Feasibility of interleaved multislice averaged magnetization inversion-recovery acquisitions of the spinal cord.

PURPOSE: To establish an interleaved multislice variant of the averaged magnetization inversion-recovery acquisitions (AMIRA) approach for 2D spinal cord imaging with increased acquisition efficiency compared with the conventional 2D single-slice approach(es), and to determine essential prerequisites for a working interleaved multislice AMIRA approach in practice. METHODS: The general AMIRA concept is based on an inversion recovery-prepared, segmented, and time-limited cine balanced SSFP sequence, generating images of different contrast. For AMIRA imaging of multiple, independent slices in a 2D interleaved fashion, a slice loop within the acquisition loops was programmed. The former non-selective inversions were replaced with slice-selective inversions with user-definable slice thickness. RESULTS: The thickness of the slice-selective inversion in 2D interleaved multislice AMIRA should be doubled compared with the manufacturer's standard setting to avoid an increased sensitivity to flow and pulsation effects particularly in the CSF. However, this solution also limits its practical applicability, as slices located at directly adjacent vertebrae cannot be imaged together. Successful interleaved two-slice AMIRA imaging for a "reference" in vivo protocol with 0.50 × 0.50 mm2 in-plane resolution and 8-mm slice thickness is demonstrated, therefore halving its acquisition time per slice from 3 min down to 1.5 min. CONCLUSION: The investigated 2D interleaved two-slice AMIRA variant facilitates spinal cord imaging that maintains similar contrast and the same resolution as the conventional 2D single-slice AMIRA approach, but does so with a halved acquisition time.

Facial soft tissue depth of a contemporary adult Greek population.

Facial approximation is a technique that involves constructing the facial muscles and applying a suitable facial soft tissue depth (FSTD) dataset. To date, several FSTD studies have been conducted for varying population groups. This study aims to establish a FSTD dataset of an adult Greek population sample for the first time. The facial depths of subjects were measured on 100 head CT scans of 50 male and 50 female subjects aged from 18 to 99. The 3D head and skull models of subjects were segmented in Amira 6.1 by using histogram method. FSTDs were measured at 22 cranial landmarks (5 mid-sagittal, 17 bilateral). The FSTD dataset was generated by considering the age and sex of subjects. The impact of age and sex on the FSTD was limited. Slight inter-population depth variations were reported. Facial asymmetry calculated between the bilateral landmarks was insignificant for both male and female subjects.

Impact of body weight and sex in selected dog breeds on the canine adrenal gland dimensions measured by computed tomographic imaging.

BACKGROUND: The present study aimed to investigate possible influences of body weight and sex on adrenal gland size in endocrinologically healthy dogs. Possible factors influencing the adrenal size are discussed in relation to a universal upper reference value from the literature of 7.4 mm as the thickness in the caudal pole of the adrenal gland. The adrenal size was measured by computed tomography (CT) from 66 normal dogs of six different breeds (Labrador Retriever (n = 16), German Shepherd (n = 10), Boxer (n = 8), Beagle (n = 14), Dachshund (n = 6) and Jack Russell Terrier (n = 12); male n = 38 (thereof neutered n = 23), female n = 28 (thereof neutered n = 17)) based on volume quantification and linear measurements using the data processing software Amira. For interbreed comparability, a ratio consisting of the third root of adrenal volume to aortic diameter (Ratio volume-aorta, RVA) was introduced. Additionally, breed-related attenuation values in contrast-enhanced CT data sets were measured. RESULTS: The measured volumes ranged from 0.34 to 1.93 cm3 for the right and from 0.39 to 2.23 cm3 for the left adrenal gland. The present study was able to demonstrate a body weight effect on the adrenal volume as well as on length and height. In terms of adrenal size, no significant differences between male and female, nor between intact and neutered dogs were obtained due to the RVA. In addition, for the weight classes, a breed independent threshold for dogs less (left 1.4; right gland 1.5) or more than 20 kg body weight (left 1.1; right gland 1.2) based on RVA was defined. Breed-related significant differences with respect to attenuation were determined only for the left adrenal gland, with lower attenuation values in large dog breeds. CONCLUSION: The present study points out the importance of weight-related data when assessing CT data of the canine adrenal gland regarding volume, size and attenuation. The use of a universal reference value for the assessment of adrenal size appears unsuitable considering weight-related volume and linear measurements. Sex seems not to affect adrenal gland size.

Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography.

We present a software-assisted workflow for the alignment and matching of filamentous structures across a three-dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labour. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite dealing with imperfect data related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time-consuming. Furthermore, an interactive visualisation of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialised, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualisation and interaction tools for each processing step to guarantee real-time response, and an optimised workflow that efficiently guides the user through datasets. The final solution presented here is the result of an iterative process with tight feedback loops between the involved computer scientists and cell biologists. LAY DESCRIPTION: Electron tomography of biological samples is used for a three-dimensional (3D) reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large-scale electron tomography can be applied to increase the reconstructed volume for the visualisation of full spindles. For this, each spindle is cut into a series of semi-thick physical sections, from which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (ie physical distortions) caused by the image acquisition process, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi-automatic solution that considers all requirements for large-scale stitching of microtubules in image stacks of consecutive sections. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large-scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts.

Exploring confocal laser scanning microscopy (CLSM) and fluorescence staining as a tool for imaging and quantifying traces of marine microbioerosion and their trace-making microendoliths.

Microscopic organisms that penetrate calcareous structures by actively dissolving the carbonate matrix, namely microendoliths, have an important influence on the breakdown of marine carbonates. The study of these microorganisms and the bioerosion traces they produce is crucial for understanding the impact of their bioeroding activity on the carbonate recycling in environments under global climate change. Traditionally, either the extracted microendoliths were studied by conventional microscopy or their traces were investigated using scanning electron microscopy (SEM) of epoxy resin casts. A visualisation of the microendoliths in situ, that is within their complex microbioerosion structures, was previously limited to the laborious and time-consuming double-inclusion cast-embedding technique. Here, we assess the applicability of various fluorescence staining methods in combination with confocal laser scanning microscopy (CLSM) for the study of fungal microendoliths in situ in partly translucent mollusc shells. Among the tested methods, specific staining with dyes against the DNA (nuclei) of the trace making organisms turned out to be a useful and reproducible approach. Bright and clearly delineated fluorescence signals of microendolithic nuclei allow, for instance, a differentiation between abandoned and still populated microborings. Furthermore, infiltrating the microborings with fluorescently stained resin seems to be of great capability for the visualisation and quantification of microbioerosion structures in their original spatial orientation. Potential fields of application are rapid assessments of endolithic bio- and ichnodiversity and the quantification of the impact of microendoliths on the overall calcium carbonate turnover. The method can be applied after CLSM of the stained microendoliths and retains the opportunity for a subsequent investigation of epoxy casts with SEM. This allows a three-fold approach in studying microendoliths in the context of their microborings, thereby fostering the integration of biological and ichnological aspects of microbial bioerosion.

Bioerosion describes the process of active erosion of hard substrates induced by the activity of living organisms. Beside numerous marine macroscopic bioeroding organisms such as sponges, annelids or bivalves, there is an astonishing 'hidden diversity' of microscopic bioeroding organisms which produce minute tunnels and chambers, for example in calcareous shells and skeletons of other marine organisms. These so-called microendoliths belong to bacteria, microalgae, foraminiferans, or fungi. Due to their lifestyle hidden inside the hard substrate, scientific investigation is often laborious and involves complex preparation techniques, electron microscopy, or even nano-computed tomography. Photo-autotrophic microendoliths (eg cyanobacteria and algae) have been studied with fluorescence microscopy using autofluorescence properties, for example of their chloroplasts. However, microendoliths of aphotic depths, mostly of fungal origin, do not show autofluorescence. With the present study we test different fluorescent dyes staining the microbioeroders 'in situ', that is, inside their microscopic tunnels, and visualise them using three-dimensional confocal laser scanning microscopy (CLSM). Very good results have been obtained with the dye Sybr Green I that stains DNA molecules and thereby the cell nuclei of the microendoliths. This method can be used, for instance, to measure the infestation rate of a given substrate by discriminating between abandoned microborings and those still inhabited by microendoliths. Another approach that was successfully tested in the course of the present study was the infiltration of the cleaned microborings with resin that was previously mixed with the fluorescent dye Safranin-O. The datasets obtained with the CLSM were used to reconstruct 3D-surface models of the microborings of three different microendoliths. Such models can be used to analyse the original spatial arrangement inside the hard substrate and to measure exact volumes. The resulting possibility to make exact quantifications is of high value for future investigations that focus on the role and proportion of microbioerosion in the (re)cycling of marine carbonates.

Validation of canine prostate volumetric measurements in computed tomography determined by the slice addition technique using the Amira program.

BACKGROUND: Prostatic diseases are common and mostly associated with enlargement of the accessory gland. Thus, determining the prostate size has become a main criterion for evaluating prostate health status. Computed tomography (CT) is recommended as a beneficial tool for evaluating prostate size, morphology and surrounding tissues. The purpose of this study was to establish an accurate procedure for volume estimation and afterwards evaluate the prostate volume in CT. Data of 95 dogs were analysed (58 male intact, 37 male neutered) using the slice addition technique with the Amira program. Accuracy of volumetric measurements by CT was validated by comparing them with those of phantoms of known volume. Patients were grouped according to age (< 4 yrs., 4-8 yrs., > 8 yrs) and prostate morphology in CT (H = homogeneous, I = inhomogeneous, C = cystic). The length of the sixth lumbar vertebra was measured to relate prostate volume to body size. This ratio was generated to compare prostate volume between the groups, irrespective of body size (ratio volume = Rv). RESULTS: A high correlation between the CT-derived and phantom volume was found. Overall, the mean prostate volume was 58.6 cm3. The mean ratio volume was 1.3 in intact male dogs, this being significantly higher than in neutered dogs (0.7). The lowest ratio volume values were found in group H for intact (Rv = 0.9) and neutered dogs (Rv = 0.6), followed by group I (intact: Rv = 1.1; neutered: Rv = 0.7) and C (intact: Rv = 1.4; neutered: Rv = 0.8). The length of the sixth lumbar vertebra was well correlated with the prostate volume (intact: r = 0.63, p < 0.001; neutered: r = 0.48, p = 0.003), while age exhibited a correlation only in intact dogs (r = 0.52, p < 0.001). CONCLUSION: The present study is pioneering in applying a slice addition technique to volumetric measurements of the prostate gland in CT, resulting in a highly precise method. Volumetric measurements of the canine prostate gland in CT images provide information about the prostate structure, castration status, age and body size of the patients. Therefore, prostate volume is a relevant parameter for evaluating prostate health status.

Evaluation of canine prostate volume in calculated tomographic images - comparison of two assessment methods.

BACKGROUND: Since most prostatic diseases are associated with the organ's enlargement, evaluation of prostatic size is a main criterion in the diagnosis of prostatic state of health. While enlargement is a non-uniform process, volumetric measurements are believed to be advantageous to any single dimensional parameter for the diagnosis of prostatomegaly. In a previous study, volume was analysed with a slice addition technique (SAT), which was validated as highly accurate. Irrespective of high accuracy, SAT represents a complex and time-consuming procedure, which limits its clinical use. Thus, demand exists for more practical volume assessment methods. In this study, the prostatic volume of 95 canine patients (58 intact males, 37 neutered males) were analysed retrospectively by using the ellipsoid formula (Formula) and an imaging "wrap" function tool (Wrap) to help assess accuracy and applicability. Accuracy was checked against phantom measurements and results were compared to SAT measurements of the same patient pool obtained from a previously published paper. Patients were grouped according to prostatic structure (H = homogeneous, I = inhomogeneous, C = cystic) and volume using the SAT (volume group = vg: 1, 2 and 3). RESULTS: High correlation between the Formula or Wrap volume and the phantom volume was found, the values being higher for the Formula. Mean Formula volumes (vg 1: 2.2 cm3, vg 2: 14.5 cm3, vg 3: 109.4 cm3, respectively) were significantly underestimated, while mean Wrap volumes (vg 1: 3.8 cm3, vg 2: 19.5 cm3, vg 3: 159.2 cm3) were statistically equivalent to SAT measurements (vg 1: 3.1 cm3, vg 2: 18.6 cm3, vg 3: 157.2 cm3, respectively). Differences between Formula and SAT volumes ranged from 22.4-31.1%, while differences between Wrap and SAT volumes were highest in small prostates (vg 1: 22.1%) and fell with increasing prostatic size (vg 3: 1.3%). CONCLUSION: The Wrap function is highly accurate, less time-consuming and complex compared to SAT and could serve as beneficial tool for measuring prostatic volume in clinical routine after further validation in future studies. The Formula method cannot be recommended as an alternative for volumetric measurements of the prostate gland due to its underestimation of volumes compared to SAT results.

A guide to analysis and reconstruction of serial block face scanning electron microscopy data.

Serial block face scanning electron microscopy (SBF-SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers - how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time-consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step-by-step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF-SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF-SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast-based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.

In vivo facial soft tissue depths of a modern adult population from Germany.

Forensic facial reconstruction may be the final option available to draw public attention in cases where the identity of an individual cannot be established by standard identification methods. Two fundamental components of all forensic facial reconstruction techniques are cranial morphology and soft tissue depth databases. The purpose of this study was to extend such databases by providing a complete set of accurate facial soft tissue thickness measurements, acquired from a contemporary adult population from Germany. The material for the study consisted of 320 (160 male, 160 female) anonymised multi-slice computerised tomography (MSCT) scans of individuals drawn from a German population. In Amira®, 3D models of the surfaces of the skull and the facial skin were semi-automatically segmented using calculated thresholds and surface extraction algorithms. Facial soft tissue depth was measured at 10 midline and 28 bilateral anatomical landmarks. The analysis of facial soft tissue thickness versus BMI, sex and age indicated that, at a number of the landmarks, facial soft tissue depth is significantly (p < 0.05) influenced by all three biometric variables. Facial soft tissue thickness increased with increasing BMI. The differences between males and females were statistically significant (p < 0.05) for almost all anatomical landmarks with the exception of a few in the region of the nasal root and orbitals. Asymmetry was noted at over half of the bilateral landmarks. The differences between the results from this sample and those obtained from comparable databases contradict the hypothesis that population specificity significantly influences facial soft tissue thickness.

Computed reconstruction of spatial ammonoid-shell orientation captured from digitized grinding and landmark data.

The internal orientation of fossil mass occurrences can be exploited as useful source of information about their primary depositional conditions. A series of studies, using different kinds of fossils, especially those with elongated shape (e.g., elongated gastropods), deal with their orientation and the subsequent reconstruction of the depositional conditions (e.g., paleocurrents and transport mechanisms). However, disk-shaped fossils like planispiral cephalopods or gastropods were used, up to now, with caution for interpreting paleocurrents. Moreover, most studies just deal with the topmost surface of such mass occurrences, due to the easier accessibility. Within this study, a new method for three-dimensional reconstruction of the internal structure of a fossil mass occurrence and the subsequent calculation of its spatial shell orientation is established. A 234 million-years-old (Carnian, Triassic) monospecific mass occurrence of the ammonoid Kasimlarceltites krystyni from the Taurus Mountains in Turkey, embedded in limestone, is used for this pilot study. Therefore, a 150×45×140 mm3 block of the ammonoid bearing limestone bed has been grinded to 70 slices, with a distance of 2 mm between each slice. By using a semi-automatic region growing algorithm of the 3D-visualization software Amira, ammonoids of a part of this mass occurrence were segmented and a 3D-model reconstructed. Landmarks, trigonometric and vector-based calculations were used to compute the diameters and the spatial orientation of each ammonoid. The spatial shell orientation was characterized by dip and dip-direction and aperture direction of the longitudinal axis, as well as by dip and azimuth of an imaginary sagittal-plane through each ammonoid. The exact spatial shell orientation was determined for a sample of 675 ammonoids, and their statistical orientation analyzed (i.e., NW/SE). The study combines classical orientation analysis with modern 3D-visualization techniques, and establishes a novel spatial orientation analyzing method, which can be adapted to any kind of abundant solid matter.

A combined MRI, histological and immunohistochemical rendering of the rhesus macaque locus coeruleus (LC) enables the differentiation of three distinct LC subcompartments.

Locus coeruleus (LC) neurons send their noradrenergic axons across multiple brain regions, including neocortex, subcortical regions, and spinal cord. Many aspects of cognition are known to be dependent on the noradrenergic system, and it has been suggested that dysfunction in this system may play central roles in cognitive decline associated with both normative aging and neurodegenerative disease. While basic anatomical and biochemical features of the LC have been examined in many species, detailed characterizations of the structure and function of the LC across the lifespan are not currently available. This includes the rhesus macaque, which is an important model of human brain function because of their striking similarities in brain architecture and behavioral capacities. In the present study, we describe a method to combine structural MRI, Nissl, and immunofluorescent histology from individual monkeys to reconstruct, in 3 dimensions, the entire macaque LC nucleus. Using these combined methods, a standardized volume of the LC was determined, and high-resolution confocal images of tyrosine hydroxylase-positive neurons were mapped into this volume. This detailed representation of the LC allows definitions to be proposed for three distinct subnuclei, including a medial region and a lateral region (based on location with respect to the central gray, inside or outside, respectively), and a compact region (defined by densely packed neurons within the medial compartment). This enabled the volume to be estimated and cell density to be calculated independently in each LC subnucleus for the first time. This combination of methods should allow precise characterization of the LC and has the potential to do the same for other nuclei with distinct molecular features.

Successive measurement errors of consecutive computed tomography for airway-related craniofacial dimensional measurements.

Background/purpose: The use of computed tomography (CT) for craniofacial measurements is common in medical imaging, but concerns about accuracy and reliability persist, especially with different CT technologies. This study assessed the accuracy of twenty-six common measurements on consecutive CT images from the same patients, using multidetector CT (MDCT) and cone-beam CT (CBCT) with two software programs (Amira and Dolphin). Materials and methods: Ten adult subjects with consecutive CBCT scans within one year were randomly selected. Another ten subjects with consecutive MDCT scans were paired with the CBCT group based on age, gender, race, occlusion, and craniofacial pattern. All digital imaging and communications in medicine (DICOM) files were randomly coded and analyzed using the two software programs. Intra-examiner reliability was assessed using the intraclass correlation coefficient. Successive measurement errors from consecutive scans for both imaging modalities and software programs were compared. Results: For most skeletal linear and angular measurements, Dolphin showed greater successive measurement errors compared to Amira. Eight of the 26 common measurements had errors greater than one unit (millimeter or degree). Despite almost perfect intra-examiner reliability for upper airway analysis, average successive measurement errors were notably high, particularly for intraoral and oropharyngeal airway volumes. The successive Dolphin measurement error for oropharyngeal airway volume on CBCT images was over three times that on MDCT images. Conclusion: Given the substantial successive measurement errors observed during consecutive CT scanning for the upper airway, this study does not support the quantitative use of CT for analyzing changes in airway dimensions for research purposes.

Formation of the junctions between lymph follicles in the Peyer's patches even before postweaning activation.

Peyer's patches (PPs), which contain an abundance of B and T cells, play a key role in inducing pivotal immune responses in the intestinal tract. PPs are defined as aggregated lymph follicles, which consist of multiple lymph follicles (LFs) that may interact with each other in a synergistic manner. LFs are thought to be spherical in shape; however, the characteristics of their structure are not fully understood. To elucidate changes in the structure of PPs as individuals grow, we generated serial 2D sections from entire PPs harvested from mice at 2, 4, and 10 weeks of age and performed a 3D analysis using a software, Amira. Although the number of LFs in PPs was not changed throughout the experiment, the volume and surface area of LFs increased significantly, indicating that LFs in PPs develop continuously by recruiting immune cells, even after weaning. In response to the dramatic changes in the intestinal environment after weaning, the development of germinal centers (GCs) in LFs was observed at 4 and 10 weeks (but not 2 weeks) of age. In addition, GCs gradually began to form away from the center of LFs and close to the muscle layer where export lymphatic vessels develop. Importantly, each LF was joined to the adjacent LF; this feature was observed even in preweaning nonactivated PPs. These results suggest that PPs may have a unique organization and structure that enhance immune functions, allowing cells in LFs to have free access to adjacent LFs and egress smoothly from PPs to the periphery upon stimulation after weaning.

Automated Segmentation and 3D Reconstruction of Different Membranes from Confocal Z-Stacks.

Confocal laser scanning microscopy (CLSM) is an advanced microscopy technique based on fluorescence technology which produces sharp images of a specimen in a single focal plane. The optical sectioning by CLSM allows to have z-stacks which can be further processed into 3D reconstructions. These then provide the option of variable perspectives and additional precise data evaluation on structural and anatomical alterations. Here, we used CLSM to image the thylakoids of cyanobacteria and the endoplasmic reticulum (ER) in moss protonemata as an example. Then, out of the confocal z-stacks, we create 3D constructions of the membranes and their alterations to present a holistic, structural view from different angles.

No cost but high performance-An alternative open source solution for 3D-visualizations in morphology.

3D-visualization has become common courtesy in science and also found its way into teaching in schools and universities. Nevertheless, the way to high performance 3D-visualization and analyses remains difficult and is often also a matter of budget. Due to the obvious advantages of presenting morphological and anatomical datasets with the help of 3D-figures, all in one software solutions usually come along with high rental and maintenance fees. For that reason, it is more than overdue to establish and use open source software solutions with all their obvious advantages - as other disciplines already do. Here we provide a modular, highly adaptive and freely available software pipeline for high performance 3D-visualizations of (not only) morphological datasets by combining features of ImageJ, MeshLab and Blender, without any additional costs. Exemplarily using serial-block face SEM data as well as serial AZAN-stained histological sections, the herein presented step-by-step protocol allows for a fast and efficient analysis, visualization and animation of large, anatomical datasets. Regardless which type of serial, morphological datasets needs to be analyzed, our open source guide provides an easy to handle and promptly adaptable solution. Therefore, our pipeline for 3D-visualization represents a valuable alternative to conventional, commercial packages. RESEARCH HIGHLIGHTS: We provide a highly modular and easy to learn open source solution for multiple 3D-visualizations. The step-by-step-guide make it easy to start, and advanced users can replace software, add others and such build your own individual software pipeline.

Preoperative 3D Image Evaluation of EC-IC Bypass by 3D Visualization Analysis Software Amira®.

Background: Careful evaluation of the preoperative imaging for extracranial-intracranial bypass performed for conditions like intracranial stenosis and Moya disease is important. The traditional use of 2D imaging has a significant limitation for neurosurgeons, primarily to determine the optimal location of the recipient artery for performing the surgical bypass. Therefore, many neurosurgeons use 3D angiograms more frequently to overcome these shortcomings. Materials And Methods: We performed the preoperative evaluation of the possibility of performing an anastomosis between the superficial temporal artery and the middle cerebral artery (STA-MCA) bypass by synthesizing images of computerized tomography (CT), magnetic resonance imaging (MRI), and digital subtraction angiography (DSA) using Amira® of 3D visualization analysis software (Thermo Scientific Co.). Results: The 3D images prepared before surgery using this software showed good agreement with the intraoperative findings. Conclusion: Preoperative image processing using tools like Amira provide optimal information for good planning and communication for performing STA-MCA bypass and may become a helpful tool.

Intraspecific variation in the Cambrian: new observations on the morphology of the Chengjiang euarthropod Sinoburius lunaris.

BACKGROUND: The Chengjiang biota from southwest China (518-million-years old, early Cambrian) has yielded nearly 300 species, of which more than 80 species represent early chelicerates, crustaceans and relatives. The application of µCT-techniques combined with 3D software (e.g., Drishti), has been shown to be a powerful tool in revealing and analyzing 3D features of the Chengjiang euarthropods. In order to address several open questions that remained from previous studies on the morphology of the xandarellid euarthropod Sinoburius lunaris, we reinvestigated the µCT data with Amira to obtain a different approach of visualization and to generate new volume-rendered models. Furthermore, we used Blender to design 3D models showing aspects of intraspecific variation. RESULTS: New findings are: (1) antennulae consist of additional proximal articles that have not been detected before; (2) compared to other appendages, the second post-antennular appendage has a unique shape, and its endopod is comprised of only five articles (instead of seven); (3) the pygidium bears four pairs of appendages which are observed in all specimens. On the other hand, differences between specimens also have been detected. These include the presence/absence of diplotergites resulting in different numbers of post-antennular appendages and tergites and different distances between the tip of the hypostome and the anterior margin of the head shield. CONCLUSIONS: Those new observations reveal intraspecific variation among Chengjiang euarthropods not observed before and encourage considerations about possible sexual dimorphic pairs or ontogenetic stages. Sinoburius lunaris is a variable species with respect to its morphological characters, cautioning that taxon-specific variabilities need to be considered when exploring new species.

Antennal-lobe neurons in the moth Helicoverpa armigera: Morphological features of projection neurons, local interneurons, and centrifugal neurons.

The relatively large primary olfactory center of the insect brain, the antennal lobe (AL), contains several heterogeneous neuronal types. These include projection neurons (PNs), providing olfactory information to higher-order neuropils via parallel pathways, and local interneurons (LNs), which provide lateral processing within the AL. In addition, various types of centrifugal neurons (CNs) offer top-down modulation onto the other AL neurons. By performing iontophoretic intracellular staining, we collected a large number of AL neurons in the moth, Helicoverpa armigera, to examine the distinct morphological features of PNs, LNs, and CNs. We characterize 190 AL neurons. These were allocated to 25 distinct neuronal types or sub-types, which were reconstructed and placed into a reference brain. In addition to six PN types comprising 15 sub-types, three LN and seven CN types were identified. High-resolution confocal images allowed us to analyze AL innervations of the various reported neurons, which demonstrated that all PNs innervating ventroposterior glomeruli contact a protocerebral neuropil rarely targeted by other PNs, that is the posteriorlateral protocerebrum. We also discuss the functional roles of the distinct CNs, which included several previously uncharacterized types, likely involved in computations spanning from multisensory processing to olfactory feedback signalization into the AL.

Moving legs: A workflow on how to generate a flexible endopod of the 518 million-year-old Chengjiang arthropod Ercaicunia multinodosa using 3D-kinematics (Cambrian, China).

Understanding the functional morphology and mobility of appendages of fossil animals is important for exploring ecological traits such as feeding and locomotion. Previous work on fossils from the 518 million-year-old Chengjiang biota of China was based mainly on two-dimensional information captured from the surface of the specimens. Only recently, µCT techniques started to reveal almost the entire, though flattened and compressed, three-dimensionally preserved morphologies of the arthropods from Chengjiang. This allows more accurate work on reconstructing the possible movement of certain structures such as the appendages. Here, we present a workflow on how to reconstruct the mobility of a limb of the early Chengjiang arthropod Ercaicunia multinodosa from the famous Chinese fossil site. Based on µCT scans of the fossil, we rendered surface models of the 13th-15th right endopods using the 3D visualization and 3D-rendering software Amira. The 3D objects then were postprocessed (Collapse Hierarchy, Unify Normals) in SAP 3D Visual Enterprise Author before being imported into the 3D animation program Autodesk Maya 2020. Using the add-on tool X_ROMM in Maya, we illustrate step-by-step on how to make the articles of the limbs swing-in toward each other. Eventually, we propose several possible limb movements of E. multinodosa, which helps to understand how this early arthropod could have moved its endopods.

Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.