Corresponding author: Eva Chatzinikolaou (
Academic editor: Alexander M. Weigand
Micro-computed tomography (micro-CT) is a high-resolution 3D-imaging technique which is now increasingly applied in biological studies focusing on taxonomy and functional morphology. The creation of virtual representations of specimens can increase availability of otherwise underexploited and inaccessible samples. The 3D model dataset can be also further processed through volume rendering and morphometric analysis. The success of micro-CT as a visualisation technique depends on several methodological manipulations, including the use of contrast enhancing staining agents, filters, scanning mediums, containers, exposure time and frame averaging. The aim of this study was to standardise a series of micro-CT scanning and 3D analysis protocols for a marine gastropod species,
This work was funded under the project ECCO (HFRI, Hellenic Foundation for Research and Innovation for the Support of Post-doctoral Researchers, project ID 343).
Hellenic Centre for Marine Research - Institute of Marine Biology, Biotechnology and Aquaculture
Micro-computed tomography (micro-CT) is a high-resolution technique, based on X-ray 3D-imaging which allows non-destructive studies of external and internal structures of a specimen. Micro-CT has been more extensively used in geological and paleontological studies, but it is now often used as a tool in biological studies focusing on taxonomy, evolution and functional morphology (
A specimen is placed between an X-ray source and an X-ray detector in the micro-CT scanner, resulting in the acquisition of a large set of 2D projections (2D images) of the object around a rotation axis which can be then reconstructed into cross-section images. A full virtual representation of the specimen is created as a 3D model, which can be interactively manipulated on screen (rotation, zoom, virtual dissection and isolation of specific features of interest) (
The success of micro-CT as a visualisation technique depends initially on achieving a sufficient contrast difference between the specimen and its surrounding medium, as well as between the different specimen tissues (e.g. hard structures versus soft tissues) (
The appropriate selection of several parameters, including voltage and current applied, scanning medium and container, use of filters and/or staining agents, exposure time, magnification, degrees of specimen rotation and rotation step, frame averaging etc., are important for the achievement of the optimum imaging result. Therefore, the aim of this study was to establish and standardise a series of micro-CT scanning and 3D analysis protocols for a marine gastropod species,
The analytical protocols have followed all the developmental stages of this gastropod, from egg capsules and embryos to juveniles and adults.
All scans were performed with a SkyScan 1172 micro-tomograph (Bruker, Kontich, Belgium) at the Hellenic Center for Marine Research (HCMR), Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC), Heraklion, Crete. The scanner uses a tungsten source and is equipped with an 11MP CCD camera (4000 x 2672 pixels), which can reach a maximal resolution of < 0.8 m m/pixel.
Adult
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium) which employs a modified Feldkamp’s back-projection algorithm. All scans were reconstructed using the same range of attenuation coefficients (0 - 0.13) in order to obtain comparable results between samples. The reconstructed images were stored as 16-bit TIFF images. Volume renderings of each specimen were created using the CTVox software (Bruker, Kontich, Belgium) in order to display the reconstructed images as a 3D object (Fig.
The cross section images were loaded into the software CT Analyser v.1.18.4.0 (CTAn, Bruker, Kontich, Belgium). The mean greyscale values of the total shell were calculated using the binary threshold module which allows for comparable measurements of the relative density of the calcified tissues (i.e. shell). For the present analyses, no absolute density values (e.g. Hounsfield units) were required, since comparability between the scans by the calculation of relative densities was considered to be sufficient. Relative grey scale density in the present study is used as a proxy for "micro-density" (i.e. density of the shell material including calcium carbonate (CaCO3) and intraskeletal organic matrix) and not to bulk density which includes porosity. The range of the greyscale histogram was 30 - 255 for all shell specimens of adult
Furthermore, the 3D analysis was performed with the custom processing plugin of the CTAn software, using the volume of interest (VOI) in each specimen in order to calculate the porosity and the structure thickness. Porosity was calculated as the percentage of the closed porosity of the shell (i.e. total volume of enclosed pores of each specimen as a percentage of the total shell volume) (Fig.
3D geometric morphometric methods were applied on the surface model of each specimen, which was created using the CTAn software. Subsequently, these surface models were loaded into the Landmark Editor software in order to add landmarks as single points and curves in specific shell areas, which will be used for the morphological shape comparisons. Comparable measurements of shape were performed by reproducing the same landmark protocol in all shell specimens. All data points were loaded into the MorphoJ software where models were adjusted in a "procrustes fit" in order to conduct a Procrustes Analyses.
The protocols for adult
Egg capsules of
Projection images were reconstructed into cross sections using SkyScan’s NRecon software (Bruker, Kontich, Belgium) which employs a modified Feldkamp’s back-projection algorithm. All scans were reconstructed using the same range of attenuation coefficients (0 - 0.064) in order to obtain comparable results. The reconstructed images were stored as 16-bit TIFF images. Volume renderings of the egg capsules were created using the CTVox software (Bruker, Kontich, Belgium) in order to display the reconstructed images as a 3D object (Fig.
The scanning protocols for the egg capsules of
Embryos of
Projection images were reconstructed into cross sections using SkyScan’s NRecon software (Bruker, Kontich, Belgium) which employs a modified Feldkamp’s back-projection algorithm. All scans were reconstructed using the same range of attenuation coefficients (0 - 1.127 for embryos and 0 - 1 for juveniles) in order to obtain comparable results. The reconstructed images were stored as 16-bit TIFF images. Volume renderings of the embryos and juveniles were created using the CTVox software (Bruker, Kontich, Belgium) in order to display the reconstructed images as a 3D object (Fig.
The scanning protocols for the embryos and juveniles of
The use of micro-CT for studying the structural and morphological properties of the gastropod shell has been proven as a valuable tool, which can be successfully applied throughout the different developmental stages of this gastropod species. In addition, the micro-CT generated volume renderings revealed the internal calcareous statolith structures of embryos and egg capsules of
The present manuscript presented some specific guidelines regarding scanning of the marine gastropod
Dr Eva Chatzinikolaou (writing of manuscript, design of protocols, implementation of scanning and 3D analysis, scientific responsible)
Kleoniki Keklikoglou (writing of manuscript, design of protocols, implementation of scanning and 3D analysis)
Volume rendering of an adult
3D model of the closed pores of the adult
Colour-coded image of adult
Volume rendering of egg capsules of
Volume rendering of gastropod embryos of
Volume rendering of a 6.5 months old juvenile