Atomic force microscopy investigation of chemically stabilized pericardium tissue

Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.     

A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils that display a banding with periodicity greater than the 67nm periodicity of native collagen. FLS fibrils can be formed in vitro by addition of alpha(1)-acid glycoprotein to an acidified solution of monomeric collagen, followed by dialysis of the resulting mixture. We have investigated the ultrastructure of FLS fibrils formed in vitro using the atomic force microscope (AFM). The majority of the fibrils imaged showed typical diameters of approximately 150nm and had a distinct banding pattern with a approximately 250nm periodicity. However, we have also observed an additional type of FLS fibril, which is characterized by a secondary banding pattern surrounding the primary bands. These results are compared with those obtained in past investigations of FLS ultrastructure carried out using the transmission electron microscope (TEM). The importance of the fibril's surface topography in TEM staining patterns is discussed. Images of FLS fibrils in various stages of assembly have also been collected, and the implications of these images in determining the mechanism of assembly and the formation of the characteristic banding pattern of the fibrils is discussed.     

Atomic force microscopy on human trabecular bone from an old woman with osteoporotic fractures

AFM images were taken of the exterior surface of a single trabecula, extracted from a human femoral head removed during surgery for a hip fracture in an old women with former fractures. The images showed a dense structure of bundled collagen fibrils banded with 67 nm periodicity. Bundles were seen to run in parallel in layers confirming the collagen structure seen by other techniques. Single collagen fibrils were seen to cross the bundles, thus forming cross-links between neighboring bundles of collagen fibrils. Some of these crossing fibrils did not have the 67 nm band pattern and their dimensions were about half compared to the neighboring collagen fibrils. Very little mineral was found on the surface of the trabecula. An AFM image of a fracture plane was also displayed. The trabecula was extracted from a region close to the hip fracture. However, there were in this case no obvious features in the images that could be linked directly to osteoporosis, but altered collagen banding and collagen protrusions may alter mechanical competence. A path to extensive studies of the nanometer scale structure of bone was demonstrated.     

Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy

Atomic force microscopy (AFM) was used in concert with transmission electron microscopy (TEM) to image magnetotactic bacteria (Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magneticum AMB-1), magnetosomes, and purified Mms6 proteins. Mms6 is a protein that is associated with magnetosomes in M. magneticum AMB-1 and is believed to control the synthesis of magnetite (Fe(3)O(4)) within the magnetosome. We demonstrated how AFM can be used to capture high-resolution images of live bacteria and achieved nanometer resolution when imaging Mms6 protein molecules on magnetite. We used AFM to acquire simultaneous topography and amplitude images of cells that were combined to provide a three-dimensional reconstructed image of M. gryphiswaldense MSR-1. TEM was used in combination with AFM to image M. gryphiswaldense MSR-1 and magnetite-containing magnetosomes that were isolated from the bacteria. AFM provided information, such as size, location and morphology, which was complementary to the TEM images.     

Mechanical heterogeneity of dentin at different length scales as determined by AFM phase contrast

In this study we sought to gain insights of the structural and mechanical heterogeneity of dentin at different length scales. We compared four distinct demineralization protocols with respect to their ability to expose the periodic pattern of dentin collagen. Additionally, we analyzed the phase contrast resulting from AFM images obtained in tapping mode to interrogate the viscoelastic behavior and surface adhesion properties of peritubular and intertubular dentin, and partially demineralized dentin collagen fibrils, particularly with respect to their gap and overlap regions. Results demonstrated that all demineralization protocols exposed the gap and overlap zones of dentin collagen fibrils. Phase contrast analyses suggested that the intertubular dentin, where the organic matrix is concentrated, generated a higher phase contrast due a higher contribution of energy dissipation (damping) than the highly mineralized peritubular region. At increasing amplitudes, viscoelasticity appeared to play a more significant contribution to the phase contrast of the images of collagen fibrils. The overlap region yielded a greater phase contrast than the more elastic gap zones. In summary, our results contribute to the perspective that, at different length scales, dentin is constituted of structural features that retain heterogeneous mechanical properties contributing to overall mechanical performance of the tissue. Furthermore, the interpretation of phase contrast from images generated with AFM tapping mode appears to be an effective tool to gain an improved understanding of the structure and property relationship of biological tissues and biomaterials at the micro- and nano-scale.     

Understanding tapping-mode atomic force microscopy data on the surface of soft block copolymers

In this paper, we focus on better understanding tapping-mode atomic force microscopy (AFM) data of soft block copolymer materials with regard to: (1) phase attribution; (2) the relationship between topography and inside structure; (3) contrast-reversal artifacts; (4) the influence of annealing treatment on topography. The experiments were performed on the surface of poly(styrene–ethylene/butylene–styrene) (SEBS) triblock copolymer acting as a model system. First, by coupling AFM with transmission electron microscopy (TEM) measurements, the phase attribution for AFM images was determined. Secondly, by imaging an atomically flat SEBS surface as well as an AFM tip-scratched SEBS surface, it was confirmed that the contrast in AFM height images of soft block copolymers is not necessarily the result of surface topography but the result of lateral differences in tip-indentation depth between soft and hard microdomains. It was also found that there is an enlarging effect in AFM images on the domain size of block copolymers due to the tip-indention mechanism. Thirdly, based on the tip-indention mechanism, tentative explanations in some detail for the observed AFM artifacts (a reversal in phase image followed by another reversal in height image) as function of imaging parameters were given. Last, it was demonstrated that the commonly used annealing treatments in AFM sample preparation of block copolymers may in some cases lead to a dramatic topography change due to the unexpected order-to-order structure transition.     

Atomic force microscopy on surfaces with a developed profile

We propose a new operation mode for an atomic force microscope (vertical mode), which can be used to measure the topography and other physical parameters of a surface with deep pores, vertical walls, and significant height differences. The measurement of height in this mode is conducted upon vertical uniform motion of the probe towards the surface. Corresponding AFM images are presented.     

Imaging sclera with hard X-ray microscopy

In this study, the organization of collagen fibrils within the sclera of the eye was investigated using the 7 keV hard X-ray microscope of the Pohang light source and compared to images from electron and atomic force microscopy. From the captured X-ray images, individual collagen fibrils were observed clearly in a spatial resolution much better than 100 nm, both in longitudinal sections and in transverse sections. Some of the collagen fibrils showed evidence of axial periodicity. In some regions of the samples, we could see cross-bridge like structures between adjacent collagen fibrils. The X-ray microscope also allowed the observation of keratocytes and the lamella structure of the scleral stroma. The X-ray microscope has some unique advantages in the nano-scale imaging of bio-samples relative to other established imaging techniques.     

Observation of angiotensin II-induced changes in fixed and live mesangial cells by atomic force microscopy

Glomerular mesangial cells (MCs) are centrally located in the glomerulus. MCs control not only glomerular filtration, but also the response to local injury, including cell proliferation and basement membrane remodeling. Angiotensin II (Ang II) plays an important role in kidney function regulation, and participates in the progression of renal damage, as well as mesangial injury. However, studies on Ang II effects on MCs have used indirect methods, such as gene and protein expression after MC injury. In this study, we visually observed structural and mechanical changes to MC after Ang II treatment using atomic force microscopy (AFM). We obtained AFM topography and deflection images of live MCs, as well as fixed MCs in liquid, before and after Ang II treatment. Real-time imaging showed the dynamic movement of live MCs induced by Ang II. Changes in MC elastic property after Ang II treatment were measured using force–distance curves. AFM images of fixed and live MCs showed that cells contracted after Ang II exposure, with the nucleus height increasing within 20 min of Ang II stimulation. Force–distance analysis showed that Ang II caused MCs to stiffen (p < 0.0001). In conclusion, we demonstrated that AFM is an effective tool for real-time monitoring of live cell responses to drugs and stimuli.     

Chemical surface composition of the polyethylene implanted by Ag ions studied by phase imaging atomic force microscopy

High density polyethylene (HDPE) has been modified by Ag⁺ ion implantation with the energy of 60 keV. The total amount of implanted silver ions was 1, 5 and 12 × 10¹⁵ ions/cm². The surface topography was observed by atomic force microscopy (AFM), while the surface composition changes were detected using phase imaging AFM. Surface topography changes were studied in detail using 3D surface parameters analyses. The average roughness decreased for the implanted HDPE indicating the flattening of the surface. Phase AFM images indicated the homogenization of the polyethylene during ion implantation, while histogram analyses confirmed the change in surface composition.     

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.     

High-speed synthetic aperture microscopy for live cell imaging

We present a high-speed synthetic aperture microscopy for quantitative phase imaging of live biological cells. We measure 361 complex amplitude images of an object with various directions of illumination covering an NA of 0.8 in less than one-thirteenth of a second and then combine the images with a phase-referencing method to create a synthesized phase image. Because of the increased depth selectivity, artifacts from diffraction that are typically present in coherent imaging are significantly suppressed, and lateral resolution of phase imaging is improved. We use the instrument to demonstrate high-quality phase imaging of live cells, both static and dynamic, and thickness measurements of a nanoscale cholesterol helical ribbon.     

Thermo-mechanical properties of the cell surface assessed by atomic force microscopy

Atomic force microscopy (AFM) in lateral force mode was applied to assess the microscale thermo-mechanical (frictional) properties of the air-dried cell surface in the wide temperature range (288-363K/15-90°C). AFM-investigated cell surface layer can be represented as a biocomposite composed of several layers including the glycocalyx, the membrane and the intercellular layer containing membrane (cortical) cytoskeleton. The cells with two different cytoskeleton structures, erythrocytes and thymocytes, were studied. Above a certain temperature (T(g)), the significant change in friction force with temperature was revealed for the both cell types whereas there was no similar change in their topography parameters. The experimentally determined value T(g) for erythrocyte samples was lower than that for thymocyte ones. Treating living cells with the cross-linking agent, glutaraldehyde, led to the weakening of the temperature dependence of air-dried cell surface frictional properties in the studied temperature range. Addition of oxidizing agent, peroxynitrite, to living cell suspensions changed the temperature dependence of air-dried cell surface frictional properties depending on cell type and peroxynitrite concentration. The obtained data indicate that the study of thermo-mechanical properties of air-dried cells with AFM in lateral force mode provides expanded information on the structural characteristics of the living cell surface layer, and sets the stage for the development of AFM-based method (with using a lateral force mode) for the cell pathology diagnostics.     

Internal reflection mode scanning near-field optical microscopy with the tetrahedral tip on metallic samples

An internal reflection mode is introduced for scanning near-field optical microscopy (SNOM) with the tetrahedral tip. A beam of light is coupled into the tip and the light specularly reflected out of the tip is detected as a photosignal for SNOM. An auxiliary STM mode is used to control the distance during the scanning process and to record the topography of the sample simultaneously with the SNOM image. Images were obtained of different metallic samples which show a contrast in the order of 10% of the total reflected photosignal. In images of metallic samples an inverted contrast is consistently obtained compared to images previously obtained of comparable samples in a transmission mode. The contrast shows a pronounced dependence on the polarization of the incident beam with respect to the orientation of the edges of the tip. In the case of gold surfaces, the photosignal as a function of distance between the tip and the surface shows a pronounced peak in the near-field range of 0–20 nm which is tentatively attributed to the excitation of surface plasmons on the gold surface. The pronounced near-field effects and the strong contrast in the near-field images and the resolution well below 50 nm are an indication of a highly efficient coupling of the incident beam to a local excitation of the tip apex which is essential for the function of the tip as a probe for SNOM. Received: 17 May 1999 / Accepted: 18 May 1999 / Published online: 21 October 1999     

Quantification of the lift height for magnetic force microscopy using 3D surface parameters

In this work, the quantitative conditions for the lift height for imaging of the magnetic field using magnetic force microscopy (MFM) were optimized. A thin cobalt film deposited on a monocrystalline silicon (1 0 0) substrate with a thickness of 55 nm and a thin nickel film deposited on a glass with a thickness of 600 nm were used as samples. The topography of the surface was acquired by tapping mode atomic force microscopy (AFM), while MFM imaging was performed in the lift mode for various lift heights. It was determined that the sensitivity of the measurements was about 10% higher for images obtained at a scan angle of 90° compared to a scan angle of 0°. Therefore, the three-dimensional surface texture parameters, i.e., average roughness, skewness, kurtosis and the bearing ratio, were determined in dependence on the lift height for a scan angle of 90°. The results of the analyses of the surface parameters showed that the influence of the substrate and its texture on the magnetic force image could be neglected for lift heights above 40 nm and that the upper lift height limit is 100 nm. It was determined that the optimal values of the lift heights were in the range from 60 to 80 nm, depending on the nature of the sample and on the type of the tip used.     

Characterization of tip size and geometry of the pipettes used in scanning ion conductance microscopy

Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the probe. In SICM the probe is a pipette, obtained by heating and pulling a glass capillary tubing. The size of the pipette tip is therefore an important parameter in SICM experiments. However, the characterization of the tip is not a consolidated routine in SICM experimental practice. In addition, potential and limitations of the different methods available for this characterization may not be known to all users. We present an overview of different methods for characterizing size and geometry of the pipette tip, with the aim of collecting and facilitating the use of several pieces of information appeared in the literature in a wide interval of time under different disciplines. In fact, several methods that have been developed for pipettes used in cell physiology can be also fruitfully employed in the characterization of the SICM probes. The overview includes imaging techniques, such as scanning electron microscopy and atomic Force microscopy, and indirect methods, which measure some physical parameter related to the size of the pipette. Examples of these parameters are the electrical resistance of the pipette filled with a saline solution and the surface tension at the pipette tip. We discuss advantages and drawbacks of the methods, which may be helpful in answering a wide range of experimental questions.     

Atomic force microscopy of Bacillus spore surface morphology

Bacillus spore surface morphology was imaged with atomic force microscopy (AFM) to determine if characteristic surface features could be used to distinguish between four closely related species; Bacillus anthracis Sterne strain, Bacillus thuringiensis var. kurstaki, Bacillus cereus strain 569, and Bacillus globigii var. niger. AFM surface height images showed an irregular topography across the curved upper surface of the spores. Phase images showed a superficial grain structure with different levels of phase contrast and significant differences in average surface morphologies among the four species. Although spores of the same species showed similarities, there was significant variability within each species. Overall, AFM revealed that spore surface morphology is rich with information, which can be used to distinguish a sample of about 20 spores from a similar number of spores of closely related species. Statistical analysis of spore morphology from a combination of amplitude and phase images for a small sample allows differentiation between, B. anthracis and its close relatives.     

Assessing the structure and function of single biomolecules with scanning transmission electron and atomic force microscopes

The scanning transmission electron microscope (STEM) and the atomic force microscope (AFM) have provided a wealth of useful information on a wide variety of biological structures. These instruments have in common that they raster-scan a probe over a sample and are able to address single molecules. In the STEM the probe is a focused electron beam that is deflected by the scan-coils. Detectors collecting the scattered electrons provide quantitative information for each sub-nanometer sized sample volume irradiated. These electron scattering data can be reconstituted to images of single macromolecules or can be integrated to provide the mass of the macromolecules. Samples need to be dehydrated for such quantitative STEM imaging. In contrast, the AFM raster-scans a sharp tip over a sample surface submerged in a buffer solution to acquire information on the sample's surface topography at sub-nanometer resolution. Direct observation of function-related structural changes induced by variation of temperature, pH, ionic strength, and applied force provides insight into the structure-function relationship of macromolecules. Further, the AFM allows single molecules to be addressed and quantitatively unfolded using the tip as nano-tweezers. The performance of these two scanning probe approaches is illustrated by several examples including the chaperonin GroEL, bacterial surface layers, protein crystals, and bacterial appendices.     

原子力显微镜探针耦合变形下的微观扫描力研究

原子力显微镜(AFM)的微探针系统是典型的微机械构件，它在接触扫描过程处于耦合变形状态.采用数值模拟方法探究恒力模式下探针耦合变形对微观扫描力信号、微观形貌信号的影响.研究表明，AFM的恒力模式扫描中，法向扫描力并不是恒定大小，与轴向扫描力存在耦合作用，在粗糙峰峰值增加阶段，二力均增加；在粗糙峰峰值减小阶段，二力均减小；该耦合作用随形貌坡度、针尖长度等增加而加强.微观形貌的测试信号和横向扫描侧向力信号受探针耦合变形影响较小，但侧向力与形貌斜率密切相关，且其极值点与形貌极值点存在位置偏差，这些结果均与原子力 关键词： 原子力显微镜 探针悬臂梁 耦合变形 扫描力