Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus—HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine–phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.     

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.     

Nano-wear of the diamond AFM probing tip under scratching of silicon, studied by AFM

In order to improve such a widely used microtribological testing procedure as surface scratching by an AFM diamond tip, an experimental study has been carried out using single-crystalline silicon as the tested material. Wear of the AFM diamond tip under scratching was observed by a decrease in the scratch depth with increasing wear cycles and by the direct imaging of the diamond tip shape using a Si₃N₄ AFM tip. It was shown that the current widely used experimental method, which assumes the diamond tip to be non-wearable, introduces uncontrollable error into the obtained values for the tested material's wear rate. The harder the tested material, the larger may be the tip wear, and, therefore, the bigger may be its effect on the obtained wear rate values. The specific wear rates for the diamond tip and a silicon wafer were estimated to be 1.4 × 10^-9 and 4.5 × 10^-4 mm³/(N m), respectively.     

High resolution imaging of surface patterns of single bacterial cells

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

Optimized hand fabricated AFM probes for simultaneous topographical and electrochemical tapping mode imaging

In this work hybrid AFM-electrochemical (SECM) probes to be used in dynamic atomic force microscopy are presented. These nanosensors are hand fabricated from gold microwires using a simple benchtop method. They display proportions close to commercially available silicon and silicon nitride cantilevers giving comparable performance in terms of resolution and imaging stability. The remarkable characteristic of these hybrid nanosensors is that they allow the coupling of 3D imaging ability and versatility of atomic force microscopy with the power of electrochemical methods. Local measurement of electrochemical-activity of a test sample consisting of gold bands functionalized by redox-labeled nanometer-sized polyethylene glycol chains has been achieved with simultaneous imaging of the 3D surface topography at high resolution. These hybrid AFM-SECM tips are capable of sensing local electrochemical currents down to ∼10 fA emphasizing the sensitivity and resolution of this technique.     

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.     

Instrumental requirements for high resolution imaging

The resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma-free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution-checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on-line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful. Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low-Z specimens, particularly in the dark field mode. Finally, some ‘non-conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.     

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.     

AFM工作台扫描控制电路的设计

在现有理论的基础上,设计出一种新型的AFM工作台扫描控制电路。介绍了该电路的设计思想,与现有的AFM控制系统相比,X、Y方向的运动采用闭环控制,提高了控制精度。文中还介绍了一种简单实用的PI调节电路,该电路具有结构简单、成本低、功耗小和控制精度高等优点。     

High accuracy FIONA-AFM hybrid imaging

Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein-DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA-AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8 nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5-10 nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.     

Lateral Displacement of an AFM Tip Observed by In-Situ TEM/AFM Combined Microscopy: The Effect of the Friction in AFM

When the lateral displacement of an AFM tip due to friction is comparable to or larger than the scan size, for example during atomic-scale friction measurement, the interpretation of the friction image is different from the situation where the scan size is much larger than the lateral displacement of the tip and the image is a simple direct mapping of the friction value. This is because, due to the lateral displacement of the tip, the tip is not at the position where the scan indicates, as can be clearly observed by an in-situ TEM/AFM combined microscopy and atomic-scale friction analysis. This lateral displacement of the tip at the nanometer scale affects the shape of the force-distance curve. We discuss the effect of the tip lateral displacement in AFM data and its normal load dependence.     

AFM picking-up manipulation of the metaphase chromosome fragment by using the tweezers-type probe

We have studied the development of a new procedure based on atomic force microscopy (AFM) for the analysis of metaphase chromosome. The aim of this study was to obtain detailed information about the specific locations of genes on the metaphase chromosome. In this research, we performed the manipulation of the metaphase chromosome by using novel AFM probes to obtain chromosome fragments of a smaller size than the ones obtained using the conventional methods, such as glass microneedles. We could pick up the fragment of the metaphase chromosome dissected by the knife-edged probe by using our tweezers-type probe.     

Preparation of TiO2 nanowire gas nanosensor by AFM anode oxidation

Applications of atomic force microscopy (AFM) to the fabrication of chemical nanosensors are presented in this paper. Using AFM cantilever as cathode, the surface of Ti thin film is oxidized to form a few tens of nanometers wide oxidized metal semiconductor wire, which works as a nanowire-based hydrogen sensor. The reaction mechanism is proposed. The AFM observations of fabrication of a TiO₂ nanowire are carried out. The sensitive characteristic of such TiO₂ nanowires to hydrogen is investigated.     

Contribution to the elucidation of the structure of the bacterial flagellum nano-motor through AFM imaging of the M-Ring

The flagellar nano-motor of bacteria is one of the most interesting and amazing natural nano-machine. Despite its discovery 30 years ago, some details of its structure and mechanisms are not yet elucidated. Several studies have revealed some important aspects of its structure and numerous data are available today; however, the inner mechanisms of the nano-motor have not been yet resolved, partially due to the lack of information about the 3D assembly, shape and interactions of the different parts in experimental environment as close as possible as the native cellular conditions. We have developed an approach using atomic force microscopy imaging in liquid media, which allows us to study part of the motor in native liquid environment. In this work, we are interested in the FliG proteins, identified as the key functional proteins of this nano-machine. We report 3D images of their assembly on surfaces, which could be representative of the so-called M-ring part of the nano-motor. These images have been acquired on both mica surfaces and on supported bilayer membranes mimetics of E. coli native membrane. The systematic analysis of the shape and the size of different recorded assemblies made us believe that the FliG organization we observed could lead to a new model for the structure and mechanism of the flagellar nano-motor.     

An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells

This communication presents the design and application of an integrated environmental perfusion chamber and stage heating blanket suitable for time-lapse video microscopy of living cells. The system consists of two independently regulated components: a perfusion chamber suitable for the maintenance of cell viability and the variable delivery of environmental factors, and a separate heating blanket to control the temperature of the microscope stage and limit thermal conduction from the perfusion chamber. Two contrasting experiments are presented to demonstrate the versatility of the system. One long-term sequence illustrates the behaviour of cells exposed to ceramic fibres. The other shows the shrinking response of cultured articular cartilage chondrons under dynamic hyper-osmotic conditions designed to simulate joint loading. The chamber is simple in design, economical to produce and permits long-term examination of dynamic cellular behaviour while satisfying the fundamental requirements for the maintenance of environmental factors that influence cell viability.     

Improvement of the pore trapping method to immobilize vital coccoid bacteria for high‐resolution AFM: a study of Staphylococcus aureus

Preparation of vital bacteria for atomic force microscope study under aqueous fluid, such as physiological buffer or bacterial growth medium, presents challenges as cells will often desorb from the supporting surface or be dislodged by the atomic force microscope tip during imaging. An established method of immobilizing coccoid bacteria is to trap cells in polycarbonate track etched filter pores. We have significantly improved this method by modifying the pore diameter of commercially available filters to correspond to the diameter of the target strain, enabling high‐resolution imaging of stationary organisms under buffer and dividing organisms under growth media.     

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.     

AFM针尖-试样面作用力连续介质方法研究

介绍了目前研究纳米接触问题的方法；根据Hamaker理论，利用连续介质方法，建立了原子力显微镜针尖和试样面接触的包括斥力项的力学模型；对模型进行了仿真，仿真结果同实验现象一致。将纳米接触问题的研究领域从非接触区域扩展到间歇接触区域。为原子力显微镜的进一步研究。实现原子操纵提供理论基础。     

Direct observation of PFPE lubricant molecules by cryogenic AFM under ultra-high vacuum

Surface lubrication is one of the essential technologies in modern magnetic disk systems and improvement of the surface lubrication is very important in the development of next generation systems. In this study, we used AFM for the direct observation of perfluoropolyether (PFPE) lubricant molecules on atomically flat surfaces. We used a cryogenic non-contact AFM to observe the molecules in a frozen state of micro-Brownian motion of PFPE segments, because the glass transition temperature of PFPE is very low. To avoid freezing a trace amount of water vapor on the sample surface at liquid nitrogen temperatures, the AFM observation was performed under ultra-high vacuum. We observed that on a gold surface the size of the molecules increases with repeated AFM scans. This is because the mechanical stimulus causes the fusion of PFPE lubricant molecules to form reversed micelles at the non-polar surface. At a hydrophilic silicon wafer surface, however, we succeeded in observing single lubricant molecules. This is because almost all PFPE lubricant molecules are fixed to the hydrophilic solid surface by polar–polar bond formation and they cannot move around on the surface and thus they cannot fuse to each other. As formation of the reversed micelle structure is a rather general phenomenon in the PFPE lubricant thin layer at non-polar surfaces, we also will discuss briefly the expected molecular structures of PFPE lubricants at the surface of the carbon overcoat of magnetic disks.     

Characterization of micellar film morphologies of semifluorinated block copolymers by AFM

The micellar morphologies of well-defined amphiphilic block copolymers composed of 1H,1H-dihydroperfluorooctyl methacrylate (FOMA) and ethylene oxide (EO) blocks with different chain lengths were effectively investigated by using tapping mode-atomic force microscopy (TM-AFM). By spin-casting chloroform solutions, well-ordered spherical micellar films could be obtained for poly(FOMA(10k)-b-EO(10k)) and poly(FOMA(20k)-b-EO(20k)) copolymers. The atomic force microscopy (AFM) height and phase image analysis indicated that dark regions of the micelles corresponded to PEO blocks and the light regions were for PFOMA blocks. The spherical micelles with PEO corona and PFOMA core were also identified by transmission electron microscopy (TEM) and X-ray photoelectron spectrometer (XPS) analysis. The core diameters of the block-copolymer aggregates were 20nm for poly(FOMA(10k)-b-EO(10k)) and 30nm for poly(FOMA(20k)-b-EO(20k)) by TM-AFM, whereas slightly lower values of 17 and 26nm were obtained from TEM. A detailed study on the inverted morphological change observed in the micelles films after annealing above glass transition temperature (T(g)) was also presented.