylsiloxane (PDMS) substrate as a function of the elongation of the structure. Stretching and releasing is remotely controlled with use of a dc actuator. Moreover, the sample is stretched symmetrically, which gives an opportunity to perform AFM scans in the same site without a time-consuming finding procedure. Electrical connections to the sample are also provided, enabling Kelvin probe force microscopy (KPFM) investigations. Additionally, we present results of AFM imaging using the stretching stage.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

Probe three-dimensional structure of soft organized surfactants at solid–liquid interface with an AFM in MAC Mode and contact mode

Atomic force microscopy provides a unique direct-visualization tool to study the three-dimensional structure of adsorbed surfactants on solid surfaces. Ionic surfactant molecules spontaneously adsorbed onto hydrophilic surfaces from aqueous solution above the critical micelle concentration (cmc) have been imaged using an atomic force microscope in magnetic ac mode (MAC Mode) and contact mode. It was found that the soft organized surfactants were highly compressible and therefore showed a wide range of corrugations depending on imaging forces. When using gentle MAC Mode, corrugations of the organized surfactants around half of the estimated height of the proposed surfactant aggregate cylinders on mica have been stably observed. Traditional contact mode operating in the pre-contact double-layer electrostatic interaction region, however, showed significantly reduced height of the organized molecules.     

Sensitivity and resonant frequency of an AFM with sidewall and top-surface probes for both flexural and torsional modes

The resonant frequencies and flexural sensitivities of an atomic force microscope (AFM) with assembled cantilever probe (ACP) are studied. This ACP comprises a horizontal cantilever, a vertical extension and two tips located at the free ends of the cantilever and the extension, which makes the AFM capable of simultaneous topography at top surface and sidewalls of microstructures especially microgears, which consequently leads to a time-saving swift scanning process. In this work, the effects of the sample surface contact stiffness and the geometrical parameters such as the ratio of the vertical extension length to the horizontal cantilever length and the distance of the vertical extension from clamped end of the horizontal cantilever on both flexural and torsional resonant frequencies and sensitivities are assessed. These geometrical effects are illustrated in some figures. The results show that the low-order vibration modes are more sensitive for low values of the contact stiffness, but the situation is reversed for high values.     

Atomic force microscopy analysis of enveloped and non-enveloped viral entry into, and egress from, cultured cells

Since its invention, the atomic force microscope has been used to image a wide variety of biological samples, including viruses. Viral entry into, and egress from, cultured cells has been extensively studied using numerous scientific techniques and to a limited extent using atomic force microscopy. One of the main structural differences that can exist between viruses is the absence, or presence, of an envelope and this factor has consequences for the mode of viral entry and egress. In this study, the entry into, and egress from, cultured cells of enveloped and non-enveloped viruses were investigated using atomic force microscopy. No significant cell surface changes were observed following infection with enveloped or non-enveloped viruses. Although roughness analysis of viral entry revealed cell smoothing post-infection, no differences between the roughness values of enveloped and non-enveloped viral entry were observed. Line analysis of viral entry revealed minor differences between cells infected with an enveloped rather than a non-enveloped virus. These differences may represent a distinction between the uptake processes of enveloped and non-enveloped viruses. Studies of viral egress revealed that infected cells were undergoing cytopathic changes. Whilst topographic, height and roughness differences clearly occurred between virally- and mock-infected cells, no significant differences were elucidated between enveloped and non-enveloped viral egress.