Electrostatic force microscopy of self-assembled peptide structures

In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO(2) surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated.     

Self-organized nanotemplating on misfit dislocation networks investigated by scanning tunneling microscopy

Self-ordering growth of nanoarrays on strained metallic interfaces is an attractive option for preparing highly ordered nanotemplates. The great potential of this natural templating approach is that symmetry, feature sizes, and density are predicted to depend on the interfacial stress in these strained layers, which can be adjusted by changing the substrate-thin film composition, temperature, and adlayer coverage. This bottom-up approach of growing nanostructured two-dimensional ordered arrays of clusters on the misfit dislocation networks of strained metallic thin films and surfaces requires a detailed understanding of the nucleation and film-adsorbate interaction processes. Here we show how high resolution, large scale, variable temperature scanning tunneling microscopy imaging can improve our understanding of these self-assembly processes.     

A comparative study of colloidal particles as imaging standards for microscopy

Colloidal particles have long been used as imaging standards for electron microscopy and, more recently, for scanning probe microscopy. We have analysed gold, polystyrene and silica colloidal particles by both transmission electron microscopy and atomic/scanning force microscopy in an attempt to determine if any can be truly used as 'standards' of shape and/or size. From the transmission electron micrographs, we have obtained precise information of the particle circumference and mean diameter. By comparing the ratio of these to the value for π, we obtained a measure of the sphericity of the particles. We have also shadowed the particles with metal at a known angle and have analysed the shadow length to determine the particles' heights and shapes. The height information obtained from the shadow length data collected from the transmission electron micrographs was then compared with that obtained by atomic/scanning force microscopy. Our results show that cleaned (washed) silica or polystyrene particles closely approach true spheres. In the case of gold particles, height data obtained from shadow lengths analysed in transmission electron micrographs show good agreement with that obtained from the atomic/scanning force microscopy images even without washing. However, the gold particles often deviate from sphericity. Based upon both the shape and the physical properties of the colloidal particles, silica would be the best choice as a standard. We also have noticed that metal shadowing of colloidal particle samples used for atomic/scanning force microscopy offers an advantage which we call a 'nanoscale metric' visible in the image directly at each particle site. This information can be important if one wishes to use samples prepared from colloidal particles simply and reliably to determine the probe shape for scanning probe microscopy from image deconvolution/restoration methods or as a calibration sample.     

An improved specimen loader for cryo-scanning electron microscopy

Cryo-electron microscopy of cryofixed samples is a well-established and accepted technique for imaging liquid-containing specimens without removing water and other volatile components. There are many steps between cryofixation and cryo-observation in the microscope, during which the sample and sample holder need to be handled. One such major step is the loading of the specimen onto the sample holder and the fixing of the sample holder onto the transfer mechanism. During this handling, the specimen is often exposed (mostly inadvertently) to moisture in the atmosphere, which results in frost deposition. The new specimen loader described here is designed to overcome the traditional tedious handling and to achieve ease in specimen loading. The modifications made are mainly towards allowing movement of the liquid freon cup, eliminating the need for a lock-screw and improving the shape of the stage holder, which makes the mounting of the specimen holder easy, thereby permitting smooth specimen loading without too much handling and with consequent reduced frost deposition.     

Large-scale, uniform DNA network on 11-mercaptoundecanoic acid modified gold (111) surface: atomic force microscopy study

Large-scale, uniform plasmid deoxyribonucleic acid (DNA) network has been successfully constructed on 11-mercaptoundecanoic acid modified gold (111) surface using a self-assembly technique. The effect of DNA concentration on the characteristics of the DNA network was investigated by atomic force microscopy. It was found that the size of meshes and the height of fibers in the DNA network could be controlled by varying the concentration of DNA with a constant time of assembly of 24 h.     

Tau binds ATP and induces its aggregation

Tau is a microtubule‐associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well‐defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self‐assembly. We found that tau's N‐terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self‐assembly into filaments. At 1 mM ATP the filaments are 4–7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP‐induced self‐assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly. Microsc. Res. Tech. 77:133–137, 2014. © 2013 Wiley Periodicals, Inc.     

Cryo-electron microscopy of helical particles TMV and T4 polyheads

Vitrified Tobacco Mosaic Virus (TMV) and T4 polyhead suspensions have been studied by cryo-electron microscopy. The order of TMV particles is preserved to distances better than 0.3 nm when vitrified. In spite of the high beam sensitivity of unstained biological material, the resolution of images extends to 1.15 nm. Radial density distributions calculated from images of TMV and the helical aggregate of TMV protein (TMVP) show that the RNA is likely visualized in the virus. However, due to the imaging conditions of unstained vitrified specimens, the position of the RNA is erroneous. While TMV and TMVP do not show any departure from cylindrical symmetry, T4 polyheads are sensitive to the thickness of the ice layer in which they are embedded. To avoid flattening, T4 polyheads have to be embedded in an ice layer thicker than their diameter.     

An algorithm selection methodology for automated focusing in optical microscopy

Autofocus systems are essential in optical microscopy. These systems typically sweep the sample through the focal range and apply an algorithm to determine the contrast value of each image, where the highest value indicates the optimal focus position. As the optimal algorithm may vary according to the images' content, we evaluate the 15 most used algorithms in the field using 150 stacks of images from four different kinds of tissue. We use four measuring criteria and two types of analysis and propose a general methodology to apply to select the best fitting algorithm for any given application. In this paper, we present the results of this evaluation and a detailed discussion of different features: the threshold used for the algorithms, the criteria parameters, the analysis used, the bit depth of the images, their magnification, and the type of tissue, reaching the conclusion that some of these parameters are more relevant to the study than others, and the implementation of the proposed methodology can lead to a fast and reliable autofocus system capable of performing an analysis and selection of algorithms with no supervision required.     

Metrics for focusing in extremely noisy scanning electron microscopy condition

Finding a best focused image in very noisy condition is an extremely difficult task for the SEM user. If a performance, which is much higher than that of an expert in focusing, can be achieved in a computer-controlled scanning electron microscope (SEM), it will be very helpful for our field due to the many possible applications. To accomplish this work, we employ a powerful metric-the covariance obtained by a special scanning method. It can select the best focused image from a series of SEM images acquired by altering the focus of the objective lens under an extremely noisy SEM image condition. The noise immunity of the present method is quantitatively evaluated, and it is further improved based on the obtained evaluation result.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.     

Potential limitations in the use of KillerRed for fluorescence microscopy

KillerRed, a bright red fluorescent protein, is a genetically encoded photosensitizer, which generates radicals and hydrogen peroxide upon green light illumination. The protein is a potentially powerful tool for selective light-induced protein inactivation and cell killing, and can also be used to study downstream effects of locally increased levels of reactive oxygen species. The initial aim of this study was to investigate whether or not KillerRed-mediated reactive oxygen species production inside peroxisomes could trigger the sequestration of these organelles into autophagosomes. Green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 was used as autophagosome marker. We observed that KillerRed also emits weak green fluorescence upon excitation at 480 nm, and this may lead to erroneous data interpretation in conditions where green fluorophores are used. We discuss this potential pitfall of KillerRed for biological imaging and formulate recommendations to avoid misinterpretation of the data.     

Digital imaging for scanning electron microscopy

The development and application of digital imaging technology has been one of the major advancements in scanning electron microscopy (SEM) during the past several years. This digital revolution has been brought about by significant progress in semiconductor technology, notably the availability of less expensive, high-density memory chips and the development of inexpensive, high-speed, analog-to-digital and digital-to-analog converters, mass storage, and high-performance central processing units. This paper reviews a number of the advantages presented by digital imaging as applied to the SEM and describes a system developed at the National Institute of Standards and Technology for this purpose.     

Interfacial ultramorphology evaluation of resin luting cements to dentin: A correlative scanning electron microscopy and transmission electron microscopy analysis

The objective of this study was to analyze the dentin‐resin cements interfacial ultramorphologies using two different methods: scanning (SEM) and transmission electron microscopy (TEM). Four commercial products were evaluated: two conventional cementing system (RelyX ARC/Adper? Scotchbond? Multi‐Purpose Plus, 3M ESPE and Clearfil Esthetic Cement/DC Bond, Kuraray) and two self‐adhesive resin cements (RelyX Unicem, 3M ESPE and Clearfil SA Cement, Kuraray). Prepolymerized resin disks (Sinfony, 3M ESPE) were cemented on oclusal dentin surfaces of 24 third human molars, simulating the indirect restorations. After 24 h, teeth were sectioned into 0.9‐mm thick slabs and processed for microscopy analyses (SEM or TEM/ n = 3). Qualitative characterization of dentin‐resin cement interface was performed. Hybrid layer formation with long and dense resin tags was observed only for RelyX ARC cementing system. Clearfil Esthetic Cement/DC Bond system revealed few and short resin tags formation, whereas no hybridization and resin tags were detected for self‐adhesive resin cements. Some interfacial regions exhibited that the self‐adhesive resin cements were not bonded to dentin, presenting bubbles or voids at the interfaces. In conclusion, TEM and SEM bonding interface analyses showed ultramorphological variations among resin cements, which are directly related to dental bonding strategies used for each resin cement tested. Microsc. Res. Tech. 76:1234–1239, 2013. © 2013 Wiley Periodicals, Inc.     

An automatic method for atom identification in scanning tunnelling microscopy images of Fe‐chalcogenide superconductors

We describe a computational approach for the automatic recognition and classification of atomic species in scanning tunnelling microscopy images. The approach is based on a pipeline of image processing methods in which the classification step is performed by means of a Fuzzy Clustering algorithm. As a representative example, we use the computational tool to characterize the nanoscale phase separation in thin films of the Fe‐chalcogenide superconductor FeSe_xTe_1‐x, starting from synthetic data sets and experimental topographies. We quantify the stoichiometry fluctuations on length scales from tens to a few nanometres.     

An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10–20 nm laterally and 10–60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Phase imaging atomic force microscopy in the characterization of biomaterials

The phase imaging atomic force microscopy is a powerful tool in surface characterization of the biomaterials, and the resulting phase image is able to detect chemical variation and reveal more detailed surface properties than the morphological image. However, the chemical‐ and morphological‐dependent phase images were still not distinguished well. In order to better understand actual occurring phase images, we examined non‐carious human maxillary incisor, microphase separated polyurethane and self‐assembling peptide nanofibres. We herein reported that phase image mainly plotted the morphological change: the phase peak corresponding to the morphological valley, and the morphological peak to the phase valley, and exhibited fine surface structures of materials. The chemical‐dependent phase contrast was generally masked by their inherent roughness. For the sample being very rough and having great phase separation, its chemical‐dependent phase contrast could be detected at the hard tapping mode (‘Amp. Ref. “set point ratio”’: ?0.4 to ?0.8), for the sample with medium roughness only at the light tapping mode (‘Amp. Ref.’: ?0.1 to ?0.4). These results will help us understand and determine actual occurring phase images of natural or fabricated biomaterials, even, other materials.     

Correction of image drift and distortion in a scanning electron microscopy

Continuous research on small‐scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high‐resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift–time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three‐order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high‐resolution electron microscopic system.     

Investigation of porous asphalt microstructure using optical and electron microscopy

Direct observations of porous asphalt concrete samples in their natural state using optical and electron microscopy techniques led to useful information regarding the microstructure of two mixes and indicated a relationship between microstructure and in situ performance. This paper presents evidence that suboptimal microstructure can lead to premature failure thus making a first step in defining well or suboptimal performing pavements with a bottom-up approach (microstructure). Laboratory and field compaction produce different samples in terms of the microstructure. Laboratory compaction using the gyratory method has produced more microcracks in mineral aggregates after the binder had cooled. Well-performing mixes used polymer-modified binders, had a more homogeneous void structure with fewer elongated voids and better interlocking of the aggregates. Furthermore, well-performing mixes showed better distribution of the mastic and better coverage of the aggregates with bitumen. Low vacuum scanning electron microscopy showed that styrene butadiene styrene polymer modification in binder exists in the form of discontinuous globules and not continuous networks. A reduction in the polymer phase was observed as a result of aging and in-service use.