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The future of three-dimensional microscopic imaging in marine biology
Abstract
Measuring biodiversity in the oceans and its modifications with time or during climate change requires the accurate description of plankton organisms from viruses to fishes. However, our taxonomic knowledge of this 70% of the Earth is extremely limited. The ultimate way to perform large-scale taxonomical analysis is to achieve full 3D imaging of every specimen collected. Novel 3D imaging techniques are undoubtedly at the forefront of such efforts, and will provide an important tool to identify, classify and generate reference models (or 3D gold standards) for rapid recognition and classification of plankton organisms during community or basin studies. This review gives an overview of recently developed 3D imaging techniques and discusses their limitations and promises.
... XMTs were realized according to the protocols reported by Endo and Frey (2008), Boistel et al. (2011a, b) and Lötters et al. (2011). In particular, we used a Viscom X8050-16 µCT scanner at the Centre for Microtomography of the University of Poitiers (France). ...
... 3D images were produced in 16bit and subsequently converted into 8-bit voxels for visualization. Three-dimensional processing and rendering was obtained after semi-automatic segmentation of the cranial skeleton (Boistel et al., 2009(Boistel et al., , 2011a using 'generate surface' and volume rendering in AVIZO 7.1 (VSG, SAS, Merignac, France, http://www.vsg3d.com). The iso-surface function was used to build a smooth 3D surface from a sub-set of selected voxels of skull. ...
... X-ray imaging techniques are non-invasive and non-damaging, except at high (sub-micron) resolution with synchrotron beams when using hydrated specimens (Boistel et al. 2009;Richards et al., 2012). Three dimensional information is available in images produced by computed tomography (CT) (Endo and Frey, 2008;Boistel et al., 2011a), scanners, and synchrotron imaging (Boistel et al., 2011a). CT is now a reference tool for three dimensional non-destructive studies, in particular for biological and paleontological specimens (Tafforeau et al., 2006;Sutton, 2008;Boistel et al., 2011a,b). ...
The Lanza’s Salamander, Salamandra lanzai, an endemism of the Italian and French slopes of the south-western Alps, was described on the basis of molecular and external morphological data. The species is similar in colouration and life history to the Alpine salamander, S. atra, but phylogenetic studies showed that it is more closely related to S. corsica, and thus these similarities are mostly due to evolutionary convergence. In order to provide a comparative basis for systematic, phylogenetic and palaeontological researches concerning the genus Salamandra, here we report a description of the skull and lower jaw morphology in S. lanzai that were studied on the basis of skeletonized, disarticulated dry preserved specimens as well as by X-ray microtomography of wet preserved specimens. The skull is about as long as wide, relatively depressed and rather flat dorsally. The skull roof represents 28 to 32 % of the skull width measured at the level of the posterior tip of the processus maxillaris posterior. The number of tooth positions varies from 21 to 29 in the premaxilla, from 28 to 47 in the maxilla, from 42 to 62 in the vomer, and from 56 to 69 in the dentary. The frontosquamosal arch is absent. The bone-by-bone description here reported represents the first attempt of providing detailed morphological information on the skull morphology of a member of genus Salamandra as well as a starting basis for including osteological information in the analysis of the phylogenetic relationships within this clade.
... A variety of software packages using sophisticated algorithms are available to subsequently reconstruct the scanned OOI in three dimensions. Given that for equivalent X-ray energy a denser structure will attenuate the beam more than a less dense structure, excellent 3D-reconstructions are produced by utilization of the natural contrast when a highly absorbing structure is surrounded by tissues with relatively weak absorbance (e.g., bones in contrast to soft tissues such as muscles; Boistel et al., 2011). However, if the density of the different soft tissues are similar the use of appropriate contrasting agents is advisable. ...
... Another limitation of CT is the restriction to measurements of the skeleton topography only, while for example coral tissue components remain undetected. To achieve a higher spatial resolution, which in turn enables imaging of delicate details, greater X-ray fluxes are required, hence, evoking an inherent trade-off between image quality and tissue damage (Boistel et al., 2011). However, sophisticated state of the art technologies in CT in combination with classical methods will enable scientists to improve their knowledge on ecosystem function. ...
... It may even foster environmental sciences as it allows for accurate biomass calculations or the establishment of taxonomic 3D-libraries in biodiversity research. The latter will enable a high throughout identification, since the 3D-nature of organisms is crucial for a reliable morphological identification (Boistel et al., 2011). In addition to the described digital imaging techniques there are numerous other 3D-based methods that become increasingly important in biological research. ...
... One solution is to generate high quality three-dimensional (3D) images of a type specimen, a 'cybertype' (Godfray, 2007). Nondestructive three-dimensional imaging techniques such as Confocal Laser Scanning Microscopy, Optical Projection Tomography, Magnetic Resonance Imaging and Micro-Computed Tomography (micro-CT), can rapidly create high-resolution morphological and anatomical data in 3D (Boistel et al., 2011;Faulwetter et al., 2013a,b). Whilst these methods have been criticised as having insufficient resolution for taxonomic purposes (e.g. ...
... This retains the type material in an intact state for the future. Some methods such as Magnetic Resonance Imaging and Optical Projection Tomography are also possible using live specimens of organisms (Boistel et al., 2011). ...
We review the current knowledge of the biodiversity of the ocean as well as the levels of decline and threat for species and habitats. The lack of understanding of the distribution of life in the ocean is identified as a significant barrier to restoring its biodiversity and health. We explore why the science of taxonomy has failed to deliver knowledge of what species are present in the ocean, how they are distributed and how they are responding to global and regional to local anthropogenic pressures. This failure prevents nations from meeting their international commitments to conserve marine biodiversity with the results that investment in taxonomy has declined in many countries. We explore a range of new technologies and approaches for discovery of marine species and their detection and monitoring. These include: imaging methods, molecular approaches, active and passive acoustics, the use of interconnected databases and citizen science. Whilst no one method is suitable for discovering or detecting all groups of organisms many are complementary and have been combined to give a more complete picture of biodiversity in marine ecosystems. We conclude that integrated approaches represent the best way forwards for accelerating species discovery, description and biodiversity assessment. Examples of integrated taxonomic approaches are identified from terrestrial ecosystems. Such integrated taxonomic approaches require the adoption of cybertaxonomy approaches and will be boosted by new autonomous sampling platforms and development of machine-speed exchange of digital information between databases.
... Furthermore, invasive techniques invariably alter the structural integrity of zoological specimens and thus do not permit studying organ systems in their natural context, ultimately resulting in a significantly restricted representation of the complexity of an organism. In contrast, digital three-dimensional (3D) imaging techniques such as computed tomography (CT) or magnetic resonance imaging (MRI) permit minimally or even entirely non-invasive analyses of whole biological specimens or parts thereof [4][5][6][7][8]. Although the application of such methods-an approach more recently labelled morphomics [9]-has so far been successfully demonstrated for the identification or description of smaller fauna [10][11][12], larger metazoan species have, for various technical or logistical reasons, thus far not been the target of this type of analysis. ...
... In recent years, several non-invasive imaging techniques have become available [4][5][6][7][8] that permit acquisition of high quality structural data in 3D from various metazoan taxa, including terrestrial and aquatic megafauna [33,[35][36][37]. In the present study, MRI was performed overnight using a high-field clinical imaging system [33]. ...
Background
In zoology, species descriptions conventionally rely on invasive morphological techniques, frequently leading to damage of the specimens and thus only a partial understanding of their structural complexity. More recently, non-destructive imaging techniques have successfully been used to describe smaller fauna, but this approach has so far not been applied to identify or describe larger animal species. Here, we present a combination of entirely non-invasive as well as minimally invasive methods that permit taxonomic descriptions of large zoological specimens in a more comprehensive manner.
Results
Using the single available representative of an allegedly novel species of deep-sea cephalopod (Mollusca: Cephalopoda), digital photography, standardized external measurements, high-field magnetic resonance imaging, micro-computed tomography, and DNA barcoding were combined to gather all morphological and molecular characters relevant for a full species description. The results show that this specimen belongs to the cirrate octopod (Octopoda: Cirrata) genus Grimpoteuthis Robson, 1932. Based on the number of suckers, position of web nodules, cirrus length, presence of a radula, and various shell characters, the specimen is designated as the holotype of a new species of dumbo octopus, G. imperator sp. nov. The digital nature of the acquired data permits a seamless online deposition of raw as well as derived morphological and molecular datasets in publicly accessible repositories.
Conclusions
Using high-resolution, non-invasive imaging systems intended for the analysis of larger biological objects, all external as well as internal morphological character states relevant for the identification of a new megafaunal species were obtained. Potentially harmful effects on this unique deep-sea cephalopod specimen were avoided by scanning the fixed animal without admixture of a contrast agent. Additional support for the taxonomic placement of the new dumbo octopus species was obtained through DNA barcoding, further underlining the importance of combining morphological and molecular datasets for a holistic description of zoological specimens.
... The identification of zoological specimens is usually based on external, internal or on the combination of external and internal morphological characteristics. The 3D nature of the different structures is important for the classification of the organisms (Boistel et al. 2011;Faulwetter et al. 2013a). The creation of 3D models at a resolution of a few microns, the accessibility of micro-CT scanners, the low cost and the use of these datasets for morphometric, functional, ecological and developmental analysis reveal the utility of this technology for zoological studies (Faulwetter et al. 2013a;Fernández et al. 2014). ...
... The creation of 3D models at a resolution of a few microns, the accessibility of micro-CT scanners, the low cost and the use of these datasets for morphometric, functional, ecological and developmental analysis reveal the utility of this technology for zoological studies (Faulwetter et al. 2013a;Fernández et al. 2014). According to Boistel et al. (2011), different scans -which are referred to as 'morphoscans' -should be included in a reliable 3D library with reference models (or gold standards) including intra-species variations and key references established by the community. ...
Micro-computed tomography (micro-CT or microtomography) is a non-destructive imaging technique using X-rays which allows the digitisation of an object in three dimensions. The ability of micro-CT imaging to visualise both internal and external features of an object, without destroying the specimen, makes the technique ideal for the digitisation of valuable natural history collections. This handbook serves as a comprehensive guide to laboratory micro-CT imaging of different types of natural history specimens, including zoological, botanical, palaeontological and geological samples. The basic European Journal of Taxonomy 522: 1-55
... However, serial sectioning is usually painstaking, time-consuming, and limited to small specimens. Advanced imaging technologies, such as magnetic resonance imaging [3] and computed tomography [4], are powerful tools for imaging internal structures; however, these instruments have limited resolution compared to light microscopy, and are much less accessible to most zoologists. ...
... This approach could also be used to evaluate changes to any morphological characteristics caused by exposure to chemicals, genetic mutation, or selection pressure. The library of 3D reference models also has the potential to facilitate the sorting and identification of collected species, and, eventually, our understanding of local fauna [4]. ...
Background:
Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested.
Results:
We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, Armadillidium vulgare. Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and A. vulgare became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, Philyra sp.
Conclusions:
In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation.
... Stoev et al. 2013, Wilhelm et al. 2011, Zimmermann et al. 2011). Hundreds, if not thousands of three-dimensional datasets of biological specimens have already been produced (Ziegler et al. 2011, Boistel et al. 2011. Through an ever-increasing effort of natural history museums to digitise their collections, this number is expected to be multiplied by several orders of magnitude over the next decades. ...
... Through an ever-increasing effort of natural history museums to digitise their collections, this number is expected to be multiplied by several orders of magnitude over the next decades. However, the existence of most of these datasets remains unknown to the research community, since they are neither accessible through a public repository for micro-CT data, nor are they documented with metadata which could be queried (Ziegler et al. 2011, Boistel et al. 2011). The development of standards and protocols for annotating and disseminating three-dimensional data as well as the creation of centralised registers to make the information retrievable remains an utmost priority for the micro-CT community working with biological specimens (Rowe and Frank 2011). ...
... As the gathered MR image data are digital by nature, advanced 3D visualization and modeling software can be used to convey complex morphological structures in a more plastic manner (Walter et al. 2010, Boistel et al. 2011, for example by embedding fully interactive, labeled 3D models of zoological specimens into the publication itself (Ziegler et al. 2010b(Ziegler et al. , c, 2011. In addition, the simultaneous display of several interactive datasets on a computer screen has become possible, for example by using the ImageJ (NIH, http://rsb.info.nih.gov/ij) ...
... Unfortunately, MRI currently does not permit to gather meaningful anatomical data from specimens smaller than about 1 mm. This void in whole specimen imaging can be filled using other imaging techniques such as micro-computed tomography (lCT), confocal laser scanning microscopy (cLSM), or optical projection tomography (OPT) (Walter et al. 2010;Boistel et al. 2011). However, in contrast to MRI, soft tissue imaging using these techniques may require considerable specimen preparation, for example through staining (Metscher 2009). ...
Magnetic resonance imaging (MRI) is a noninvasive imaging technique that today constitutes one of the main pillars of preclinical and clinical imaging. MRI’s capacity to depict soft tissue in whole specimens ex vivo as well as in vivo, achievable voxel resolutions well below (100 µm)3, and the absence of ionizing radiation have resulted in the broad application of this technique both in human diagnostics and studies involving small animal model organisms. Unfortunately, MRI systems are expensive devices and have so far only sporadically been used to resolve questions in zoology and in particular in zoomorphology. However, the results from two recent studies involving systematic scanning of representative species from a vertebrate group (fishes) as well as an invertebrate taxon (sea urchins) suggest that MRI could in fact be used more widely in zoology. Using novel image data derived from representative species of numerous higher metazoan clades in combination with a comprehensive literature survey, we review and evaluate the potential of MRI for systematic taxon scanning. According to our results, numerous animal groups are suitable for systematic MRI scanning, among them various cnidarian and arthropod taxa, brachiopods, various molluscan taxa, echinoderms, as well as all vertebrate clades. However, various phyla in their entirety cannot be considered suitable for this approach mainly due to their small size (e.g., Kinorhyncha) or their unfavorable shape (e.g., Nematomorpha), while other taxa are prone to produce artifacts associated either with their biology (e.g., Echiura) or their anatomy (e.g., Polyplacophora). In order to initiate further uses of MRI in zoology, we outline the principles underlying various applications of this technique such as the use of contrast agents, in vivo MRI, functional MRI, as well as magnetic resonance spectroscopy. Finally, we discuss how future technical developments might shape the use of MRI for the study of zoological specimens.
... As the gathered MR image data are digital by nature, advanced 3D visualization and modeling software can be used to convey complex morphological structures in a more plastic manner (Walter et al. 2010, Boistel et al. 2011, for example by embedding fully interactive, labeled 3D models of zoological specimens into the publication itself (Ziegler et al. 2010b(Ziegler et al. , c, 2011. In addition, the simultaneous display of several interactive datasets on a computer screen has become possible, for example by using the ImageJ (NIH, http://rsb.info.nih.gov/ij) ...
... Unfortunately, MRI currently does not permit to gather meaningful anatomical data from specimens smaller than about 1 mm. This void in whole specimen imaging can be filled using other imaging techniques such as micro-computed tomography (lCT), confocal laser scanning microscopy (cLSM), or optical projection tomography (OPT) (Walter et al. 2010;Boistel et al. 2011). However, in contrast to MRI, soft tissue imaging using these techniques may require considerable specimen preparation, for example through staining (Metscher 2009). ...
... Investigations into the effect of environmental variability on sediment-organism interactions have mostly been limited to two-dimensions [10][11][12] and methods to infer the three dimensional structure of burrow networks, such as burrow castings 13,14 and camera insertion 15 , are comparatively invasive 16 . More recently, the use of high-resolution micro-focus computed tomography (μ-CT) has been used to image, in three dimensions, individual organisms 17,18 , their behaviour 19 , and any associated biogenic structures 20 , allowing quantitative examination of burrow characteristics 21 . The majority of these descriptive studies, however, do not consider abiotic or biotic factors that may influence the morphology of biogenic structures, making it difficult to understand the extent of intraspecific variation and generalise the full functional contribution of species. ...
The morphology and architecture of structures formed by sediment-dwelling invertebrates, such as excavations or burrows, are often assumed to be characteristic of a given species, consistent across a range of environmental conditions, and used to categorise species contributions to ecosystem functioning. However, very few investigations use non-invasive high-resolution techniques capable of determining fine scale variations in burrow form and complexity, or consider whether or not the form of the burrow is context dependent. Here, we provide replicate high-resolution micro-focus computed tomography data for the complete burrow systems of the Opheliid polychaete, Armandia cirrhosa, across a range of salinity and habitat conditions. These data provide reference models which can be used by ecologists investigating intraspecific variation in species traits and organism-sediment interactions and, more generally, by those tasked with pattern and shape recognition of objects that are morphologically highly variable and which adjust their architecture with changing circumstance or context.
... High resolution digital 3D imaging techniques such as photogrammetry, confocal laser scanning microscopy, optical projection tomography, magnetic resonance imaging and microcomputed tomography (micro-CT), can rapidly create highresolution morphological and anatomical data in 3D (e.g. Boistel et al., 2011;Faulwetter et al., 2013a;b;Nguyen et al., 2014;Medina et al., 2020). Multispectral imaging has also been used to produce high resolution images of the colours and shapes of organisms (e.g. ...
Ocean Census is a new Large-Scale Strategic Science Mission aimed at accelerating the discovery and description of marine species. This mission addresses the knowledge gap of the diversity and distribution of marine life whereby of an estimated 1 million to 2 million species of marine life between 75% to 90% remain undescribed to date. Without improved knowledge of marine biodiversity, tackling the decline and eventual extinction of many marine species will not be possible. The marine biota has evolved over 4 billion years and includes many branches of the tree of life that do not exist on land or in freshwater. Understanding what is in the ocean and where it lives is fundamental science, which is required to understand how the ocean works, the direct and indirect benefits it provides to society and how human impacts can be reduced and managed to ensure marine ecosystems remain healthy. We describe a strategy to accelerate the rate of ocean species discovery by: 1) employing consistent standards for digitisation of species data to broaden access to biodiversity knowledge and enabling cybertaxonomy; 2) establishing new working practices and adopting advanced technologies to accelerate taxonomy; 3) building the capacity of stakeholders to undertake taxonomic and biodiversity research and capacity development, especially targeted at low- and middle-income countries (LMICs) so they can better assess and manage life in their waters and contribute to global biodiversity knowledge; and 4) increasing observational coverage on dedicated expeditions. Ocean Census, is conceived as a global open network of scientists anchored by Biodiversity Centres in developed countries and LMICs. Through a collaborative approach, including co-production of science with LMICs, and by working with funding partners, Ocean Census will focus and grow current efforts to discover ocean life globally, and permanently transform our ability to document, describe and safeguard marine species.
... Three-dimensional reconstructions of skeleton and swimbladder were performed at the Centre for Microtomography of the University of Poitiers (France) to compare males (n = 1) and females (n = 2) sonic apparata. X-ray microtomography was performed on all specimens according to the protocols reported by Boistel et al. (Boistel et al., 2011) and Zanette et al. (Zanette et al., 2014) using an RXsolutions (Annecy, France) EasyTom XL Duo microtomograph. ...
This study investigates the sounds and the anatomy of the sound‐producing organ in the male and female sand‐dwelling cusk‐eel Parophidion vassali. Although both sexes have similar external phenotype, they can be distinguished by their sonic apparatus and sounds. As in many Ophioidei, Parophidion vassali presents a panel of highly derived characters. Fish possess three pairs of sonic muscles, and males have mineralized swimbladder caps on which inserts the ventral sonic muscle, a neural arch that pivots, a stretchable swimbladder fenestra, an osseous swimbladder plate and a rounded pressure‐release membrane in the caudal swimbladder. Females, however, do not possess anterior swimbladder caps, a swimbladder fenestra and the caudal rounded membrane. Males possess the unusual ability to produce sounds starting with a set of low amplitude pulses followed by a second set with higher amplitudes clearly dividing each sound unit into two parts. Females do not vary their sound amplitude in this way: they produce shorter sounds and pulse periods but with a higher peak frequency. Morphology and sound features support the sound‐producing mechanism is based on a rebound system (i.e. quick backward snap of the anterior swimbladder). Based on features of the sounds from tank recordings, we have putatively identified the sound of male Parophidion vassali at sea. As these species are ecologically cryptic, we hope this work will allow assessment and clarify the distribution of their populations. This study investigates the sounds and the anatomy of the sound‐producing organ in the dwelling cusk‐eel. Males produce sounds starting with a set of low amplitude pulses followed by a second set with higher amplitudes clearly dividing each sound unit into two parts. Females produce shorter sounds and pulse periods but with a higher peak frequency. Differences in sounds correspond to differences in sound‐producing mechanisms.
... In the last two decades, major leaps have been made in the development of taxonomic approaches and methodologies, including DNA-based taxonomy and (meta-) barcoding, "-omic" techniques, imaging tools, and integrative approaches linking different types of taxonomic information (Dayrat, 2005;Boistel et al., 2011;Raupach et al., 2016;Paulus et al., 2021). Many of these methods, despite their apparent advantage in facilitating and accelerating species delimitation, have been slow to find their way into deep-sea taxonomy. ...
Correct identification of species is required to assess and understand the biodiversity of an ecosystem. In the deep sea, however, this is only possible to a limited extent, as a large part of the fauna is undescribed and the identification keys for most taxa are inadequate or missing. With the progressive impact of climate change and anthropogenic activities on deep-sea ecosystems, it is imperative to define reliable methods for robust species identification. In this study, different techniques for the identification of deep-sea species are tested, including a combination of morphological, molecular (DNA barcoding, and proteomic fingerprinting), biogeographical and ecological modeling approaches. These are applied to a family of isopods, the Haploniscidae, from deep waters around Iceland. The construction of interactive identification keys based on the DELTA format (DEscription Language for TAxonomy) were a major pillar of this study, the evaluation of which was underpinned by the application of the supplementary methods. Overall, interactive keys have been very reliable in identifying species within the Haploniscidae. Especially in a deep-sea context, these types of keys could become established because they are easy to adapt and flexible enough to accommodate newly described species. Remarkably, in this study, the interactive key enabled identification of a supposedly new species within the Haploniscidae that was later verified using both molecular genetic – and proteomic methods. However, these keys are limited given that they are based on purely morphological characteristics, including where species with strong ontogenetic or sexual dimorphism occur as both genders are not always described. In this case, integrative taxonomy is the method of choice and the combination presented here has been shown to be very promising for correct identification of deep-sea isopods.
... The frog's general state was unaffected by the lesion, and it was found to eat and pass feces normally until further investigation 1 month later. Whole-body computed tomography (CT) was performed using a Easytom XL Duo microtomographic device (RX Solutions, Chavanod, France) with an acceler-ating tension of 150 kV and an intensity of 201 lA according to the protocols described for reducing radiation damage (Boistel et al., 2011;Zanette et al., 2013;Immel et al., 2016). The conscious frog was placed in a ventilated plastic box for the CT scan; 1,440 slices were acquired with an exposure time of 0.08 sec and no averaging. ...
A wild-caught mature female marsh frog (Pelophylax ridibundus) was presented for a left-sided dorsal swelling. No other abnormalities were detected during the examination. Fine-needle aspiration was non-diagnostic. Baseline hematological and biochemical analyses were within normal reference ranges, and medical imaging did not provide further useful information. Surgical exploration under general anesthesia was performed. The swelling was confirmed to be a digestive tract hernia protruding through a breach of the dorsal muscles and coelomic membrane. Reduction of the hernia was made difficult by the presence of multiple adhesions between the herniated tissue and the coelomic membrane, suggesting a relatively chronic lesion. A traumatic cause was hypothesized. One month later, the animal was considered healed and released back into the wild. Hernias in amphibians are poorly reported in the literature and historically are only found ventrally. To the best of our knowledge, this is the first report of a dorsal hernia in a captive or a wild amphibian.
... Currently, neuroscientists and medical personnel as well as paleontologists heavily rely on structural imaging techniques. Computed tomography (CT), which is a significant exploratory tool in biology (9,10), is one of the most useful techniques for digitizing bone structures and making endocranial models from them (11). The resulting digital endocasts reflect the morphology of the brain (12)(13)(14)(15)(16) and the associated structures, such as the vascular system (17) and the cranial nerves (18). ...
Computed tomography (CT) is one of the most useful techniques for digitizing bone structures and making endocranial models from the neurocranium. The resulting digital endocasts reflect the morphology of the brain and the associated structures. Our first aim was to document the methodology behind creating detailed digital endocasts of canine skulls. We created digital endocasts of the skulls of 24 different dog breeds and 4 wild canids for visualization and teaching purposes. We used CT scanning with 0.323 mm × 0.322 mm × 0.6 mm resolution. The imaging data were segmented with 3D Slicer software and refined with Autodesk Meshmixer. Images were visualized in 3D Slicer and surface models were converted to 3D PDFs to provide easier interactive access, and 3D prints were also generated for visualization purposes. Our second aim was to analyze how skull length and width relate to the surface areas of the prepiriform rhinencephalic, prefrontal, and non-prefrontal cerebral convexity areas of the endocasts. The rhinencephalic area ratio decreased with a larger skull index. Our results open the possibility to analyze the relationship between the skull and brain morphology, and to link certain features to behavior, and cognition in dogs.
... In addition, one juvenile that had been preserved in ethanol was rinsed and rehydrated in phosphate-buffered saline (PBS) for 24 h before being fixed in 5% formaldehyde for 24 h, and then treated with PMA. X-ray microtomography of all specimens was performed according to the protocols reported by Boistel et al. (2011) and Zanette et al. (2014) using an RXsolutions (Annecy, France) EasyTom XL Duo microtomograph. ...
The few works on audition in sharks and rays concern only adult specimens. We report the hearing abilities in the dogfish Scyliorhinus canicula at different stages, from embryos that still have their yolk sac inside their egg, to juveniles. Hearing development corresponds to an increase in the frequency range from 100−300 Hz in early pre‐hatching stages to 100–600 Hz in juveniles. Modifications in hearing abilities correspond to the development of the brain, the increase of the volume of the membranous labyrinth, the growth of the sensory epithelium, and the development of stereocilia in addition to kinocilium before hatching. This work offers solid insights into the development of hearing abilities that usually can only be inferred from the anatomy of vertebrates or after birth/hatching. It shows also that shark can be sensitive to background noise during development.
... In order to visualize metazoan soft tissues digitally, non-invasively, and in 3D only a limited number of techniques can be employed, in particular clinical or preclinical MRI (Boistel et al. 2011;Zanette et al. 2014;Ziegler et al. 2011a. Apart from research on various other Metazoa, in particular preclinical MRI has in the past successfully been used to obtain soft part information from living as well as fixed echinoderm specimens (Ziegler & Angenstein 2007;Ziegler et al. , 2008Ziegler et al. , 2010cSigl et al. 2013), but also from a fossil taxon (Mietchen et al. 2008). ...
Recent studies have shown that micro-computed tomography (µCT) must be considered one of the most suitable techniques for the non-invasive, three-dimensional (3D) visualization of metazoan hard parts. In addition, µCT can also be used to visualize soft part anatomy non-destructively and in 3D. In order to achieve soft tissue contrast using µCT based on X-ray attenuation, fixed specimens must be immersed in staining solutions that include heavy metals such as silver (Ag), molybdenum (Mo), osmium (Os), lead (Pb), or tungsten (W). However, while contrast-enhancement has been successfully applied to specimens pertaining to various higher metazoan taxa, echinoderms have thus far not been analyzed using this approach. In order to demonstrate that this group of marine invertebrates is suitable for contrast-enhanced µCT as well, the present study provides results from an application of this technique to representative species from all five extant higher echinoderm taxa. To achieve soft part contrast, freshly fixed and museum specimens were immersed in an ethanol solution containing phosphotungstic acid and then scanned using a high-resolution desktop µCT system. The acquired datasets show that the combined visualization of echinoderm soft and hard parts can be readily accomplished using contrast-enhanced µCT in all extant echinoderm taxa. The results are compared with µCT data obtained using unstained specimens, with conventional histological sections, and with data previously acquired using magnetic resonance imaging, a technique known to provide excellent soft tissue contrast despite certain limitations. The suitability for 3D visualization and modeling of datasets gathered using contrast-enhanced µCT is illustrated and applications of this novel approach in echinoderm research are discussed.
... To overcome these problems, some biologists have started using microfocus X-ray computed tomography (microCT) imaging 6,7,8,9,10,11,12,13,14,15 . In X-ray CT, the specimen is irradiated with X-rays from various angles that are generated from an X-ray source moving around the sample, and the transmitted X-rays are monitored by a detector that also moves around the sample. ...
... A number of 3D imaging techniques can be employed to study animal morphology and anatomy non-destructively or even entirely non-invasively (Ziegler et al. 2008, Boistel et al. 2011, Ziegler 2012, Zanette et al. 2014, Gutiérrez et al. 2018. These technologies include magnetic resonance imaging (MRI), a technique based on the principle of nuclear magnetic resonance (NMR) as well as computed tomography (CT), which relies on differences in X-ray attenuation of biological tissues (Table 1). ...
Research on molluscan specimens is increasingly being carried out using high-throughput molecular techniques. Due to their efficiency, these technologies have effectively resulted in a strong bias towards genotypic analyses. Therefore, the future large-scale correlation of such data with the phenotype will require a significant increase in the output of morphological studies. Three-dimensional (3D) scanning techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) can achieve this goal as they permit rapidly obtaining digital data non-destructively or even entirely non-invasively from living, fixed, and fossil samples. With a large number of species and a relatively complex morphology, the Mollusca would profit from a more widespread application of digital 3D imaging techniques. In order to provide an overview of the capacity of various MRI and CT techniques to visualize internal and external structures of molluscs, more than twenty specimens ranging in size from a few millimeters to well over one meter were scanned in vivo as well as ex vivo. The results show that all major molluscan organ systems can be successfully visualized using both MRI and CT. The choice of a suitable imaging technique depends primarily on the specimen's life condition, its size, the required resolution, and possible invasiveness of the approach. Apart from visual examples derived from more than two dozen scans, the present article provides guidelines and best practices for digital 3D imaging of a broad range of molluscan taxa. Furthermore, a comprehensive overview of studies that previously have employed MRI or CT techniques in malacological research is given.
... In this technique, 2D section images and 3D modelling is achieved through multiple images taken at each section plan, without dissecting or mechanically damaging the tissues (Fernández et al. 2014;Lenihan et al. 2014). During the recent years, micro-CT has seen a steep increase of usage in taxonomy and systematics (Faulwetter et al. 2013a) as the 3D nature of the different internal and external structures are important for the classification of the organisms (Boistel et al. 2011). In particular, micro-CT has recently been applied on soft-bodied invertebrates, based on the use of specific high molecular weight stains that enhance contrast between soft tissues. ...
The ozobranchid leech, Ozobranchus margoi (Apáthy, 1890), parasite of the loggerhead turtle, Caretta caretta (Linnæus), was described through a multilevel approach including three different techniques: scanning electronic microscopy, histology and micro-CT. New insights are provided concerning the structure of the sensilla on the body, the eyes with emphasis on the structure of photoreceptors, the digestive system and the reproductive organs.
... Computed tomography (CT) allows the reconstruction of high-quality 3D models of both hard and soft tissues that can be used for different purposes, such as anatomical and biomechanical studies. It thus constitutes an important exploratory tool in biology and opens a range of new possible investigations (Boistel et al. 2011a;Carril et al. 2015). ...
Brain endocasts obtained from computed tomography (CT) are now widely used in the field of comparative neuroanatomy. They provide an overview of the morphology of the brain and associated tissues located in the cranial cavity. Through anatomical comparisons between species, insights on the senses, the behavior, and the lifestyle can be gained. Although there are many studies dealing with mammal and bird endocasts, those performed on the brain endocasts of squamates are comparatively rare, thus limiting our understanding of their morphological variability and interpretations. Here, we provide the first comparative study of snake brain endocasts in order to bring new information about the morphology of these structures. Additionally, we test if
the snake brain endocast encompasses a phylogenetic and/or an ecological signal. For this purpose, the digital endocasts of 45 snake specimens, including a wide diversity in terms of phylogeny and ecology, were digitized using CT, and compared both qualitatively and quantitatively. Snake endocasts exhibit a great variability. The different methods performed from descriptive characters, linear measurements and the outline curves provided complementary information. All these methods have shown that the shape of the snake brain endocast contains, as in mammals and birds, a phylogenetic signal but also an ecological one. Although phylogenetically related taxa share several similarities between each other, the brain endocast morphology reflects some notable ecological trends: e.g. (i) fossorial species possess both reduced optic tectum and pituitary gland; (ii) both fossorial and marine species have cerebral hemispheres poorly developed laterally; (iii) cerebral hemispheres and optic tectum are more developed in arboreal and terrestrial species.
... https://3d.si.edu/) have established open source 3D model data bases, but the production (via 3D printing) or utilization (via VR) has been limited due to a lack of user friendly, low cost systems to realize these models. Various methods of scanning have being used to create these databases, however efficient, precise, and comparable 3D digital model generation is still a major area of development for Digital manufacturing and moreover the Digital specimen [8]. ...
... Synchrotron x-ray imaging. Specimen ZRC 1.10828 was fixed in 3.7% formalin, transferred to 75% alcohol and then rehydrated before being placed in an 8-cm polypropylene tube and embedded in Agarose at 3% (without air bubbles), for synchrotron x-ray imaging (Boistel et al., 2011a(Boistel et al., , 2011b. We used the ID17 long (144 m) imaging beamline of the European Synchrotron Radiation Facility (Boistel et al., 2009;Loetters et al., 2011) with a large field of view and monochromatic beam. ...
Barbourula busuangensis Taylor and Noble, 1924 is a poorly known basal aquatic frog from the Philippines. Here we describe some features of the head anatomy of B. busuangensis, with a focus on jaw and eye musculature and the cranial skeleton, based on 3D reconstructions from serial histological sections of a metamorphosed but not fully grown (subadult) female and two fully grown adults, one of which was investigated by synchrotron x-ray imaging. Examination of two different post-metamorphic stages allowed tentative assessment of the development of some characters from the subadult to the adult condition, e.g., shape and size of the parahyoid or arrangement of some jaw adductors. The subadult specimen of B. busuangensis also possesses some unique cranial characters, including a salamander-like arrangement of the jaw adductors, which is obscured in fully grown adults, and a well-developed basal (= basicranial) articulation resembling that of temnospondyls. The facial nerve of B. busuangensis does not fuse with the trigeminal ganglion. Head anatomy confirms that Barbourula is a basal anuran, and comparison with other basal taxa suggests that B. busuangensis most closely resembles the hypo-ossified taxa Alytes and Bombina, and Discoglossus with respect to degree of ossification.
... Puesto que las imágenes en 2D aún presentan algunas limitaciones para la identificación a nivel específico, actualmente se están desarrollando nuevas tecnologías para la obtención de imágenes en 3D con alta definición (imagen acústica (FishTV), estereoscopia, tomografía (resonancia magnética, rayos X), microscopios confocales y holografía), las cuales permiten rotar la imagen y observar estructuras claves para la identificación (i.e., antenas y apéndices entre otras), y obtener medidas volumétricas. Actualmente se ha proyectado para el futuro, hacer una biblioteca de referencia de varios grupos de zooplancton en 3D, y hasta ahora se tiene registros digitalizados de aproximadamente 350-400 organismos en modelos 3D, en su mayoría peces, aunque todavía se requiere un programa específico para su procesamiento (Benfield et al., 2007;Boistel et al., 2011). ...
The study of zooplankton communities is very important in the context of the biology, ecology, and conservation of the marine ecosystem. However, research on zooplankton implies a highly costly process in terms of time and effort, requiring trained personnel for identification, counting and measuring of various taxonomic groups, including biomass estimates. At present, there is an increasing interest for implementing new techniques allowing automatic analyses of zooplankton by means of informatics technology and assessment of digitized images. In this work, the state of the art of some methodologies applied to digital images of zooplankton is revised describing the general methods used, such as ZooScan-ZooProcess and ZooImage, and an analysis of their results for identification, a comparison with traditional methods, and recommendations to improve the identification process. Additionally, a brief summary of the experiences with these systems is presented, focused on the analysis of spatial and temporal variability of the zooplankton community by means of its size spectrum and tests for deriving biomass estimates. Finally, a list of capacities and limitations of the methods is discussed using recent literature, as well as the perspectives of development and applications of this new technology can offer to the scientific community of the Latin American region.
... Relatively few studies apply non-invasive interrogation of intact sedimentary media [10][11][12][13] , despite significant advances in optical and clinical imaging technology 14 . High-resolution micro-focus computed tomography (μ-CT) offers a way of not only imaging the organisms themselves 15,16 but also visualising the structure of a whole sediment core in three dimensions to allow quantitative examination of organismal burrowing 17 . Experimental details are given in Hale et al. 18 . ...
The architecture of biogenic structures can be highly influential in determining species contributions to major soil and sediment processes, but detailed 3-D characterisations are rare and descriptors of form and complexity are lacking. Here we provide replicate high-resolution micro-focus computed tomography (μ-CT) data for the complete burrow systems of three co-occurring, but functionally contrasting, sediment-dwelling inter-tidal invertebrates assembled alone, and in combination, in representative model aquaria. These data (≤2,000 raw image slices aquarium(-1), isotropic voxel resolution, 81 μm) provide reference models that can be used for the development of novel structural analysis routines that will be of value within the fields of ecology, pedology, geomorphology, palaeobiology, ichnology and mechanical engineering. We also envisage opportunity for those investigating transport networks, vascular systems, plant rooting systems, neuron connectivity patterns, or those developing image analysis or statistics related to pattern or shape recognition. The dataset will allow investigators to develop or test novel methodology and ideas without the need to generate a complete three-dimensional computation of exemplar architecture.
... Improved automated taxonomic categorisation, as seen in the last decade, has been the result of two different lines of development: (1) novel hardware instrumentation, like 3D imaging, allows that more information is gathered on plankton cells, thus enlarging the portfolio of features that can be used in automated classification (Embleton et al. 2003;Culverhouse et al. 2006;Boistel et al. 2011), and (2) new developments in software have led to improved image processing and classification algorithms (Benfield et al. 2007;Schulze et al. 2013). ...
... However, a morphological description of a new species does not have to rely on drawings only. Improvements in resolution, suitability, and quality of modern imaging techniques, among them the confocal laser scanning microscopy (CLSM) (Fig. 2), magnetic resonance imaging (MRI), and micro-CT, have led to a remarkable increase in morphological studies that buildup massive amounts of digital raw data during the last years (see reviews in Ziegler et al. 2010;Boistel et al. 2011;Faulwetter et al. 2013;Sombke et al. 2015). Such new high-throughput analyses allow the digitalization of complete biological specimens or specific diagnostic structures (Handschuh et al. 2013;Lenihan et al. 2014Akkari et al. 2015), acting as supplementary traits and making morphological descriptions of a species significantly more comprehensive and the identification of individuals easier. ...
The correct classification of organisms based on specific rules is essential in biological sciences. Traditionally, morphological characteristics such as size, shape, color, and anatomical structures have been used to identify and classify species. However, as consequence of the tremendous advances in molecular technologies during the last years, new approaches have become available for taxonomic research. Various modern high-throughput technologies allow the detailed characterization of the genome, proteome, metabolome as well as the morphology of an organism. Furthermore, the open access storage of such comprehensive data sets as part of an uprising digital cybertaxonomy enables highly fascinating digital dimensions for modern taxonomy, including the buildup of virtual collections as well as data sets for 3D printing techniques that can be used to replicate complete voucher specimens or at least important diagnostic characters. As a result of these advances, we are now able to document, describe, and identify species much more comprehensively than just a few years ago. In this review we provide an overview about the technical advances in taxonomic research in recent years and discuss their power and limitations.
... Improved automated taxonomic categorisation, as seen in the last decade, has been the result of two different lines of development: (1) novel hardware instrumentation, like 3D imaging, allows that more information is gathered on plankton cells, thus enlarging the portfolio of features that can be used in automated classification (Embleton et al. 2003;Culverhouse et al. 2006;Boistel et al. 2011), and (2) new developments in software have led to improved image processing and classification algorithms (Benfield et al. 2007;Schulze et al. 2013). ...
Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time-consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on "imaging-in-flow," i.e., the ability to acquire microscopic images of planktonic cells in a flow-through mode. Imaging-in-flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e.g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging-in-flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off-axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape-based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic-based imaging-in-flow for similar looking organisms in the nanoplankton range. © 2014, by the American Society of Limnology and Oceanography, Inc.
... The latter has gained interest with the increasing development of valuable new automated imaging techniques, which facilitate visualization, processing and analysis of 3D images (see also Zanette et al., 2013). Those methods include X-ray micro Computed Tomography (µCT) scanning Cnudde et al., 2011), magnetic resonance imaging (MRI) (tysZka et al., 2005;pohlmann et al., 2007), Optical Projection Tomography (OPT) (sharpe et al., 2002), absorption and phase-contrast synchrotron X-ray imaging (BetZ et al., 2007;Boistel et al., 2011) and Light Sheet (based) Fluorescence Microscopy (LSFM) (santi, 2011;Buytaert et al., 2012;desCamps et al., 2012). ...
The use of high resolution, three-dimensional visualization has been receiving growing interest within life sciences, with non-invasive imaging tools becoming more readily accessible. Although initially useful for visualizing mineralized tissues, recent developments are promising for studying soft tissues as well. Especially for micro-CT scanning, several X-ray contrast enhancers are performant in sufficiently contrasting soft tissue organ systems by a different attenuation strength of X-rays. Overall visualization of soft tissue organs has proven to be possible, although the tissue-specific capacities of these enhancers remain unclear. In this study, we tested several contrast agents for their usefulness to discriminate between tissue types and organs, using three model organisms (mouse, zebrafish and Xenopus). Specimens were stained with osmium tetroxide (OsO4), phosphomolybdic acid (PMA) and phosphotungstic acid (PTA), and were scanned using high resolution microtomography. The contrasting potentials between tissue types and organs are described based on volume renderings and virtual sections. In general, PTA and PMA appeared to allow better discrimination. Especially epithelial structures, cell-dense brain regions, liver, lung and blood could be easily distinguished. The PMA yielded the best results, allowing discrimination even at the level of cell layers. Our results show that those staining techniques combined with micro-CT imaging have good potential for use in future research in life sciences. © 2014, Societe Royale Zoologique de Belgique. All rights reserved.
... Puesto que las imágenes en 2D aún presentan algunas limitaciones para la identificación a nivel específico, actualmente se están desarrollando nuevas tecnologías para la obtención de imágenes en 3D con alta definición (imagen acústica (FishTV), estereoscopia, tomografía (resonancia magnética, rayos X), microscopios confocales y holografía), las cuales permiten rotar la imagen y observar estructuras claves para la identificación (i.e., antenas y apéndices entre otras), y obtener medidas volumétricas. Actualmente se ha proyectado para el futuro, hacer una biblioteca de referencia de varios grupos de zooplancton en 3D, y hasta ahora se tiene registros digitalizados de aproximadamente 350-400 organismos en modelos 3D, en su mayoría peces, aunque todavía se requiere un programa específico para su procesamiento (Benfield et al., 2007;Boistel et al., 2011). ...
Los estudios de comunidades de zooplancton son de gran importancia para la biología, ecología y conservación del ecosistema marino. Sin embargo, estas investigaciones implican un proceso de alto costo en términos de tiempo y esfuerzo, requiriendo personal entrenado para la identificación, conteo y medición de numerosos grupos taxonómicos, así como para la estimación de biomasa. Actualmente, existe un creciente interés por la implementación de nuevas técnicas que permitan automatizar los análisis, incorporando tecnología informática aplicada al examen automático de imágenes digitalizadas. En este trabajo se revisa el estado del conocimiento de algunas metodologías, basadas en el estudio de imágenes digitalizadas de zooplancton. Se presenta una descripción general de los métodos conocidos, tales como ZooScan-ZooProcess y Zoolmage, se analizan sus resultados para la identificación de distintos zooplancteres, se comparan con el método tradicional y se hacen propuestas para mejorar su aplicación. Adicionalmente, se resumen las experiencias con estos sistemas en el análisis de patrones de variabilidad espacial y temporal de la comunidad zooplanctónica, basados en el espectro de tamaño y pruebas para la estimación de biomasa de zooplancton. Finalmente, se elabora un listado de capacidades y limitaciones registradas en la bibliografía reciente, y se discute sobre las perspectivas de desarrollo y aplicación que ofrece esta metodología para la comunidad científica en Latinoamérica.
... The list of methodologies comprises optical projection tomography [18], magnetic resonance imaging [19], synchrotron-radiation micro-computed tomography [20,21], autofluorescence imaging [22], and microcomputed tomography [23]. Recent reviews provide a comprehensive overview of non-invasive imaging techniques and their application to terrestrial and marine organisms [24,25]. Because of its high speed, low cost, ease of use, and the achievable high dataset resolution, micro-computed tomography (mCT) has recently evolved to become an important imaging tool in zoological studies. ...
In spite of the high relevance of lumbricid earthworms ('Oligochaeta': Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (μCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that μCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.
... Three-dimensional visualization. Three-dimensional processing and rendering was accomplished after semi-automatic segmentation of the skeleton (Boistel et al., 2011) using "generate surface" and "volume rendering" in AvizoV R 7.01 (VSG, Visualization Sciences Group, Burlington, MA). ...
The comparative vertebral morphology of different chamaeleonid genera has been generally neglected and some aspects such as the comparative anatomy of the neck region remain poorly known. The atlas and axis of all chamaeleonid genera (Brookesia, Rieppeleon, Archaius, Rhampholeon, Nadzikambia, Bradypodion, Chamaeleo, Calumma, Furcifer, Kinyongia, and Trioceros) are studied here. Considerable morphological differences are revealed. Additionally, some taxa exhibit sexual dimorphism in the atlas and axis. An extremely long, divided posterodorsal process is present in males of the Trioceros johnstoni + Trioceros jacksonii clade. The solid and well-developed morphology of the posterodorsal process in males of this taxon could reflect its competitive behavior-males fight with their horns and attempt to dislodge one another from branches during encounters. An additional area of insertion for the cervical musculature may indicate an incremental cervical musculature mass and cross sectional area that can add extra support and stability to the head and assist during combat involving lateral pushing. This character is not present in females. Heterochronic processes have played a role in the evolution of chamaeleonids, as evidenced in many characters of the atlas-axis complex. A new hypothesis of an anterior shifting of synapophyses of the axis is erected and a new derived anatomical structure of the parietal of Chamaeleo calyptratus is described (the processus parietalis inferior). The presence of the processus parietalis inferior is associated with the evolution of the dorsally elevated parietal crest. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.
... This limitation prevents a deeper insight into the ecology of organ size variation and the ontogeny of organs. To overcome this restriction, non-lethal and non-invasive methods are needed (Boistel et al., 2011). A method that does not involve euthanizing the animal, while using the regenerative potential of most starfish, was introduced by Sanford et al. (2009), who found that sampling of only one arm in the starfish Pisaster ochraceus (Brandt, 1835) can be used to determine organ indices. ...
Starfish (Echinodermata: Asteroidea) are present in most benthic ocean habitats and play an important ecological
role as keystone species or by dominating through sheer individual numbers. In order to assess nutritional
and reproductive states in ecological studies on asteroids, invasive techniques to calculate organ indices are conventionally
used. We present a non-invasive method that enables imaging andmorphometric measurements in
starfish in vivo.We used a clinical 1.5 T magnetic resonance imaging (MRI) scanner to produce sectional images
of three starfish species and employed these image stacks to generate 3D models of the pyloric ceca, gonads and
the endoskeleton. In comparison to pre-clinicalMRI scanners, that provide higher resolutions, clinicalMRI is not
limited to small objects, but allows the investigation of larger samples such as the starfish used in the present
study. Volume data from MRI-based 3D reconstructions were compared to conventional invasive measurement
techniques as well as high resolution MRI scans and were tested for inter-observer effects. Here we show that
MRI is a suitablemethod for precise imaging and volumetric measurements in fixed and living marine specimens.
Compared to othermethods, it allows not only the production of time series data on single individuals as well as populations, but also non-destructive analyses of valuable specimens, such as museum material.
... X-ray tomography has been applied in large and medium-sized fish osteological studies. The advent of synchrotron X-ray microtomography expands this application into fine structures and soft tissue morphology without any staining in millimetercentimeter-sized specimens (Pasco-Viel et al, 2010;Boistel et al, 2011). Synchrotron X-rays have the advantage of high coherence suited to phase contrast imaging, an intense photon flux, and a monochromatic and parallel beam with micron/sub-micron spatial resolution. ...
Sinocyclocheilus is a cave-dwelling cyprinid genus endemic to southwest China. Several species possess a conspicuous horn on their head, which has been suggested as a constructive troglomorphic trait but lacks substantial evidence. We used non-invasive, high spatial resolution synchrotron X-ray microtomography to investigate the three-dimensional (3D) morphology of the horn of Sinocyclocheilus hyalinus, one of eight such troglobiotic species. 3D renderings demonstrated the osteological components, which were comprised of a rear wall comprised of the supraoccipital bone, a remaining frontal wall with numerous fenestrae, and the bottom continuous with the parietal and epiotic. A horn cavity occurred within the horn. The fenestrae in the frontal wall were continuous in the horn cavity and showed elaborate channeling, and were, connected to the cranial cavity by soft tissue. We tentatively called this configuration the "otocornual connection" due to its anatomic and putative functional similarity to the otolateralic connection in clupeids and loricariids, which provide an indirect pathway to enhance perception of underwater sound signals. This study provides a functional morphology context for further histological and physiological investigations of such horn structures in Sinocyclocheilus cavefish, and we suggest that the horn might enhance acoustic perception to compensate for visual loss in subterranean life, which warrants future physiological examination as lab-reared S. hyalinus become available.
Sinocyclocheilus is a cave-dwelling cyprinid genus endemic to southwest China. Several species possess a conspicuous horn on their head, which has been suggested as a constructive troglomorphic trait but lacks substantial evidence. We used non-invasive, high spatial resolution synchrotron X-ray microtomography to investigate the three-dimensional (3D) morphology of the horn of Sinocyclocheilus hyalinus, one of eight such troglobiotic species. 3D renderings demonstrated the osteological components, which were comprised of a rear wall comprised of the supraoccipital bone, a remaining frontal wall with numerous fenestrae, and the bottom continuous with the parietal and epiotic. A horn cavity occurred within the horn. The fenestrae in the frontal wall were continuous in the horn cavity and showed elaborate channeling, and were, connected to the cranial cavity by soft tissue. We tentatively called this configuration the "otocornual connection" due to its anatomic and putative functional similarity to the otolateralic connection in clupeids and loricariids, which provide an indirect pathway to enhance perception of underwater sound signals. This study provides a functional morphology context for further histological and physiological investigations of such horn structures in Sinocyclocheilus cavefish, and we suggest that the horn might enhance acoustic perception to compensate for visual loss in subterranean life, which warrants future physiological examination as lab-reared S. hyalinus become available.
... As phytoplankton cells are highly structured objects, conventional microscopy is not adequate for taxonomic recognition of many species as it demands constant refocusing to view the whole body especially under high magnifications. Consequently, 3D imaging is one of the best ways to provide detailed morphological information such that automated identification and classification of these microorganisms may be possible [4,5]. Among those 3D microscopic imaging technologies, laser scanning confocal microscopy (LSCM) is a wellestablished and widely used technology that could capture profiles of small biological particles. ...
This paper reports a light sheet fluorescence imaging flow cytometer for 3D sectioning of phytoplankton. The instrument developed has the inherent advantages of high cell counting throughput and high spatial resolution information derived from flow cytometry and light sheet microscopy. The throughput of the instrument is quantified by the sample volume flow rate of 0.5 μl/min with a spatial resolution as achieved by light sheet microscopy. Preliminary results from 3D morphology of the internal chlorophyll-a structure of two dinoflagellates species show promising application potentials of the method for phytoplankton taxonomy of selected species and species groups.
... These techniques rely on different physical principles, for example, MRI on nuclear magnetic resonance and CT as well as μCT on the application of x-rays. As a rule, the images produced by the respective scanners result in different types of datasets that may depict soft or hard tissue or a combination of both (see Boistel et al. (2011), Johnston (2011), and Laforsch et al. (2012 for further information about imaging techniques). Common to all these modalities is that they can be used to generate 3D datasets that are composed of many individual volume elements (voxels, basically 3D pixels). ...
Following the application of rapid imaging techniques in human diagnostics and neuropsychological research, the study of animal anatomy is currently under considerable transformation because of the increased use of these techniques. Scanning modalities such as computed tomography and magnetic resonance imaging allow gathering digital anatomical data of entire specimens non-invasively and at high speed, while novel visualization software permits actively exploring and manipulating these datasets in real-time and in three dimensions. More importantly, digital data acquisition and computation pave the way towards high-throughput anatomical studies encompassing hundreds or even thousands of zoological specimens. The drastic increase in the availability and computation of digital data has led to significant knowledge gain in other biological disciplines, suggesting that similar progress could be made in animal anatomy. The full potential of this novel approach to studying the internal and external features of fossil and living animals could be exploited by applying image processing machine learning, and pattern recognition algorithms that are currently being employed in and developed for medical imaging as well as other fields of computer vision. We outline how biodiversity research, taxonomy, and comparative anatomical studies can be accelerated using entire-specimen scanning and three-dimensional visualization protocols. In addition, we describe how computational algorithms performing shape recognition and shape analysis can be adapted to their application in large-scale anatomical studies in zoology. Finally, we mention some of the obstacles that a widespread application of the approach proposed here is currently facing and discuss which improvements would be desirable in order to overcome these obstacles.
... frontal, sagittal or transversal) that can be processed digitally to expedite analysis of biological samples. this is the case for X-ray micro Computed tomography (µCt) scanning (Masschaele et al., 2007; Cnudde et al., 2011), Light sheet (based) Fluorescence Microscopy (LsFM) (santi, 2011; Buytaert et al., 2012), Optical Projection tomography (OPt) (sharpe et al., 2002), standard and phase-contrast synchrotron X-ray imaging (Betz et al., 2007; Boistel et al., 2011) and magnetic resonance imaging (MRI) (Tyszka et al., 2005; Pohlmann et al., 2007). LsFM uses laser light sheets to illuminate a fluorescent and transparent sample, while OPt uses light rays to image a transparent sample. ...
Many tools are currently available to investigate and visualize soft and hard tissues in animals both in high-resolution and three dimensions. The most popular and traditional method is based on destructive histological techniques. However, these techniques have some specific limitations. In order to avoid those limitations, various non-destructive approaches have surfaced in the last decades. One of those is micro-CT-scanning. In the best conditions, resolution achieved in micro-CT currently approaches that of standard histological protocols. In addition to bone, soft tissues can also be made visible through micro-CT-scanning. However, discriminating between structures of the same tissue and among different tissue types remains a challenge. An alternative approach, which has not yet been explored to its full potential for comparative anatomy studies, is Orthogonal-Plane Fluorescence Optical Sectioning (OPFOS) microscopy or tomography, also known as (Laser) Light Sheet based Fluorescence Microscopy (LSFM). In this study, we compare OPFOS with light microscopy, applying those techniques to the model organism Xenopus laevis. The potential of both methods for discrimination between different types of tissues, as well as different structures of the same tissue type, is tested and illustrated. Since the histological sections provided a better resolution, adjacent structures of the same tissue type could be discerned more easily compared to our OPFOS images. However, we obtained a more naturally-shaped 3D model of the musculoskeletal system of Xenopus laevis with OPFOS. An overview of the advantages and disadvantages of both techniques is given and their applicability for a wider scope of biological research is discussed.
... The female specimen of R. melanus was fixed in 3.7% formaldehyde solution and placed in a small polypropylene tube for X-ray phase contrast synchrotron microtomography (Betz et al., 2007;Boistel et al., 2011). Images were taken with an effective pixel resolution of 14.8m at 967mm sample-detector distance. ...
Solifugae are an understudied group of relatively large arachnids with well over 1000 species distributed on almost all major continents. These highly active predators utilize their large chelicerae for feeding, defense, burrowing and mating. We investigated the differences in cheliceral morphology and performance of two ecologically divergent species from North Africa; the cursorial Galeodes sp. and the burrowing Rhagodes melanus. Morphological data show differences in aspect ratio between the two species. Bite force measurements show Rhagodes (N=11) to be a much stronger biter than Galeodes (N=8), in terms of both absolute maximum force (Rhagodes 5.63 N, Galeodes 2.12 N) and force relative to cheliceral size. Synchrotron microtomographs of one specimen for each species reveal large differences in physiological cross-sectional area (PCSA) and estimated muscle stress, resulting in a much higher muscle stress in Rhagodes. This species also showed a longer muscle fiber length. Muscle volume and PCSA were found to differ between the two chelicerae in the two scanned specimens. Whereas Rhagodes reflects this morphological asymmetry in having a higher bite force in the right chelicera, Galeodes shows no such bias.
High-resolution 3D imaging of species with exoskeletons such as shell-bearing mollusks typically involves destructive steps. Nondestructive alternatives are desirable since samples can be rare and valuable, and destructive steps are time-consuming and may distort the tissue. Here, we show for the first time that propagation-based phase-contrast X-ray imaging can significantly increase contrast in mollusks with intact shells. By using the recently upgraded monochromator at the SPring-8 BL20B2 synchrotron beamline, we imaged six species of mollusks, showing that X-ray phase contrast enhances soft-tissue contrast. Features that are almost invisible in conventional attenuation-based micro-computed tomography (micro-CT) are clearly reproduced with phase-contrast imaging under the same scan conditions. Furthermore, this method can reveal features such as growth rings in the shell and differentiate between calcite and aragonite crystal forms. Phase-contrast imaging can thus serve as a compelling alternative when destructive methods are not an option.
Members of the shrimp genus StenopusLatreille, 1819 generally inhabit shallow-water coral reefs. Individuals are colorful, with many of them highly sought in the aquarium trade. A recent biodiversity cruise to the Saya de Malha Banks in the western Indian Ocean collected a new species of Stenopus from rather deep waters, at 192–216 m depth. This species is unusual in having a long rostrum overreaching the scaphocerite and a red and white color pattern, unique for Stenopus. A badly damaged specimen from French Polynesia likely belongs to the new species. The characteristics of the new species are illustrated by X-ray microcomputed tomography images (uCT imaging), line drawings, and color photographs. A key is also provided for the 12 known species of Stenopus.
Magnetic resonance imaging (MRI) is a potent tool utilised in several areas of science. The present study contains a short outline of the function of this technique which was provided. Such applications cover the use of MRI in aquatic biology, in fish physiology, in the imaging of the zoological specimens deposited in the museums and in food processing. In the second part of the chapter, a review of the possible aspects where the MRI technique is possible to be used in Iraq was given. Judging from the recent political settlements in Iraq, it seems that the natural history specimens deposited in the Baghdad and Basrah natural history museums are the first issues to be taken into thoughtfulness in operating an MRI technique to save whatever’s left of the specimens after the destruction that occurred in Iraq in the last 15 years. It is also the aim of archiving in a digital library the current fauna of Iraq for the next generations. The other avenues where MRI can be applied in Iraq are the physiological studies on the anadromous fish species such as the clupeid fish Tenualosa ilisha and the future food processing industries.
Otophysan fish are identified as a natural group and have been studied as such since the first half of the 19th century. This very speciose group largely dominates the vertebrate diversity of inland waters today. In the 1980’s passionate debates concerned the otophysan radiation and notably their primary habitat: are they primitively freshwaters or marine animals? To answer we need to better understand the phylogenetic relationships including those of the fossils and thus we need to enhance our knowledge on key bony structures such as the Weberian Apparatus. In this context we propose a CT scan approach, for a specimen of †Chanoides macropoma. In the last years, palaeontology benefited from the developments of phase contrast X-ray microtomography. We expect great advances to investigate the Weberian apparatus and its evolution in Otophysan fishes
Light-sheet fluorescencemicroscopy (LSFM) is a non-invasive opticalmethod for the observation of living specimens. Although this concept was established a century ago it is only during the last decade that instruments suitable for biological applications have been developed, thereby circumventing some of the limitations of established fluorescence imaging techniques such as confocal laser scanning microscopy. LSFM utilizes a sheet of laser light to illuminate only a thin slice of a fluorescently labeled sample. A wide-field fluorescence microscope, placed perpendicular to the light-sheet, serves to collect the fluorescence signal and image the observed region by means of a camera. This side-on illumination configuration features several advantages, including intrinsic optical sectioning without the need of spatial filtering as employed in confocal microscopy, excellent signal-to-noise ratio, high temporal resolution, and drastically reduced overall photobleaching and phototoxicity inside living specimens. Moreover, the non-conventional geometry of LSFM opens up a completely new way of sample mounting, enabling convenientmulti- view image acquisition for 3Dimaging by simple rotation of the sample within the medium-filled chamber. Although LSFM was developed originally for the observation of large organisms such as zebrafish embryos, this method can be adapted to a large range of samples from macroscopic specimens like corals or copepods to microscopic organisms like tintinnids.
Brooding in ophiuroids (Echinodermata: Ophiuroidea) is a highly-developed reproduction strategy and has evolved independently several times. In South African waters, brooding occurs both in the very small amphiurid Amphipholis squamata, which is hermaphroditic, and in the large ophiodermatid Ophioderma wahlbergii, which is gonochoric. For the first time, microcomputed tomography was applied to visualise and compare brooding and brooded young of these two brittle star species from two distantly related families. The scanned A. squamata brooded six young, whereas O. wahlbergii brooded 13 young. Volume measurements of the adults revealed that A. squamata dedicated about 25% of its disc volume to brooding and O. wahlbergii about 14%. Although the brooded young of O. wahlbergii are thought to be among the largest of all known ophiuroids, young of A. squamata were four times larger in relation to adult body disc volume. Other than differences in the sizes of brooded young, these distantly related species have evolved very similar brooding adaptations. All juveniles were found lying with the mouth upward, pressed against the mother's bursal wall, strengthening the hypothesis that the bursal wall acts as a nutritive organ. In both species, the large size of the young can only be explained by maternal nutrient supply. Both brittle star species, therefore, are regarded as truly viviparous.
Fluorescence microscopy allows for the recording of large data sets in high resolution. The analysis of cells and organs in the three dimensional space over time and the exact description of their development is of utmost importance, e.g. in order to discriminate pathological from normal trends during the development of organisms. Oftentimes the human eye does not suffice to observe these trends in the recorded data, since the images are degraded by noise and blur and first need to be reconstructed in their three dimensional shape. The main problems with the recording of larger organisms are the absorption of light in axial direction, as well as noise, diffraction and blurring of light due to the system and object dependent point spread function. The recorded data is incomplete and can only be reconstructed by using prior knowledge. The reconstruction of the original data from the recordings alone is therefore an ill-posed problem.
A new microscopy technology, the light sheet fluorescence microscopy (LSFM), has been developed in the past ten years in order to make the recording of small living animals at cellular resolution over time feasible. The object is recorded from multiple views by rotating and translating the robustly embedded sample. Although a similar setting is known for multiview reconstruction of computer tomography or stereo recordings, well-established techniques from these fields cannot be directly applied to the LSFM reconstruction. However, it is advisable to divide the reconstruction problem into two smaller subproblems, which has also been done in the two applications mentioned above. The two subproblems are registration and fusion of the registered data sets.
In this work a new gray-value based and a new point set based registration algorithm were developed for the registration of microscopic data. The particular challenge for the algorithms are the noise, blur and missing parts of the data. For these challenges a robust quality metric is necessary in order to measure the goodness of the registration result. The "Structural Similarity Index Measure" showed particularly good results for the task of registering LSFM images. Nevertheless a precise and validated registration is only possible with the help of additionally included point markers. Position invariant descriptors for single points can be computed by group integration on the point markers. These descriptors are further used to determine point correspondences between several recordings of the same object and to register them.
For the final data fusion we developed a spatially variant method, which uses the spherical markers to estimate the local point spread function. The most plausible reconstruction of the original object is computed using the estimation for the point spread function, an appropriate noise model and an assumption about the smoothness of the original data. The image restoration problem is formulated in a Bayesian framework and solved using variational methods. We showed in experiments that in comparison to other state-of-the-art methods, our method significantly reduces the noise and sharpens the final reconstructed image.
Apart from the specialized solution for the LSFM data, we also show successful applications of our methods on blind deconvolution of standard microscopy recordings, as well as algorithms for the comparison of structural similarity of proteins.
The monotypic Mimophis mahfalensis is an opisthoglyphous snake endemic of Madagascar. The goal of this study is to clarify the presence of toxin-secreting oral glands through a multidisciplinary approach. We thus provide data on the internal anatomy of the head of M. mahfalensis' and demonstrate the presence of Duvernoy's glands. Furthermore, we refer to two cases of human bitten by this species and compared this with the reported case of mild envenomation mentioned by Domergue (Arch Inst Pasteur Madagascar 56(1):299–311, 1989). Symptoms included brief slight local pain and minor bleeding.
An accessible workflow is presented to create interactive, rotational scanning electron micrographs (rSEM). These information-rich animations facilitate the study and communication of complex morphological structures exemplified here by male arthropod genitalia. Methods are outlined for the publication of rSEMs on the web or in journal articles as SWF files. Image components of rSEMs were archived in MorphBank to ensure future data access. rSEM represents a promising new addition to the toolkit of a new generation of digital taxonomy.
Continuous improvements in the resolution of three-dimensional imaging have led to an increased application of these techniques in conventional taxonomic research in recent years. Coupled with an ever increasing research effort in cybertaxonomy, three-dimensional imaging could give a boost to the development of virtual specimen collections, allowing rapid and simultaneous access to accurate virtual representations of type material. This paper explores the potential of micro-computed tomography (X-ray micro-tomography), a non-destructive three-dimensional imaging technique based on mapping X-ray attenuation in the scanned object, for supporting research in systematics and taxonomy. The subsequent use of these data as virtual type material, so-called "cybertypes", and the creation of virtual collections lie at the core of this potential. Sample preparation, image acquisition, data processing and presentation of results are demonstrated using polychaetes (bristle worms), a representative taxon of macro-invertebrates, as a study object. Effects of the technique on the morphological, anatomical and molecular identity of the specimens are investigated. The paper evaluates the results and discusses the potential and the limitations of the technique for creating cybertypes. It also discusses the challenges that the community might face to establish virtual collections. Potential future applications of three-dimensional information in taxonomic research are outlined, including an outlook to new ways of producing, disseminating and publishing taxonomic information.
Chameleons are highly specialized and mostly arboreal lizards characterized by a suite of derived characters. The grasping feet and tail are thought to be related to the arboreal life-style of chameleons. Yet, specializations for grasping are thought to trade-off with running ability. Indeed, previous studies have demonstrated a trade-off between running and clinging performance with faster species being poorer clingers. Here we investigate the presence of trade-offs by measuring running and grasping performance in four species of chameleon belonging to two different clades (Chamaeleo and Bradypodion). Within each clade we selected a largely terrestrial and a more arboreal species to test whether morphology and performance are related to habitat use. Our results show that habitat drives the evolution of morphology and performance but that some of these effects are specific to each clade. Terrestrial species in both clades show poorer grasping performance than more arboreal species and have smaller hands. Moreover, hand size best predicts gripping performance suggesting that habitat use drives the evolution of hand morphology through its effects on performance. Arboreal species also had longer tails and better tail gripping performance. No differences in sprint speed were observed between the two Chamaeleo species. Within Bradypodion, differences in sprint speed were significant after correcting for body size, yet the arboreal species were both better sprinters and had greater clinging strength. These results suggest that previously documented trade-offs may have been caused by differences between clades (i.e. a phylogenetic effect) rather than by design conflicts between running and gripping per se.
ABSTRACT: In the future, if marine science is to achieve any progress in addressing biological diversity of ocean plankton, then it needs to sponsor development of new technology. One requirement is the development of high-resolution sensors for imaging field-collected and in situ specimens in a non-invasive manner. The rapid automatic categorisation of species must be accompanied by the creation of very large distributed databases in the form of high-resolution 3D rotatable images of
species, which could become the standard reference source for automatic identification. These 3D images will serve as classification standards for field applications, and (in adjusted optical quality) as training templates for image analysis systems based on statistical and other pattern-matching processes. This paper sets out the basic argument for such developments and proposes a long-term
solution to achieve these aims.
A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital
scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant
zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides
“digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global
cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body
axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's
cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
A fundamental understanding of the interaction between physical and biological factors that regulate plankton species composition requires, first of all, detailed and sustained observations. Only now is it becoming possible to acquire these types of observations, as we develop and deploy instruments that can continuously monitor individual organisms in the ocean. Our research group can measure and count the smallest phytoplankton cells using a submersible flow cytometer (FlowCytobot), in which optical properties of individual suspended cells are recorded as they pass through a focused laser beam. However, FlowCytobot cannot efficiently sample or identify the much larger cells (10 to >100 μm) that often dominate the plankton in coastal waters. Because these larger cells often have recognizable morphologies, we have developed a second submersible flow cytometer, with imaging capability and increased water sampling rate (typically, 5 mL seawater analyzed every 20 min), to characterize these nano- and microplankton. Like the original, Imaging FlowCytobot can operate unattended for months at a time; it obtains power from and communicates with a shore laboratory, so we can monitor results and modify sampling procedures when needed. Imaging FlowCytobot was successfully tested for 2 months in Woods Hole Harbor and is presently deployed alongside FlowCytobot at the Martha's Vineyard Coastal Observatory. These combined approaches will allow continuous long-term observations of plankton community structure over a wide range of cell sizes and types, and help to elucidate the processes and interactions that control the life cycles of individual species. © 2007, by the American Society of Limnology and Oceanography, Inc.
A two-vessel exercise was conducted over the southern flank of Georges Bank during the onset of vernal stratification in May 1992. The Video Plankton Recorder (VPR), a towed video system, was used to map out the fine-scale distributions of zooplankton to a depth of 70 m along a trackline which described a regular grid (3.5 × 4.5 km) in Lagrangian space. A second vessel following a parallel course conducted Multiple Opening/Closing Net and Environmental Sensing System (MOCNESS) sampling during the last section of the grid, which provided an opportunity to compare data from the two systems. Both the VPR and the MOCNESS provided similar data on the taxonomic composition of the plankton which was numerically dominated by copepods (Calanus, Pseudocalanus, Oithona), pteropods (Limacina) and larvaceans (Oikopleura). The absence of rare (
IN conventional radiography, X-rays which pass through an object along
different paths are differentially absorbed, and the intensity pattern
of the emerging beam records the distribution of absorbing materials
within the sample. An alternative approach is phase-contrast
radiography, which instead records variations of the phase of the
emerging radiation. Such an approach offers improved contrast
sensitivity, especially when imaging weakly absorbing samples.
Unfortunately, current phase-contrast imaging techniques1-11
generally require highly monochromatic plane-wave radiation and
sophisticated X-ray optics, so their use is greatly restricted. Here we
describe and demonstrate a simplified scheme for phase-contrast imaging
based on an X-ray source having high spatial (but essentially no
chromatic) coherence. The method is compatible with conventional
polychromatic micro-focus X-ray tube sources, is well suited to large
areas of irradiation, can operate with a lower absorbed dose than
traditional X-ray imaging techniques, and should find broad application
in clinical, biological and industrial settings.
In the future, if marine science is to achieve any progress in addressing biological diversity of ocean plankton, then it needs to sponsor development of new technology. One requirement is the development of high-resolution sensors for imaging field-collected and in situ specimens in a non-invasive manner. The rapid automatic categorisation of species must be accompanied by the creation of very large distributed databases in the form of high-resolution 3D rotatable images of species, which could become the standard reference source for automatic identification. These 3D images will serve as classification standards for field applications, and (in adjusted optical quality) as training templates for image analysis systems based on statistical and other pattern-matching processes. This paper sets out the basic argument for such developments and proposes a long-term solution to achieve these aims.
Phase objects are readily imaged through Fresnel diffraction in the hard x-ray beams of third-generation synchrotron radiation sources such as the ESRF, due essentially to the very small angular size of the source. Phase objects can lead to spurious contrast in x-ray diffraction images (topographs) of crystals. It is shown that this contrast can be eliminated through random phase plates, which provide an effective way of tailoring the angular size of the source. The possibilities of this very simple technique for imaging phase objects in the hard x-ray range are explored experimentally and discussed. They appear very promising, as shown in particular by the example of a piece of human vertebra, and could be extended to phase tomography.
Coherent properties of the x‐ray beam delivered at the ESRF allow the observation of very weak perturbations of the wave front, resulting in the phase contrast. A straightforward experimental setup for phase contrast imaging is proposed and used to record holographic images from organic samples of 10–100 μm at energy 10–50 keV with the contrast up to 50%–100%. The theory of phase contrast imaging is considered and some theoretical estimations are made to reveal the performance of the proposed technique in terms of resolution, sensitivity, geometrical requirements, and energy range applicability. It is found that for carbon‐based fibers a detectable size with 2% contrast is 0.1 μm for 10 keV and -1 μm for 100 keV. It is demonstrated that the fine interference structure of the image is very sensitive to the shape, density variation, and internal structure of the sample. Some prospects for the practical use and future development of the new coherent techniques such as phase contrast microscopy, microtomography, holography, and interferometry at high energies are also discussed. © 1995 American Institute of Physics.
Because the refractive index for hard x rays is slightly different from unity, the optical phase of a beam is affected by transmission through an object. Phase images can be obtained with extreme instrumental simplicity by simple propagation provided the beam is coherent. But, unlike absorption, the phase is not simply related to image brightness. A holographic reconstruction procedure combining images taken at different distances from the specimen was developed. It results in quantitative phase mapping and, through association with three-dimensional reconstruction, in holotomography, the complete three-dimensional mapping of the density in a sample. This tool in the characterization of materials at the micrometer scale is uniquely suited to samples with low absorption contrast and radiation-sensitive systems. © 1999 American Institute of Physics.
This paper describes a 3-D reconstruction method which allows accurate measurements of volume, surface area and other morphometric measurements of three-dimensional biological objects, without removing them from the sea. It represents a novel approach based on multiple views (eight resulted to be sufficient) from underwater video images and a new image processing procedure (MOD3D), whose application has met the basic requirements (i.e. to work on images recorded in turbid waters, with nonuniform lighting, to investigate large areas and in reasonable time, etc.) imposed when operating in the marine environment with simple, easy-to-use and nonprofessional equipment. It is a noninvasive, nondestructive and in the field fast method, thus suitable for sampling also at relevant depth, whose applicability has specifically been set up for a range of growth forms from massive to submassive and irregularly shaped. The accuracy of the method was assessed using models with three levels of 3-D complexity: simple, moderate and complex morphology. A high accuracy of volume measurements made through MOD3D image analysis software was achieved when compared with the laboratory water displacement method, which represents the most accurate method for volume measurement, with an overall mean percent error of about 1.7% (S.D. 2.2%). For all three levels of morphologic complexity, no significant differences (p>0.05) were found. Volume measurements obtained in field based on geometric approximation resulted rough, with significant differences from the MOD3D values (p<0.05). The geometric approximation was lower than MOD3D for simple and moderate morphology, and variable for complex morphology. For all three models, MOD3D values for surface area computation were consistently lower (mean error 13%) than the foil-wrapping values (p<0.05), due to overlap error when foil wrapping. Two applications were made with the bryozoan Pentapora fascialis and the coral Cladocora caespitosa to quantify carbonate standing stock and biomass of these two carbonate framework builders, whose importance has been recently recognised among the temperate sublittoral benthic species. Time required for the 3-D reconstruction method (about 3 h) makes it suitable for routine application particularly for relatively large area investigations, with irregularly shaped objects on rough substrate and several biological objects within the area.
The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed fragment recruitment, addressed questions of geno
Traditional comparative morphological analyses and subsequent three-dimensional reconstructions suffer from a number of drawbacks. This is particularly evident in the case of soft tissue studies that are technically demanding, time-consuming, and often prone to produce artefacts. These problems can partly be overcome by employing non-invasive, destruction-free imaging techniques, in particular micro-computed tomography or magnetic resonance imaging.
Here, we employed high-field magnetic resonance imaging techniques to gather numerous data from members of a major marine invertebrate taxon, the sea urchins (Echinoidea). For this model study, 13 of the 14 currently recognized high-ranking subtaxa (orders) of this group of animals were analyzed. Based on the acquired datasets, interactive three-dimensional models were assembled. Our analyses reveal that selected soft tissue characters can even be used for phylogenetic inferences in sea urchins, as exemplified by differences in the size and shape of the gastric caecum found in the Irregularia.
The main focus of our investigation was to explore the possibility to systematically visualize the internal anatomy of echinoids obtained from various museum collections. We show that, in contrast to classical preparative procedures, magnetic resonance imaging can give rapid, destruction-free access to morphological data from numerous specimens, thus extending the range of techniques available for comparative studies of invertebrate morphology.
The aim of this study was to introduce high-resolution computed tomography (CT) of breast tumours using the diffraction-enhanced x-ray imaging (DEI) technique and to compare results with radiological and histo-pathological examinations. X-ray CT images of tumour-bearing breast tissue samples were acquired by monochromatic synchrotron radiation (SR). Due to the narrow beam and a large sample-to-detector distance scattering is rejected in the absorption contrast images (SR-CT). Large contrast enhancement is achieved by the use of the DEI-CT method, where the effects of refraction and scatter rejection are analysed by crystal optics. Clinical mammograms and CT images were recorded as reference material for a radiological examination. Three malignant and benign samples were studied in detail. Their radiographs were compared with optical images of stained histological sections. The DEI-CT images map accurately the morphology of the samples, including collagen strands and micro-calcifications of dimensions less than 0.1 mm. Histo-pathological examination and reading of the radiographs were done independently, and the conclusions were in general agreement. High-resolution DEI-CT images show strong contrast and permit visualization of details invisible in clinical radiographs. The radiation dose may be reduced by an order of magnitude without compromising image quality, which would make possible clinical in vivo DEI-CT with future compact SR sources.
We consider the phase retrieval problem in 3-D holotomography for strongly absorbing objects. Holotomography combines phase retrieval from Fresnel diffraction patterns with tomographic reconstruction to reconstruct the 3-D refractive index distribution. The main interest is the increase in sensitivity of up to three orders of magnitude compared to standard, absorption based tomography. Most existing algorithms are based upon linearization of the forward problem. This is motivated by the large problem size, since it yields computationally efficient solutions. Here, the mixed approach is used, which allows for both strong absorption and long propagation distances. Previous implementations have shown promising results, but in practice often suffer from strong low frequency artifacts. To address this problem, we introduce a homogeneous object assumption through a regularizing term based upon the absorption image. This allows the homogeneous object assumption to be introduced only in the low frequency range. The proportionality constant between absorption and refractive index is assumed to be known. The regularizing parameter is found using the standard L-curve technique. The benefits of our approach are illustrated using data measured at the European Synchrotron Radiation Facility. Low frequency noise in the reconstruction is alleviated, but the result is only quantitative in the areas of the sample where the homogeneous object assumption is fulfilled.
The fish order Cypriniformes is one of the most diverse ray-finned fish groups in the world with more than 3000 recognized species. Cypriniformes are characterized by a striking distribution of their dentition: namely the absence of oral teeth and presence of pharyngeal teeth on the last gill arch (fifth ceratobranchial). Despite this limited localisation, the diversity of tooth patterns in Cypriniformes is astonishing. Here we provide a further description of this diversity using X-ray microtomography and we map the resulting dental characters on a phylogenetic tree to explore evolutionary trends.
We performed a pilot survey of dental formulae and individual tooth shapes in 34 adult species of Cypriniformes by X-ray microtomography (using either conventional X-ray machine, or synchrotron microtomography when necessary) or by dissecting. By mapping morphological results in a phylogenetic tree, it emerges that the two super-families Cobitoidea and Cyprinoidea have followed two distinct evolutionary pathways. Furthermore, our analysis supports the hypothesis of a three-row dentition as ancestral for Cyprinoidea and a general trend in tooth row reduction in most derived lineages. Yet, this general scheme must be considered with caution as several events of tooth row gain and loss have occurred during evolutionary history of Cyprinoidea.
Dentition diversity in Cypriniformes constitutes an excellent model to study the evolution of complex morphological structures. This morphological survey clearly advocates for extending the use of X-ray microtomography to study tooth morphology in Cypriniformes. Yet, our survey also underlines that improved knowledge of Cypriniformes life traits, such as feeding habits, is required as current knowledge is not sufficient to conclude on the link between diet and dental morphology.
X-ray radiographic absorption imaging is an invaluable tool in medical diagnostics and materials science. For biological tissue samples, polymers or fibre composites, however, the use of conventional X-ray radiography is limited due to their weak absorption. This is resolved at highly brilliant X-ray synchrotron or micro-focus sources by using phase-sensitive imaging methods to improve the contrast. However, the requirements of the illuminating radiation mean that hard-X-ray phase-sensitive imaging has until now been impractical with more readily available X-ray sources, such as X-ray tubes. In this letter, we report how a setup consisting of three transmission gratings can efficiently yield quantitative differential phase-contrast images with conventional X-ray tubes. In contrast with existing techniques, the method requires no spatial or temporal coherence, is mechanically robust, and can be scaled up to large fields of view. Our method provides all the benefits of contrast-enhanced phase-sensitive imaging, but is also fully compatible with conventional absorption radiography. It is applicable to X-ray medical imaging, industrial non-destructive testing, and to other low-brilliance radiation, such as neutrons or atoms
In conventional projection radiography, cartilage and other soft tissues do not produce enough radiographic contrast to be distinguishable from each other. Diffraction-enhanced imaging (DEI) uses a monochromatic x-ray beam and a silicon crystal analyzer to produce images in which attenuation contrast is greatly enhanced and x-ray refraction at tissue boundaries can be detected. The aim of this study was to test the efficacy of conventional x-ray tube-based DEI for the detection of soft tissues in experimental samples.
Cadaveric human tali (normal and degenerated) and a knee and thumb were imaged with DEI using a conventional x-ray tube and DEI setup that included a double-silicon crystal monochromator and a silicon crystal analyzer positioned between the imaged object and the detector.
Diffraction-enhanced images of the cadaveric tali allowed the visualization of cartilage and its specific level of degeneration for each specimen. There was a significant correlation between the grade of cartilage integrity as assessed on the tube diffraction-enhanced images and on their respective histologic sections (r = 0.97, P = .01). Images of the intact knee showed the articular cartilage edge of the femoral condyle, even when superimposed by the tibia. In the thumb image, it was possible to visualize articular cartilage, tendons, and other soft tissues.
DEI based on a conventional x-ray tube allows the visualization of skeletal and soft tissues simultaneously. Although more in-depth testing and optimization of the DEI setup must be carried out, these data demonstrate a proof of principle for further development of the technology for future clinical imaging.
Comparative, functional, and developmental studies of animal morphology require accurate visualization of three-dimensional structures, but few widely applicable methods exist for non-destructive whole-volume imaging of animal tissues. Quantitative studies in particular require accurately aligned and calibrated volume images of animal structures. X-ray microtomography (microCT) has the potential to produce quantitative 3D images of small biological samples, but its widespread use for non-mineralized tissues has been limited by the low x-ray contrast of soft tissues. Although osmium staining and a few other techniques have been used for contrast enhancement, generally useful methods for microCT imaging for comparative morphology are still lacking.
Several very simple and versatile staining methods are presented for microCT imaging of animal soft tissues, along with advice on tissue fixation and sample preparation. The stains, based on inorganic iodine and phosphotungstic acid, are easier to handle and much less toxic than osmium, and they produce high-contrast x-ray images of a wide variety of soft tissues. The breadth of possible applications is illustrated with a few microCT images of model and non-model animals, including volume and section images of vertebrates, embryos, insects, and other invertebrates. Each image dataset contains x-ray absorbance values for every point in the imaged volume, and objects as small as individual muscle fibers and single blood cells can be resolved in their original locations and orientations within the sample.
With very simple contrast staining, microCT imaging can produce quantitative, high-resolution, high-contrast volume images of animal soft tissues, without destroying the specimens and with possibilities of combining with other preparation and imaging methods. Such images are expected to be useful in comparative, developmental, functional, and quantitative studies of morphology.
Phase-sensitive X-ray imaging methods can provide substantially increased contrast over conventional absorption-based imaging, and therefore new and otherwise inaccessible information. Differential phase-contrast (DPC) imaging, which uses a grating interferometer and a phase-stepping technique, has been integrated into TOMCAT, a beamline dedicated to tomographic microscopy and coherent radiology experiments at the Swiss Light Source. Developments have been made focusing on the fast acquisition and post-processing of data to enable a high-throughput of samples, with obvious advantages, also through increasing the efficiency of the detecting system, of helping to reduce radiation dose imparted to the sample. A novel aquarium design allows a vertical rotation axis below the sample with measurements performed in aqueous environment. Optimization of the data acquisition procedure enables a full phase volume (1024 x 1024 pixels x 1000 projections x 9 phase steps, i.e. 9000 projections in total) to be acquired in 20 min (with a pixel size of 7.4 microm), and the subsequent post-processing has been integrated into the beamline pipeline for sinogram generation. Local DPC tomography allows one to focus with higher magnification on a particular region of interest of a sample without the presence of local tomography reconstruction artifacts. Furthermore, 'widefield' imaging is shown for DPC scans for the first time, enabling the field of view of the imaging system to be doubled for samples that are larger than the magnification allows. A case study is illustrated focusing on the visualization of soft tissue features, and particularly the substantia nigra of a rat brain. Darkfield images, based on local X-ray scattering, can also be extracted from a grating-based DPC scan: an example of the advantages of darkfield contrast is shown and the potential of darkfield X-ray tomography is discussed.
Stereotaxic atlases of the mouse brain are important in neuroscience research for targeting of specific internal brain structures during surgical operations. The effectiveness of stereotaxic surgery depends on accurate mapping of the brain structures relative to landmarks on the skull. During postnatal development in the mouse, rapid growth-related changes in the brain occur concurrently with growth of bony plates at the cranial sutures, therefore adult mouse brain atlases cannot be used to precisely guide stereotaxis in developing brains. In this study, three-dimensional stereotaxic atlases of C57BL/6J mouse brains at six postnatal developmental stages: postnatal day (P) 7, P14, P21, P28, P63 and in adults (P140-P160) were developed, using diffusion tensor imaging (DTI) and micro-computed tomography (CT). At present, most widely-used stereotaxic atlases of the mouse brain are based on histology, but the anatomical fidelity of ex vivo atlases to in vivo mouse brains has not been evaluated previously. To account for ex vivo tissue distortion due to fixation as well as individual variability in the brain, we developed a population-averaged in vivo magnetic resonance imaging adult mouse brain stereotaxic atlas, and a distortion-corrected DTI atlas was generated by nonlinearly warping ex vivo data to the population-averaged in vivo atlas. These atlas resources were developed and made available through a new software user-interface with the objective of improving the accuracy of targeting brain structures during stereotaxic surgery in developing and adult C57BL/6J mouse brains.
Despite a growing need, oceanographers are limited by existing technological constrains and are unable to observe aquatic microbes in their natural setting. In order to provide a simple and easy to implement solution for such studies, a new Thin Light Sheet Microscope (TLSM) has been developed. The TLSM utilizes a well-defined sheet of laser light, which has a narrow (23 micron) axial dimension over a 1 mm × 1 mm field of view. This light sheet is positioned precisely within the depth of field of the microscope’s objective lens. The technique thus utilizes conventional microscope optics but replaces the illumination system. The advantages of the TLSM are two-fold: First, it concentrates light only where excitation is needed, thus maximizing the efficiency of the illumination source. Secondly, the TLSM maximizes image sharpness while at the same time minimizing the level of background noise. Particles that are not located within the objective’s depth of field are not illuminated and therefore do not contribute to an out-of-focus image. Images from a prototype system that used SYBR Green I fluorescence stain in order to localize single bacteria are reported. The bacteria were in a relatively large and undisturbed volume of 4ml, which contained natural seawater. The TLSM can be used for fresh water studies of bacteria with no modification. The microscope permits the observation of interactions at the microscale and has potential to yield insights into how microbes structure pelagic ecosystems.
Can conscious processing be inferred from neurophysiological measurements? Some models stipulate that the active maintenance of perceptual representations across time requires consciousness. Capitalizing on this assumption, we designed an auditory paradigm that evaluates cerebral responses to violations of temporal regularities that are either local in time or global across several seconds. Local violations led to an early response in auditory cortex, independent of attention or the presence of a concurrent visual task, whereas global violations led to a late and spatially distributed response that was only present when subjects were attentive and aware of the violations. We could detect the global effect in individual subjects using functional MRI and both scalp and intracerebral event-related potentials. Recordings from 8 noncommunicating patients with disorders of consciousness confirmed that only conscious individuals presented a global effect. Taken together these observations suggest that the presence of the global effect is a signature of conscious processing, although it can be absent in conscious subjects who are not aware of the global auditory regularities. This simple electrophysiological marker could thus serve as a useful clinical tool.
Male songbirds learn their songs from an adult tutor when they are young. A network of brain nuclei known as the 'song system' is the likely neural substrate for sensorimotor learning and production of song, but the neural networks involved in processing the auditory feedback signals necessary for song learning and maintenance remain unknown. Determining which regions show preferential responsiveness to the bird's own song (BOS) is of great importance because neurons sensitive to self-generated vocalisations could mediate this auditory feedback process. Neurons in the song nuclei and in a secondary auditory area, the caudal medial mesopallium (CMM), show selective responses to the BOS. The aim of the present study is to investigate the emergence of BOS selectivity within the network of primary auditory sub-regions in the avian pallium.
Using blood oxygen level-dependent (BOLD) fMRI, we investigated neural responsiveness to natural and manipulated self-generated vocalisations and compared the selectivity for BOS and conspecific song in different sub-regions of the thalamo-recipient area Field L. Zebra finch males were exposed to conspecific song, BOS and to synthetic variations on BOS that differed in spectro-temporal and/or modulation phase structure. We found significant differences in the strength of BOLD responses between regions L2a, L2b and CMM, but no inter-stimuli differences within regions. In particular, we have shown that the overall signal strength to song and synthetic variations thereof was different within two sub-regions of Field L2: zone L2a was significantly more activated compared to the adjacent sub-region L2b.
Based on our results we suggest that unlike nuclei in the song system, sub-regions in the primary auditory pallium do not show selectivity for the BOS, but appear to show different levels of activity with exposure to any sound according to their place in the auditory processing stream.
High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.