Ecomorphology of the pectoral girdle in anurans (Amphibia, Anura): Shape diversity and biomechanical considerations.

Frogs and toads (Lissamphibia: Anura) show a diversity of locomotor modes that allow them to inhabit a wide range of habitats. The different locomotor modes are likely to be linked to anatomical specializations of the skeleton within the typical frog Bauplan. While such anatomical adaptations of the hind limbs and the pelvic girdle are comparably well understood, the pectoral girdle received much less attention in the past. We tested for locomotor-mode-related shape differences in the pectoral girdle bones of 64 anuran species by means of micro-computed-tomography-based geometric morphometrics. The pectoral girdles of selected species were analyzed with regard to the effects of shape differences on muscle moment arms across the shoulder joint and stress dissipation within the coracoid. Phylogenetic relationships, size, and locomotor behavior have an effect on the shape of the pectoral girdle in anurans, but there are differences in the relative impact of these factors between the bones of this skeletal unit. Remarkable shape diversity has been observed within locomotor groups indicating many-to-one mapping of form onto function. Significant shape differences have mainly been related to the overall pectoral girdle geometry and the shape of the coracoid. Most prominent shape differences have been found between burrowing and nonburrowing species with headfirst and backward burrowing species significantly differing from one another and from the other locomotor groups. The pectoral girdle shapes of burrowing species have generally larger moment arms for (simulated) humerus retractor muscles across the shoulder joint, which might be an adaptation to the burrowing behavior. The mechanisms of how the moment arms were enlarged differed between species and were associated with differences in the reaction of the coracoid to simulated loading by physiologically relevant forces.

Measurement error in µCT-based three-dimensional geometric morphometrics introduced by surface generation and landmark data acquisition.

Computed-tomography-derived (CT-derived) polymesh surfaces are widely used in geometric morphometric studies. This approach is inevitably associated with decisions on scanning parameters, resolution, and segmentation strategies. Although the underlying processing steps have been shown to potentially contribute artefactual variance to three-dimensional landmark coordinates, their effects on measurement error have rarely been assessed systematically in CT-based geometric morphometric studies. The present study systematically assessed artefactual variance in landmark data introduced by the use of different voxel sizes, segmentation strategies, surface simplification degrees, and by inter- and intra-observer differences, and compared their magnitude to true biological variation. Multiple CT-derived surface variants of the anuran (Amphibia: Anura) pectoral girdle were generated by systematic changes in the factors that potentially influence the surface geometries. Twenty-four landmarks were repeatedly acquired by different observers. The contribution of all factors to the total variance in the landmark data was assessed using random-factor nested permanovas. Selected sets of Euclidean distances between landmark sets served further to compare the variance among factor levels. Landmark precision was assessed by landmark standard deviation and compared among observers and days. Results showed that all factors, except for voxel size, significantly contributed to measurement error in at least some of the analyses performed. In total, 6.75% of the variance in landmark data that mimicked a realistic biological study was caused by measurement error. In this landmark dataset, intra-observer error was the major source of artefactual variance followed by inter-observer error; the factor segmentation contributed < 1% and slight surface simplification had no significant effect. Inter-observer error clearly exceeded intra-observer error in a different landmark dataset acquired by six partly inexperienced observers. The results suggest that intra-observer error can potentially be reduced by including a training period prior to the actual landmark acquisition task and by acquiring landmarks in as few sessions as possible. Additionally, the application of moderate and careful surface simplification and, potentially, also the use of case-specific optimal combinations of automatic local thresholding algorithms and parameters for segmentation can help reduce intra-observer error. If landmark data are to be acquired by several observers, it is important to ensure that all observers are consistent in landmark identification. Despite the significant amount of artefactual variance, we have shown that landmark data acquired from microCT-derived surfaces are precise enough to study the shape of anuran pectoral girdles. Yet, a systematic assessment of measurement error is advisable for all geometric morphometric studies.

Standard method for microCT-based additive manufacturing quality control 4: Metal powder analysis.

X-ray micro computed tomography (microCT) can be applied to analyse powder feedstock used in additive manufacturing. In this paper, we demonstrate a dedicated workflow for this analysis method, specifically for Ti6Al4V powder typically used in commercial powder bed fusion (PBF) additive manufacturing (AM) systems. The methodology presented includes sample size requirements, scan conditions and settings, reconstruction and image analysis procedures. We envisage this method will support standardization in powder analysis in the additive manufacturing community. This is aimed at ultimately improving the quality of additively manufactured parts, through the identification of impurities and defects in powders. •MicroCT analysis of metal powders for additive manufacturing•Method describes a standard workflow simplifying usage of the technique•Sample requirements and image analysis workflow is described.

Standard method for microCT-based additive manufacturing quality control 1: Porosity analysis.

MicroCT is a well-established technique that is used to analyze the interior of objects non-destructively, and it is especially useful for void or porosity analysis. Besides its widespread use, few standards exist and none for additive manufacturing as yet. This is due to the inherent differences in part design, sizes and geometries, which results in different scan resolutions and qualities. This makes direct comparison between different scans of additively manufactured parts almost impossible. In addition, different image analysis methodologies can produce different results. In this method paper, we present a simplified 10 mm cube-shaped coupon sample as a standard size for detailed analysis of porosity using microCT, and a simplified workflow for obtaining porosity information. The aim is to be able to obtain directly comparable porosity information from different samples from the same AM system and even from different AM systems, and to potentially correlate detailed morphologies of the pores or voids with improper process parameters. The method is applied to two examples of different characteristic types of voids in AM: sub-surface lack of fusion due to improper contour scanning, and tree-like pores growing in the build direction. This standardized method demonstrates the capability for microCT to not only quantify porosity, but also identify void types which can be used to improve AM process optimization.

Standard method for microCT-based additive manufacturing quality control 3: Surface roughness.

The use of microCT of 10 mm coupon samples produced by AM has the potential to provide useful information of mean density and detailed porosity information of the interior of the samples. In addition, the same scan data can be used to provide surface roughness analysis of the as-built surfaces of the same coupon samples. This can be used to compare process parameters or new materials. While surface roughness is traditionally done using tactile probes or with non-contact interferometric techniques, the complex surfaces in AM are sometimes difficult to access and may be very rough, with undercuts and may be difficult to accurately measure using traditional techniques which are meant for smoother surfaces. This standard workflow demonstrates on a coupon sample how to acquire surface roughness results, and compares the results from roughly the same area of the same sample with tactile probe results. The same principle can be applied to more complex parts, keeping in mind the resolution limit vs sample size of microCT.

Standard method for microCT-based additive manufacturing quality control 2: Density measurement.

MicroCT is best known for its ability to detect and quantify porosity or defects, and to visualize its 3D distribution. However, it is also possible to obtain accurate volumetric measurements from parts - this can be used in combination with the part mass to provide a good measure of its average density. The advantage of this density-measurement method is the ability to combine the density measurement with visualization and other microCT analyses of the same sample. These other analyses may include detailed porosity or void analysis (size and distribution) and roughness assessment, obtainable with the same scan data. Simple imaging of the interior of the sample allows the detection of unconsolidated powder, open porosity to the surface or the presence of inclusions. The CT density method presented here makes use of a 10 mm cube sample and a simple data analysis workflow, facilitating standardization of the method. A laboratory microCT scanner is required at 15 µm voxel size, suitable software to allow sub-voxel precise edge determination of the scanned sample and hence an accurate total volume measurement, and a scale with accuracy to 3 digits. •MicroCT-based mean density measurement method.•Accurate volume measurement and scale mass.•10 mm cube sample allows standardization and automation of workflow.

Temperature-dependent in-plane structure formation of an X-shaped bolapolyphile within lipid bilayers.

Polyphilic compound B12 is an X-shaped molecule with a stiff aromatic core, flexible aliphatic side chains, and hydrophilic end groups. Forming a thermotropic triangular honeycomb phase in the bulk between 177 and 182 °C but no lyotropic phases, it is designed to fit into DPPC or DMPC lipid bilayers, in which it phase separates at room temperature, as observed in giant unilamellar vesicles (GUVs) by fluorescence microscopy. TEM investigations of bilayer aggregates support the incorporation of B12 into intact membranes. The temperature-dependent behavior of the mixed samples was followed by differential scanning calorimetry (DSC), FT-IR spectroscopy, fluorescence spectroscopy, and X-ray scattering. DSC results support in-membrane phase separation, where a reduced main transition and new B12-related transitions indicate the incorporation of lipids into the B12-rich phase. The phase separation was confirmed by X-ray scattering, where two different lamellar repeat distances are visible over a wide temperature range. Polarized ATR-FTIR and fluorescence anisotropy experiments support the transmembrane orientation of B12, and FT-IR spectra further prove a stepwise "melting" of the lipid chains. The data suggest that in the B12-rich domains the DPPC chains are still rigid and the B12 molecules interact with each other via π-π interactions. All results obtained at temperatures above 75 °C confirm the formation of a single, homogeneously mixed phase with freely mobile B12 molecules.

Use of Isotope-Edited FTIR to Derive a Backbone Structure of a Transmembrane Protein.

Solving structures of membrane proteins has always been a formidable challenge, yet even upon success, the results are normally obtained in a mimetic environment that can be substantially different from a biological membrane. Herein, we use noninvasive isotope-edited FTIR spectroscopy to derive a structural model for the SARS coronavirus E protein transmembrane domain in lipid bilayers. Molecular-dynamics-based structural refinement, incorporating the IR-derived orientational restraints points to the formation of a helical hairpin structure. Disulfide cross-linking and X-ray reflectivity depth profiling provide independent support of the results. The unusually short helical hairpin structure of the protein might explain its ability to deform bilayers and is reminiscent of other peptides with membrane disrupting functionalities. Taken together, we show that isotope-edited FTIR is a powerful tool to analyze small membrane proteins in their native environment, enabling us to relate the unusual structure of the SARS E protein to its function.

Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering.

Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed ß-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide's reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer.

Quantitative biological imaging by ptychographic x-ray diffraction microscopy.

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of approximately 1.3x10(5) Gy, and were shown to scale with photon fluence.

A novel heavy-atom label for side-specific peptide iodination: synthesis, membrane incorporation and X-ray reflectivity.

Structural parameters, such as conformation, orientation and penetration depth of membrane-bound peptides and proteins that may function as channels, pores or biocatalysts, are of persistent interest and have to be probed in the native fluid state of a membrane. X-ray scattering in combination with heavy-atom labeling is a powerful and highly appropriate method to reveal the position of a certain amino acid residue within a lipid bilayer with respect to the membrane normal axis up to a resolution of several Angstrøm. Herein, we report the synthesis of a new iodine-labeled amino acid building block. This building block is intended for peptide incorporation to provide high intensities for electron density difference analysis of X-ray reflectivity data and improve the labeling potential for the lipid bilayer head-group and water region. The novel building block as well as the commercially available non-iodinated analogue, required for X-ray scattering, was implemented in a transmembrane peptide motif via manual solid-phase peptide synthesis (SPPS) following the fluorenylmethyloxycarbonyl (Fmoc)-strategy. The derived peptides were reconstituted in lipid vesicles as well as in highly aligned multilamellar lipid stacks and investigated via circular dichroism (CD) and X-ray reflectivity. Thereby, it has been revealed that the bulky iodine probe neither causes conformational change of the peptide structure nor lamellar disordering of the membrane complexes.

X-ray structure analysis of free-standing lipid membranes facilitated by micromachined apertures.

Silicon and Teflon substrates have been structured by wet etching and a focused ion beam (FIB) to obtain very defined, clean apertures. Planar, free-standing lipid membranes (black lipid membranes (BLM)) with enhanced long-term stability have been prepared on these apertures by the methods of Montal and Müller(1,2) as well as Müller and Rudin.(3) The stability and geometric control enables the use of X-ray analysis of free-standing single bilayers. With the presented setup, simultaneous structural and electrophysiological measurements will become feasible.