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Recent studies of biomacromolecules by atomic force microscopy are summarized. The resolution and reproducibility of AFM when applied to soft biosamples has been gradually improved and AFM is now recognized as a most promising technology in nanometer scale observation and manipulation of biological structures.
The newly developed atomic force microscope (AFM) provides a unique window to the microworld of cells, subcellular structures, and biomolecules. The AFM can image the three-dimensional structure of biological specimens in a physiological environment. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. The process of image acquisition is such that the AFM can also measure forces at the molecular level. In addition, the AFM can interact with the sample, thereby manipulating the molecules in a defined manner--nanomanipulation! The AFM has been used to image living cells and the underlying cytoskeleton, chromatin and plasmids, ion channels, and a variety of membranes. Dynamic processes such as crystal growth and the polymerization of fibrinogen and physicochemical properties such as elasticity and viscosity in living cells have been studied. Nanomanipulations, including dissection of DNA, plasma membranes, and cells, and transfer of synthetic structures have been achieved. This review describes the operating principles, accomplishments, and the future promise of the AFM.
We used an atomic force microscope to investigate silicon nitride tip interactions with various materials (copper, nickel, silicon carbide) as a function of pH. The electrolyte used was 10(-3) M NaCl and the interactions observed through force versus distance curves (attraction or repulsion) depended on the pH value. Interaction forces calculation was derived from force versus distance curve data and the results are discussed in terms of electrostatic interactions using Zeta potential theory.
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In atomic force microscope studies of molecular thin films, a defined jump of the tip through the film is often observed once a certain threshold force has been exceeded. Here, we present a theory to describe this film rupture and to relate microscopic parameters to measurable quantities. We assume that the tip has to overcome an activation energy before the film ruptures. A universal relation between the force dependence of the activation energy and the approaching velocity of the tip is derived. Two complementary models for calculating the activation energy are presented: a continuum nucleation model and a discrete molecular model. Both models predict a narrow distribution of yield forces in agreement with experimental results.
In atomic force microscope studies of thin films often a defined jump of the tip through the film is observed once a certain threshold force has been exceeded. In particular, on lipid bilayers this is regularly observed. In a previous paper [H.-J. Butt and V. Franz, Phys. Rev. E 66, 031601 (2002)] we presented two complementary models to describe film rupture. The aim of this study was to verify these models. Experiments were done with solid supported bilayers consisting of dioleoyloxypropyl-trimethylammonium chloride (DOTAP) and dioleoylphosphatidylserine (DOPS) in aqueous solutions and with propanol. Both models describe experimental results adequately. In particular, a narrow distribution of yield forces and an increase of the mean yield force with increasing loading rate is correctly predicted. For the lipid bilayers spreading pressures of roughly 20 mN/m (DOTAP) and 5 mN/m (DOPS) were measured. Line tensions for the edge of a lipid bilayer ranged between 3 (DOTAP) and 6 pN (DOPS).
A near contact atomic force microscope operated at low-temperature is used for vertical manipulation of selected single atoms from the Si(111)-(7 x 7) surface. The strong repulsive short-range chemical force interaction between the closest atoms of both tip apex and surface during a soft nanoindentation leads to the removal of a selected silicon atom from its equilibrium position at the surface without additional perturbation of the (7 x 7) unit cell. Deposition of a single atom on a created vacancy at the surface is achieved as well. These manipulation processes are purely mechanical, since neither bias voltage nor voltage pulse is applied between probe and sample. Differences in the mechanical response of the two nonequivalent adatoms of the Si(111)-(7 x 7) with the load applied is also detected.
Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.
Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.
PURPOSE: To analyze the surface optics of 4 currently available intraocular lenses (IOLs) with atomic force microscopy. SETTING: Licryl Laboratory, University of Calabria, Rende, Italy. METHODS: The surface roughness and topography of poly(methyl methacrylate) (PMMA), silicone, hydrophobic, and hydrophilic acrylic IOLs were evaluated with atomic force microscopy in contact mode. The analysis was performed in a liquid environment using cantilevers with a 0.01 Newtonw/meter nominal elastic constant. Measurements were made over areas of 10 microm2 on different locations of the posterior optic surface of the IOL. RESULTS: Atomic force microscopy permitted high-resolution imaging of IOL optic surface characteristics. Surface topography showed different features with respect to the lens biomaterial. The root-mean-square roughness of the IOL optic surface was significantly different between lenses of various materials (P < .001). The hydrophobic acrylic and silicone IOLs had the lowest mean surface roughness, 3.8 nm +/- 0.2 (SD) and 4.0 +/- 0.5 nm, respectively, and the 2 PMMA IOLs had the highest mean surface roughness, 6.6 +/- 0.3 nm and 7.0 +/- 0.6 nm. CONCLUSIONS: Atomic force microscopy was effective and accurate in analyzing IOL optics. The surface topography of IOLs may vary with different manufacturing processes.
PURPOSE: To analyze the surface roughness of porcine corneas after excimer laser ablation with and without the smoothing procedure by means of atomic force microscopy. METHODS: Excimer laser photorefractive keratectomy (PRK) was performed on eight porcine corneas. Immediately following the procedure, smoothing was performed on four corneas using a viscous solution of 0.25% sodium hyaluronate. The corneas were examined in balanced salt solution after fixation in 2.5% glutaraldehyde solution using atomic force microscopy. Quantitative analysis of the ablated stromal surface topography was performed using the section analysis module of the atomic force microscopy software. Repeated measurements were made over small areas (< or =50 microm2) near the center of each ablation, with a vertical resolution of <1 nm. RESULTS: Images of the ablated stromal surface showed undulations and granule-like features on the ablated surface of the specimens. The specimens on which the smoothing procedure was performed (root-mean-square [RMS] rough: 0.152 +/- 0.014 microm) were more regular (P < .001) than those on which PRK alone was performed (RMS rough: 0.229 +/- 0.018 microm). CONCLUSIONS: Atomic force microscopy analysis requires a simpler preparation of the specimens with respect to that necessary for scanning electron microscopy; for this reason, atomic force microscopy techniques are more reliable for the study of biological surfaces and prove to be a feasible method to establish the differences when comparing different laser techniques. Our investigations highlight that although the laser cut of scanning-spot excimer laser systems is precise in removing even the smallest amounts of tissue, the smoothing technique may still be useful to reduce post-ablation roughness.
The ability of the atomic force microscope to measure forces with subnanonewton sensitivity at nanometer-scale lateral resolutions has led to its use in the mechanical characterization of nanomaterials. Recent studies have shown that the atomic force microscope can be used to measure the elastic moduli of suspended fibers by performing a nanoscale three-point bending test, in which the center of the fiber is deflected by a known force. We extend this technique by modeling the deflection measured at several points along a suspended fiber, allowing us to obtain more accurate data, as well as to justify the mechanical model used. As a demonstration, we have measured a value of 78 +/- 17 GPa for Young's modulus of bacterial cellulose fibers with diameters ranging from 35 to 90 nm. This value is considerably higher than previous estimates, obtained by less direct means, of the mechanical strength of individual cellulose fibers.
The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of purple membrane while imaging in buffer solution. At forces > or = 0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic purple membrane surface was revealed. When the stylus was retracted, the scanned membrane area was relabeled with antibodies within 10 min. The extracellular surface of purple membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the purple membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.
Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.1 and 1. 5 nm and a vertical resolution of approximately 0.1 nm. For all protein surfaces, TMAFM and CMAFM topographs were in excellent agreement. TMAFM was capable of imaging the fragile polypeptide loop connecting the transmembrane alpha-helices E and F of bacteriorhodopsin in its native extended conformation. The standard deviation (SD) of averages calculated from TMAFM topographs exhibited an enhanced minimum (between 0.1 and 0.9 nm) that can be assigned to the higher noise of the raw data. However, the SD difference, indicating the flexibility of protein subunits, exhibited an excellent agreement between the two imaging modes. This demonstrates that the recently invented imaging-mode TMAFM has the ability to faithfully record high-resolution images and has sufficient sensitivity to contour individual peptide loops without detectable deformations.
Atomic force microscopy (AFM) was applied to study uncoated virus particles and RNA prepared by stripping of tobacco mosaic virions (TMV) with mild alkali or urea and dimethylsulfoxide. We found that AFM is an appropriate method to study ribonucleoprotein and free RNA structures. Images of entire tobacco mosaic virions, partially uncoated TMV particles with protruding RNA molecule from one or both ends and individual RNA molecules are presented.
Biological samples can be investigated with atomic force microscopy (AFM) in either of two imaging modes: contact mode or tapping mode. Applications of fluid tapping mode AFM in biology and medical sciences are constantly growing and the data obtained with this technique are improving, especially in terms of resolution. Even dynamic processes can be observed almost as they would occur in vivo. AFM is a proven imaging technique that has recently gained attention in biomaterials due to its ability to analyze surface properties, non-destructively at nanometer-level resolution in ambient air or in fluid environments.
In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.
Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) measurements have been shown to be consistent with the presence of nanofibrils in the cocoon silk of Bombyx mori and the dragline silk of Nephila clavipes. The transverse dimensions and correlation lengths range from >> 59 to 220 nm and in the axial direction from >> 80 to 230 nm. Also, the two-dimensional Fourier transforms of the height profiles of AFM topographic images of interior surfaces of B. mori follow a power law approximately the same as that for the Porod region of the SAXS data. In this manner, the AFM can be used to help remove ambiguity about the scatterers responsible for SAXS patterns.