Tilted Specimens Research Articles

Structure and Fracture Visualization of Tilted ABS Specimens Processed via Fused Filament Fabrication Additive Manufacturing

Fused filament fabrication (FFF) technique is one of the most frequently used additive manufacturing (AM) technologies for printing ABS and many other thermoplastic materials. The anisotropy of the mechanical properties of 3D-printed parts manufactured by FFF technology is still of major concern when using this technique. Thus, the component’s orientation, build strategy and printing parameters affect the mechanical properties, and failure mechanisms are of crucial importance. This research aims to partly fill this gap by studying the structure and mechanical behavior of FFF-ABS specimens, and by performing fracture surface analysis by the three-point bend flexural test. A series of tests were conducted to determine the flexural properties of tilted specimens at 0°, 15°, 30°, 45°, 60° and 75° inclination angles relative to the machine platform. The work describes manufacture method of the specimens, experimental procedures, and outcomes from the mechanical and structural characterizations of the FFF-ABS specimens. Overall, two main failure modes were observed for the tested specimens: (1) inter-layer/ inter-raster bond failure (typical for upright specimens) and (2) intra-layer/trans-raster failure (typical for on-edge specimens). A mixed inter-layer/ intra-layer mode was found for the specimens tilted in-between the 15o and 60o range.

Effect of specimen notch quality on the essential work of fracture of ductile polymer films

The essential work of fracture approach has been employed to analyse the effect of notch sharpening on the fracture toughness of a semicrystalline multiphase ethylene-propylene block copolymer. Double edge notched tension specimens were sharpened using different techniques: femtolaser ablation, razor blade sliding at room and liquid nitrogen temperatures, saw cutting, plastically deformed saw cutting, and scalpel sliding. The notch sharpening techniques provide notches of different quality in relation to both the notch tip radius and the plastic deformation in front of the crack tip. The best quality notches were produced by the femtolaser ablation technique, which provides very sharp notches without plastic deformation ahead of the crack tip. The effects of the non-collinearity of the notches and tilted specimens on the testing machine grips were also analysed. The shape of the registered stress–displacement curves shows differences, but only in the range comprised between the displacements corresponding to the maximum stress and the onset of crack initiation. A larger crack tip radius and/or larger extent of plastic deformation in front of the notch root leads to larger values of the displacement at the onset of crack initiation, resulting in higher values for the specific essential work of fracture. Yet on the other hand, the values of the slope of the essential work of fracture plot remain unchanged. A lower value for the specific essential work of fracture was obtained for the specimens sharpened using the femtolaser ablation technique.

Computational Tools to Improve Visualization by Cryo-Electron Tomography

Cryo-electron tomography (CryoET) can directly visualize frozen, hydrated macromolecules inside cells without staining artifacts or fluorescent tags at nanometer resolution. However, imaging tilted specimens is limited to ±60° tilt, causing a “missing wedge” of data that induces smearing artifacts in tomograms. Furthermore, each image is subject to a defocusing artifact known as the contrast transfer function (CTF) of the electron microscope. Lastly, high noise and low contrast make feature identification difficult and error prone. Although contrast is typically improved by denoising (filtering), the tomographic reconstruction process introduces additional artifacts that can be accentuated when filters are applied to tomograms in 3-D.We developed tools for CTF correction in cryoET tiltseries and to fill in the missing wedge. We implemented them in EMAN2, a widely used software package for cryo elctron microscopy data processing. Using our first too, we applied CTF correction on cryoET tiltseries of viral particles of known structure recorded with a direct electron detector (DE20). Images collected in “movie-mode” were first corrected for drift. We achieve a resolution improvement from ∼25A to ∼13A. Our second tool “fills in” the missing wedge with reprojections of tomographic data from angular directions not accessible experimentally in the electron microscope. Results on viral particle data show decreased missing wedge artifacts (smearing). We also applied pre-reconstruction denoising filters directly to 2-D images in tiltseries, which improved the accuracy of automated particle picking (measured against manual particle picking, considered ground truth).Lastly, we applied pre-reconstruction denoising filters to tiltseries of mammalian cells to minimize artifacts and enhance the reliability of feature identification. For example, in platelets, microtubules and the cell membrane become clearer with pre-filtration, compared to the standard post-reconstruction filtering approach, and mitochondria become more easily distinguishable from granules.

Substrate Effects on Structural Studies using Cryo-Em

In electron microscopy, glow discharge has been widely used to render the carbon film hydrophilic for better adhesion of aqueous solution. However, the target macromolecules (e.g. proteins) may also interact with the charge on the carbon film, which may introduce preferred orientation and/or some other artifacts. A promising alternative is to use two-dimensional (2D) crystal as a substrate. This method has been successfully employed to study DNA molecules, proteoliposomes, and multiprotein complexes. Here we will use liposomes (i.e. lipid vesicles) as a model system to study the effect of substrate on targeted structures. Four types of substrates will be studied: untreated carbon film, glow-discharged carbon film, holey carbon film, and 2D streptavidin crystal. Liposomes, doped with a few copies of biotinylated lipid and osmotically swollen to ensure a spherical shape, will be allowed to attach to the substrate before blotting and rapid freezing of the specimen. Then cryo-electron microscopy of tilted specimens and cryo-electron tomography will be employed, and the resulted images and tomographs will be compared with spherical liposome models. The deformation of the liposomes will be used to quantify the effect of the substrate on the targeted structure.

CTF determination and correction for low dose tomographic tilt series

The resolution of cryo-electron tomography can be limited by the first zero of the microscope's contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.

Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging

Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.

Morphology and structure of poly( p-dioxanone)

The morphology of solution grown single crystals of poly( p-dioxanone) was investigated. Different crystallization conditions (solvent, precipitant agent and temperature) were tested. Dendritic growth, screw dislocations and striations were observed. Crystals usually exhibited a lozenge morphology, whose apex angle changed with the crystallization conditions. In all cases, however, a single crystal electron diffraction pattern was recorded. Spherulitic morphologies were obtained by evaporation of formic acid solutions. Lamellae gave rise to well resolved electron diffraction patterns that allowed determination of the main packing characteristics. Patterns of tilted specimens helped establish an orthorhombic unit cell, whose parameters indicate a deviation from the all trans molecular conformation. Quantum mechanical calculations were performed on small model compounds to study the conformational preferences. Simulated diffraction patterns were consistent with a unit cell containing four repeat units and a P2 12 12 1 space group. Molecular packing suggests the existence of different kinds of folds for an assumed adjacent reentry. Polyethylene decoration hardly highlighted a crystal sectorization. Also enzymatic degradation of lamellar crystals was evaluated by using a Pseudomonas cepacia lipase.

Measurement of electron transmission through tilted thick specimens with an ultrahigh voltage electron microscope

Electron transmission through tilted epoxy-resin film specimens with thicknesses of 1 and 5 microm has been measured under different accelerating voltages and diameters of the objective aperture in a 3 MV ultrahigh voltage electron microscope. Measurements show that the linearity between the logarithmic electron transmission and effective thickness of the tilted specimens holds valid only for some smaller effective thicknesses. However, the higher accelerating voltage leads to a wider linear attenuation range. The linear range can reach 7 microm for the 3 MeV incident electrons. Moreover, the measurements were analyzed with the electron-scattering theory. Actually, the high-energy electrons are capable of penetrating much thicker specimens, but many electrons elastically scattered at large angles cannot be collected as useful signals because of being blocked by the objective aperture in the mode of amplitude contrast. The need for further investigations was suggested to determine theoretically the electron transmission through a thick low-Z specimen and its exponential attenuation range.

The contrast-imaging function for tilted specimens

A theoretical description of the contrast-imaging function is derived for tilted specimens that exhibit weak-phase object characteristics. We show that the tilted contrast-imaging function (TCIF) is a linear transformation, which can be approximated by the convolution operation for small tilt angles or for small specimens. This approximation is not valid for electron tomography, where specimen tilts are above 60° and specimen dimensions amount to some 10 μm. The approximation also breaks down for electron crystallography, where atomic resolution is to be achieved. Therefore, we do not make this approximation and propose a generalized algorithm for inverting the TCIF. The implications of our description are discussed in the context of electron tomography, single particle analysis, and electron crystallography, and the improved resolution is quantitatively demonstrated.

3D reconstruction and processing of volumetric data in cryo-electron tomography

A software package is presented that was primarily developed for electron tomography in biological research. It comprises routines for preprocessing micrographs, CTF-correction of images of untilted and tilted specimens, alignment of tilt series, 3D reconstruction, spatial averaging of paracrystalline specimens, alignment of single particles or components of larger macromolecular assemblies, correlation averaging, multivariate statistical analysis, classification, and volume reassembly, in which molecular components in raw tomograms are replaced by averaged versions with higher signal-to-noise ratio. The emphasis for image analysis is placed on the processing of large sets of individual molecular volumes. The design philosophy of the software is both simplicity and interoperability, i.e. to write standalone programs for simple tasks that are combined through shell scripting to provide more complex functionality, and to communicate with other software via common image formats. This paper gives an overview of the current state of the programs and some applications to cryo-electron tomography.

CTF determination and correction in electron cryotomography

Electron cryotomography (cryoET) has the potential to elucidate the structure of complex biological specimens at molecular resolution but technical and computational improvements are still needed. This work addresses the determination and correction of the contrast transfer function (CTF) of the electron microscope in cryoET. Our approach to CTF detection and defocus determination depends on strip-based periodogram averaging, extended throughout the tilt series to overcome the low contrast conditions found in cryoET. A method for CTF correction that deals with the defocus gradient in images of tilted specimens is also proposed. These approaches to CTF determination and correction have been applied here to several examples of cryoET of pleomorphic specimens and of single particles. CTF correction is essential for improving the resolution, particularly in those studies that combine cryoET with single particle averaging techniques.

The 5 Å Structure of Heterologously Expressed Plant Aquaporin SoPIP2;1

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.

Accurate determination of local defocus and specimen tilt in electron microscopy

Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10 nm, whereas tilt axis and tilt angle are determined with an error of about 2° and 1°, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6°, respectively.

Observation of Microbial Structures in Rocks Using 3D Light Microscopy

Real-time 3D light microscopy provides a large depth of field and high resolution which have considerable advantages for the discovery and examination of microbial structures in rocks. In highly translucent specimens, real-time 3D observation in transmitted light offers a greater capability for rapid recognition of microbial features than do other 3D imaging methods such as light microscopy of serial sections or tilted specimens, scanning electron microscopy, stereology, confocal microscopy, or computed tomography. Application of this new technique to samples of Precambrian, Permian, Cretaceous, and Pleistocene age reveals an intriguing variety of probable microbial structures, particularly in sparry calcites. A direct view of the true spatial relationships among microbial structures and between microbial structures and mineral substrates provides essential clues for deciphering the controls on and timing of microbial activity relative to chemical and mechanical processes in rocks.

Orientation contrast imaging of microstructures in rocks using forescatter detectors in the scanning electron microscope

AbstractWe have developed a system using ‘forescatter detectors’ for backscattered imaging of specimen surfaces inclined at 50–80° to the incident beam (inclined-scanning) in the SEM. These detectors comprise semiconductor chips placed below the tilted specimen. Forescatter detectors provide an orientation contrast (OC) image to complement quantitative crystallographic data from electron backscatter patterns (EBSP). Specimens were imaged using two detector geometries and these images were compared to those collected with the specimen surface normal to the incident beam (normal-scanning) using conventional backscattered electron detector geometries and also to an automated technique, orientation imaging microscopy (OIM). When normal-scanning, the component of the BSE signal relating to the mean atomic number (z) of the material is an order of magnitude greater than any OC component, making OC imaging in polyphase specimens almost impossible. Images formed in inclined-scanning, using forescatter detectors, have OC and z-contrast signals of similar magnitude, allowing OC imaging in polyphase specimens.OC imaging is purely qualitative, and by repeatedly imaging the same area using different specimen-beam geometries, we found that a single image picks out less than 60% of the total microstructural information and as many as 6 combined images are required to give the full data set. The OIM technique is limited by the EBSP resolution (1–2°) and subsequently misses a lot of microstructural information. The use of forescatter detectors is the most practical means of imaging OC in tilted specimens, but it is also a powerful tool in its own right for imaging microstructures in polyphase specimens, an essential asset for geological work.

Angular‐dependent energy distributions for backscattered electrons—Calculation of the surface ionization

AbstractAn equation for angular‐dependent energy distributions of backscattered electrons is presented. Extensive Monte Carlo simulations lead to a simple description for the energy distribution. A comparison of the new expression with experimentally derived curves, Monte Carlo results, and an analytical model is performed. The newly derived energy distributions are used for the calculation of the surface ionization ϕ0 for oblique angle of incidence. For normal incidence, a comparison with several published experimental data leads to a mean value for the deviation of < 4%. For tilted specimens the results are compared with findings of other authors.

Electron-crystallographic Refinement of the Structure of Bacteriorhodopsin

Using electron diffraction data corrected for diffuse scattering together with additional phase information from 30 new images of tilted specimens, an improved experimental density map has been calculated for bacteriorhodopsin. The atomic model has then been rebuilt into this new map with particular attention to the surface loops. All the residues from 7 to 227 as well as ten lipid molecules are now included, although a few amino acid residues in three of the six surface loops, about half of the lipid hydrophobic chains and all of the lipid head groups are disordered. The model has then been refined against the experimental diffraction amplitudes to an R-factor of 28% at 3.5 Å resolution with strict geometry (0.005 Å bond length deviation) using the improvement of the “free” phase residual between calculated and experimental phases from images as an objective criterion of accuracy. For the refinement some new programs were developed to restrain the number of parameters, to be compatible with the limited resolution of our data. In the final refined model of the protein (2BRD), compared with earlier co-ordinates (1BRD), helix D has been moved towards the cytoplasm by almost 4 Å, and the overall accuracy of the co-ordinates of residues in the other six helices has been improved. As a result the positions of nearly all the important residues in bacteriorhodopsin are now well determined. In particular, the buried, protonated Asp115 is 7 Å from, and so not in contact with, the retinal and Met118 forms a cap on the pocket occupied by the β-ionone ring. No clear density exists for the side-chain of Arg82, which forms a central part of the extracellular half-channel. The only arginine side-chain built into good density is that of Arg134 at the extracellular end of helix E, the others being disordered near one of the two surfaces. The interpretation of the end of helix F on the extracellular surface is now clearer; an extra loose helical turn has been built bringing the side-chain of Glu194 close to Arg134 to form a probable salt bridge. The model provides an improved framework for understanding the mechanism of the light-driven proton pumping. A number of cavities that could contain water molecules were found by searching the refined model, most of them above or below the Schiff base in the half-channels leading to the two surfaces. The ordered and disordered regions of the structure are described by the temperature factor distribution.

Oblique section 3-D reconstruction from thin sectioned biological specimens

Three dimensional (3-D) image reconstruction of plastic embedded and sectioned tissue has become an important tool for imaging complex biological structures in situ. Most 3-D imaging methods combine multiple views of tilted specimens which leads to radiation damage and mass loss. On the other hand oblique section 3-D reconstruction (OSR) can produce a 3-D image from a single view because an oblique section through a 2- or 3-D periodic specimen contains 3-D information which can be folded into a 3-D image. The resolution achievable in an OSR depends on section thickness and is lowest in the direction perpendicular to the section plane. In OSR there is no missing cone or wedge in the 3-D image because of tilt angle restrictions, but finite section thickness limits the vertical resolution. Two OSR methodologies have been developed, one called Crystallographic Serial Section Reconstruction or CSSR, and another called Super Lattice Reconstruction or SLR.

Trans-Golgi network (TGN) of different cell types: three-dimensional structural characteristics and variability.

The trans-Golgi network (TGN) is generally considered as a distinct and permanent structural compartment of the Golgi apparatus of various cell types. To verify this postulate we examined and compared the three-dimensional characteristics of the TGNs of 14 different mammalian cell types as presented in our various publications since 1979 when we initially described the trans-tubular network of Sertoli cells. In all these studies we used low and high voltage electron microscopes on thin or thick sections of tissues fixed with glutaraldehyde and postfixed with reduced osmium. The sections were stained with uranyl acetate and lead citrate. Stereopairs, prepared from photographs of tilted specimens, permitted a direct observation of the three-dimensional structure of the various elements of the Golgi apparatus. The TGNs are multilayered and extensive in cells which do not form large typical secretory granules (Sertoli cells, nonciliated cells of ductuli efferentes, spinal ganglion cells) but have an extensive lysosomal system. The TGN is absent in cells forming very large secretory granules (secretory cells of seminal vesicles and lactating mammary glands). The TGNs are small in cells producing small to medium-size secretory granules and/or appear as residual fragments on the trans aspect of the Golgi stacks (e.g., mucous cells of Brunner's gland, pancreatic acinar cells, etc.). In cells with multiple and extensive TGNs, a continuity of these tubular networks with the two or three transmost saccules of the stack is observed but there are seemingly no connections between the TGNs. Whenever the TGNs are present, they do not form a continuous structure along the Golgi ribbon. However, they do present, in all cases, configurations suggestive of desquamation and renewal. The structure of the TGN varies considerably from one cell type to another, being extensive in cells not showing typical secretory granules but having an extensive lysosomal system, while in secretory cells showing small or large secretory granules the TGN is either small or even entirely absent.