Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

Analysis of dynamic cantilever behavior in tapping mode atomic force microscopy

Tapping mode atomic force microscopy (AFM) provides phase images in addition to height and amplitude images. Although the behavior of tapping mode AFM has been investigated using mathematical modeling, comprehensive understanding of the behavior of tapping mode AFM still poses a significant challenge to the AFM community, involving issues such as the correct interpretation of the phase images. In this paper, the cantilever's dynamic behavior in tapping mode AFM is studied through a three dimensional finite element method. The cantilever's dynamic displacement responses are firstly obtained via simulation under different tip‐sample separations, and for different tip‐sample interaction forces, such as elastic force, adhesion force, viscosity force, and the van der Waals force, which correspond to the cantilever's action upon various different representative computer‐generated test samples. Simulated results show that the dynamic cantilever displacement response can be divided into three zones: a free vibration zone, a transition zone, and a contact vibration zone. Phase trajectory, phase shift, transition time, pseudo stable amplitude, and frequency changes are then analyzed from the dynamic displacement responses that are obtained. Finally, experiments are carried out on a real AFM system to support the findings of the simulations. Microsc. Res. Tech. 78:935–946, 2015. © 2015 Wiley Periodicals, Inc.     

Atomic force microscopy of histological sections using a chemical etching method

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.     

Atomic force microscopy applied to study macromolecular content of embedded biological material

We demonstrate that atomic force microscopy represents a powerful tool for the estimation of structural preservation of biological samples embedded in epoxy resin, in terms of their macromolecular distribution and architecture. The comparison of atomic force microscopy (AFM) and transmission electron microscopy (TEM) images of a biosample (Caenorhabditis elegans) prepared following to different types of freeze-substitution protocols (conventional OsO4 fixation, epoxy fixation) led to the conclusion that high TEM stainability of the sample results from a low macromolecular density of the cellular matrix. We propose a novel procedure aimed to obtain AFM and TEM images of the same particular organelle, which strongly facilitates AFM image interpretation and reveals new ultrastructural aspects (mainly protein arrangement) of a biosample in addition to TEM data.     

Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy

Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three‐dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory‐intensive and time‐consuming. In this work, we propose a parallel version of the well‐known Richardson–Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two‐dimensional and three‐dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Visualization of single proteins from stripped native cell membranes: A protocol for high‐resolution atomic force microscopy

Atomic force microscopy (AFM) proved to be able to obtain high‐resolution three‐dimensional images of single‐membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high‐resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane. Microsc. Res. Tech. 76:723–732, 2013. © 2013 Wiley Periodicals, Inc.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

A relocated technique of atomic force microscopy (AFM) samples and its application in molecular biology

A kind of simple atomic force microscopy (AFM) relocated technique, which takes advantage of homemade sample locator system, is used for investigating repeatedly imaging of some specific species on the whole substrate (over 1 x 1 cm2) with resolution about 400 nm. As applications of this sample locator system, single extended DNA molecules and plasmid DNA network are shown in different AFM operational modes: tapping mode and contact mode with different tips after the substrates have been moved.     

Scanning tunnelling and atomic force microscopy performed with the same probe in one unit

The technique demonstrated here provides features of both scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). The metallic probe acts to record current variations and sense forces from the same sample area simultaneously. Thus, separate images may be recorded, in registry. The collected data allows real space correlations between some electrical properties and the geometric structure of a sample surface. The same tip is used since the geometry and condition of the tip can effect the data recordings. Platinum alloys, tungsten and graphite tips have been employed successfully. An AFM lever can respond to surface contact forces, within the elastic limits of the sample, while electric current is sensed by the tip of the lever. The usefulness of this experimental procedure is tested here by an application to semiconducting samples of Ag-doped CdTe in air and in paraffin oil media.     

Evaluation of surface topography changes in three NiTi file systems using rotary and reciprocal motion: An atomic force microscopy study

Aim: To evaluate the surface topography changes in three nickel‐titanium (NiTi) file systems using either rotary or reciprocal motion using atomic force microscopy (AFM), and to determine the effect of scanning area on the AFM results in this study. Methodology: Five points on a F2 Protaper file, R25 Reciproc file, and a Primary file from WaveOne systems were scanned preoperatively in 1 × 1 and 5 × 5 µm² with an AFM device that can scan an intact (not sectioned) file. One standardized resin block was used for each instrument, according to the manufacturer's recommendations. Points were re‐scanned postoperatively using the same AFM and settings. Root‐mean‐square (RMS) and roughness average (Ra) values were obtained. The preoperative and postoperative surface topographies were compared separately in terms of RMS and Ra values. The surface topography change scores were analyzed using Kruskal–Wallis and Mann–Whitney U tests using a 0.10 significance level. Results: There were no significant differences preoperatively among the NiTi file systems in 1 × 1 or 5 × 5 µm² areas. Postoperatively, the WaveOne Primary had more surface irregularities (significant for 5 × 5 µm² scan in Ra evaluation). Conclusions: Three‐dimensional AFM images of instrument surfaces showed topographic irregularities preoperatively and postoperatively. AFM results differ depending on the scanning area and file used. Microsc. Res. Tech. 77:177–182, 2014. © 2013 Wiley Periodicals, Inc.     

Automated image analysis of atomic force microscopy images of rotavirus particles

A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.     

A vision-based, 3D reconstruction technique for scanning electron microscopy: direct comparison with atomic force microscopy

High-resolution, detailed 3D reconstructions of biological specimens obtained from scanning electron microscopy stereo-micrographs and proprietary software were compared with Tapping-Mode AFM datasets of the same fields. The reconstruction software implements several original solutions including a neural adaptive point-matching technique, the ability to build an irregular triangulated mesh rather than a regular orthogonal grid, and the ability to re-map one of the original images exactly onto the reconstructed surface. The technique was applied to human nerve tissue to obtain 1,424 x 968-pixel, texture-mapped datasets, which were subsequently compared against 512 x 512-pixel AFM datasets from the same viewfields. Accounting for the inherent differences of the two techniques, direct comparison revealed an excellent visual match. The correspondence was also quantified by calculating the cross-correlation coefficient between corresponding altimetric profiles in SEM and AFM data, which consistently exceeded a figure of 0.9, with a rate of point mismatch in the order of 0.01%. Research is still underway to improve the robustness of the technique when applied to arbitrary images     

Effects of temperature and humidity on atomic force microscopy dimensional measurement

The influence of environmental factors on dimensional measurements of atomic force microscopy (AFM) was investigated experimentally. Measurements were taken with environmental control over a whole AFM chamber and a local sample chamber to highlight the influence of working conditions on the instrument itself. Both temperature and humidity were found to have a significant impact on pitch measurements of a two‐dimensional grating. The effect of temperature on the behavior of the microscope itself is generally larger than the thermal expansion or contraction of the sample. The effect of humidity was further determined to be relevant to the scan direction and velocity. For precise AFM dimensional measurements, the possible influences of temperature and humidity must be carefully considered. Microsc. Res. Tech. 78:562–568, 2015. © 2015 Wiley Periodicals, Inc.     

A large-sample atomic force microscope observing in both air and liquid

A large-sample atomic force microscope (AFM) that allows high resolution observation in both air and liquid has been developed. With a unique beam tracking method, laser beam is capable of reflecting off the same spot on the AFM cantilever throughout raster scan over the entire scan area, either operating in air or in liquid environment. Incorporating the stand-alone AFM probe unit with an automated large sample stage, wide-scan-range imaging can be realized with high resolution and slight distortion. In addition, an image stitching method is utilized to build a broad merged image with range up to millimeters while keeping nanometer order resolution. By using a large-volume liquid bath, large and massive sample can be observed in liquid with this AFM system. Several typical experiments have been carried out to demonstrate the imaging ability and stability of this AFM. Topographic structures of gold pattern on a glass substrate are scanned at two different places on the same specimen surface. The porosity of a sheet of filter paper is then characterized in both air and water. Finally, larger-area AFM image of anodic aluminum oxide template in oxalic acid is on spot obtained by merging several individually scanned images together. Experiments show that this AFM system can offer high resolution and wide range AFM images even for large samples with remarkable capabilities in various environments.     

Scanning probe microscopy studies of cereal seed storage protein structures

Scanning probe microscopes (SPMs) share a number of common features which give the techniques advantages over conventional light and electron microscopy. First, high resolution, up to the atomic level, is possible in certain cases, and second, they are nondestructive, requiring no staining or coating and the images can be obtained in the hydrated state or under water. Scanning probe microscopes, particularly scanning tunnelling microscopes (STM) and atomic force microscopes (AFM), have been used to study food-related systems, ranging from relatively large structures such as starch granules to the organisation of secondary structures in proteins and the interaction of proteins. The seed storage proteins (gluten) of wheat are responsible for the viscous and elastic properties of wheat doughs that allow them to be used for a wide range of different food products. Using AFM and STM, images of individual and groups of proteins have been obtained in both the dry and hydrated states. The ability to work in liquid environments allows the conformation of proteins to be determined under conditions approaching “native.” The AFM and STM have been used to image both gliadins and glutenins and to study their aggregative behaviour in relation to gluten and dough systems.     

Direct probing of solvent-induced charge degradation in polypropylene electret fibres via electrostatic force microscopy

Electrostatic force microscopy was used to directly probe solvent‐induced charge degradation in electret filter media. Electrostatic force gradient images of individual polypropylene electret fibres were used to quantify the extent of charge degradation caused by the immersion of the fibres into isopropanol. Electrostatic force gradient images were obtained by monitoring the shifts in phase and frequency between the oscillations of the biased atomic force microscopy (AFM) cantilever and those of the piezoelectric driver. Electrostatic force microscopy measurements were performed using non‐contact scans at a constant tip‐sample separation of 75 nm with varied bias voltages applied to the cantilever. Mathematical expressions, based on the capacitance of the tip‐sample system, were used to model the phase and frequency shifts as functions of the applied bias voltage to the tip and the offset voltage due to the fibre's charge. Quantitative agreement between the experimental data and the simplified model was observed.     

Atomic force microscopy of histological sections using a new electron beam etching method

In order to examine histological sections of the rat vomeronasal epithelium with the atomic force microscope (AFM), we developed an electron beam etching method that improves the resolution of AFM images. This method results in AFM images comparable to those obtained with the transmission electron microscope (TEM). Ultrathin tissue sections embedded in epoxy resin were observed before and after the treatment with electron beam radiation. Before electron beam treatment, epithelial structures such as the microvilli surface, dendritic processes, the supporting cell layers and the neuronal cell layers were all visible using the AFM. However, only a few subcellular structures could also be resolved. The AFM images were not as clear as those obtained with the TEM. After electron beam treatment, however, the resolution of AFM images was greatly improved. Most of the subcellular structures observed in TEM images, including the inner membrane of mitochondria, ciliary-structure precursor body, junctional complexes between the neurons and supporting cells, and individual microvilli were now visible in the AFM images. The electron beam treatment appeared to melt the embedding resin, bringing subcellular structures into high relief. The result of this study suggests that electron beam etching of histological samples may provide a new method for the study of subcellular structure using the AFM.     

Developments for inverted atomic force microscopy

Atomic force microscopy (AFM) has been used to study a wide range of systems. Chemically and biologically modified probes have extended AFM by coupling chemical and biological information with the physical measurements. In an effort to further expand the capabilities of modified AFM probes, previous studies investigated the use of an inverted AFM design (i-AFM), wherein a microfabricated tip array is used to image a cantilever-supported sample. This report details developments in cantilever and tip array fabrication which are aimed at improving the applicability and performance of this i-AFM design. Using an epoxy-based procedure, commercial cantilevers were modified with a series of standard substrates, including template-stripped gold, highly oriented pyrolytic graphite, and mica. The samples on these cantilevers were imaged with i-AFM, and lateral force images are obtained. This paper demonstrates the first use of i-AFM for measuring friction.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.