Background
Molecular methods of species delineation are rapidly developing and widely considered as fast and efficient means to discover species and face the 'taxonomic impediment’ in times of biodiversity crisis. So far, however, this form of DNA taxonomy frequently remains incomplete, lacking the final step of formal species description, thus enhancing rather than reducing impediments in taxonomy. DNA sequence information contributes valuable diagnostic characters and –at least for cryptic species – could even serve as the backbone of a taxonomic description. To this end solutions for a number of practical problems must be found, including a way in which molecular data can be presented to fulfill the formal requirements every description must meet. Multi-gene barcoding and a combined molecular species delineation approach recently revealed a radiation of at least 12 more or less cryptic species in the marine meiofaunal slug genus Pontohedyle (Acochlidia, Heterobranchia). All identified candidate species are well delimited by a consensus across different methods based on mitochondrial and nuclear markers.

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How to describe a cryptic species? Practical challenges of molecular taxonomy

By providing two examples, the option for embedding 3D models in electronic versions of life science publications is presented. These examples, presumably representing the first such models published, are developmental stages of an evertebrate (Patella caerulea, Mollusca) and a vertebrate species (Psetta maxima, Teleostei) obtained from histological section series reconstruction processed with the software package Amira. These surface rendering models are particularly suitable for a PDF file because they can easily be transformed to a file format required and components may be conveniently combined and hierarchically arranged. All methodological steps starting from specimen preparation until embedding of resulting models in PDF files with emphasis on conversion of Amira data to the appropriate 3D file format are explained. Usability of 3D models in PDF documents is exemplified and advantages over 2D illustrations are discussed, including better explanation capabilities for spatial arrangements, higher information contents, and limiting options for disguising results by authors. Possibilities for additional applications reaching far beyond the examples presented are suggested. Problems such as long-term compatibility of file format and hardware plus software, editing and embedding of files, file size and differences in information contents between printed and electronic version will likely be overcome by technical development and increasing tendency toward electronic at the cost of printed publications. Since 3D visualization plays an increasing role in manifold disciplines of science and appropriate tools for the popular PDF format are readily available, we propose routine application of this way of illustration in electronic life science papers.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jemt.20618

Embedding 3D models of biological specimens in PDF publications.

In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system. The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations.

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A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

With the latest release of the S2PLOT graphics library, embedding interactive, 3-dimensional (3-d) scientific figures in Adobe Portable Document Format (PDF) files is simple, and can be accomplished without commercial software. In this paper, we motivate the need for embedding 3-d figures in scholarly articles. We explain how 3-d figures can be created using the S2PLOT graphics library, exported to Product Representation Compact (PRC) format, and included as fully interactive, 3-d figures in PDF files using the movie15 LaTeX package. We present new examples of 3-d PDF figures, explain how they have been made, validate them, and comment on their advantages over traditional, static 2-dimensional (2-d) figures. With the judicious use of 3-d rather than 2-d figures, scientists can now publish, share and archive more useful, flexible and faithful representations of their study outcomes.

The article you are reading does not have embedded 3-d figures. The full paper, with embedded 3-d figures, is recommended and is available as a supplementary download from PLoS ONE (File S2).

https://researchbank.swinburne.edu.au/file/ed80fd31-c92d-4619-8826-37220f83caf9/1/PDF (Published version).pdf

Embedding and publishing interactive, 3-dimensional, scientific figures in Portable Document Format (PDF) files.

Advances in digital data acquisition, analysis, and storage have revolutionized the work in many biological disciplines such as genomics, molecular phylogenetics, and structural biology, but have not yet found satisfactory acceptance in morphology. Improvements in non-invasive imaging and three-dimensional visualization techniques, however, permit high-throughput analyses also of whole biological specimens, including museum material. These developments pave the way towards a digital era in morphology. Using sea urchins (Echinodermata: Echinoidea), we provide examples illustrating the power of these techniques. However, remote visualization, the creation of a specialized database, and the implementation of standardized, world-wide accepted data deposition practices prior to publication are essential to cope with the foreseeable exponential increase in digital morphological data.

/pdf/opportunities-and-challenges-for-digital-morphology-35kxlzgx7k.pdf

Opportunities and challenges for digital morphology

Recent increases in performance of personal computer hard- and software enabled a variety of 3D graphical applications, such as surface visualizations of biological specimens. This indirectly recalled an old morphological method back to life, the investigation of soft part anatomy by light microscopical serial section analysis. A practical guide covering all procession steps beginning with anesthetization leading to the fi nal goal, 3D visualization of specimens, is provided. Most useful for 3D procession—of particularly small specimens—are ribbon-forming serial resin (= “semithin”) sections. A reliable method for achievement of ribbon formation is described in detail for the fi rst time. Contact cement is applied only to the cutting surface of the block, which represents a modifi cation of an old protocol. Details on the materials and tools, such as embedding media (epoxy resins) and knives (Ralph glass or special diamond knife) used and general handling for the entire procedure are given and critically evaluated. 3D procession is explained for the software AMIRA® . The major processing steps, from section image capturing until refi ning of surfaces, are explained. Based on the experience of the author, practical aids that cannot be found in the user’s guide of the software or elsewhere for facilitating the process are given. These include preliminary calculation of resolution, calibration and strategies for facilitating the process and improvement of results. The interpolate function is emphasized as most useful for completion of segmentation and also correction works. Visualization examples are followed by an estimate of work expenditure for graphical processes involved

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Bernhard Ruthensteiner

Papers

Soft Part 3D visualization by serial sectioning and computer reconstruction