Architecture of Anoteropora latirostris (Bryozoa, Cheilostomata) and implications for their biomineralization.

Cheilostome Bryozoa Anoteropora latirostris, a colonial marine invertebrate, constructs its skeleton from calcite and aragonite. This study presents firstly correlated multi-scale electron microscopy, micro-computed tomography, electron backscatter diffraction and NanoSIMS mapping. We show that all primary, coarse-grained platy calcitic lateral walls are covered by fine-grained fibrous aragonite. Vertical lateral walls separating autozooid chambers have aragonite only on their distal side. This type of asymmetric mineralization of lateral walls results from the vertical arrangement of the zooids at the growth margins of the colony and represents a type of biomineralization previously unknown in cheilostome bryozoans. NanoSIMS mapping across the aragonite-calcite interface indicates an organic layer between both mineral phases, likely representing an organic template for biomineralization of aragonite on the calcite layer. Analysis of crystallographic orientations show a moderately strong crystallographic preferred orientation (CPO) for calcite (7.4 times random orientation) and an overall weaker CPO for aragonite (2.4 times random orientation) with a high degree of twinning (45%) of the aragonite grains. The calculated Young's modulus for the CPO map shows a weak mechanical direction perpendicular to the colony's upper surface facilitating this organism's strategy of clonal reproduction by fragmentation along the vertical zooid walls.

Microscopic dual-energy CT (microDECT): a flexible tool for multichannel ex vivo 3D imaging of biological specimens.

Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 µm. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (https://github.com/microDECT/DECTDec). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research.

Tailored order: the mesocrystalline nature of sea urchin teeth.

We investigated the pattern of crystal co-orientation at different length scales, together with variations in chemical composition and nanomechanical properties in the teeth of the modern sea urchin Paracentrotus lividus with electron backscatter diffraction (EBSD), electron probe microanalysis, energy-dispersive X-ray spectroscopy and nanoindentation testing. Modern sea urchin teeth are Mg-dominated calcite composite materials. They are distinctly harder than inorganically precipitated calcite. Some parts exceed even the hardness of dolomite. The teeth show a structuring of their mechanical properties that can be correlated to variations in major element chemical composition, such that their hardness is positively correlated to their magnesium contents. Mg/Ca ratio in Paracentrotus lividus varies between 10 and 26mol.%. Nanohardness of the tooth scatters between 3.5 and >8GPa compared to values of 3.0±0.2, 7.3±0.1 and 9.2±0.9GPa measured on the (104) planes of inorganic calcite, dolomite and magnesite, respectively. High-resolution EBSD shows that major structural units and subunits of the tooth of Paracentrotus lividus are tilted to each other by ∼3-5° and 1-2°, respectively. This indicates that the tooth is not a single crystal. With EBSD we can show that the tooth of the sea urchin Paracentrotus lividus is a hierarchically assembled biological mesocrystal with a mosaic texture. In comparison to the misorientation spread of 0.5° of calcite grown from solution, misorientation in the tooth varies between 2° and 4°. Thus, the self-sharpening feature of the tooth is enabled by a close interplay of its highly evolved micro- to nanostructure, structural unit size variations with a varying degree of crystal orientation, chemical structuring of the mineral component and a gradation of incorporated organic polymers.

Ribbons of semithin sections: an advanced method with a new type of diamond knife.

Complete series of semithin sections are imperative for 3-D reconstruction, but with traditional microtomy techniques it is difficult and time-consuming to trace stained and labeled structures. In the present study we introduce a method for making and collecting ribbons of semithin sections with a new, commercial available diamond knife (histo-jumbo-diamond knife, Diatome AG, Biel, Switzerland). The special feature of the diamond knife is the large water bath (boat) into which a glass slide can be dipped. The method has distinct advantages and the handling is simple. The resin block is trimmed into a truncated pyramid. Contact glue is applied to the leading face of the pyramid, which makes sections stick together to form a ribbon. Following sectioning, the ribbons are mounted onto glass slides and aligned in parallel. Stretching out and drying the ribbons on a hot plate is the final step of the method. Major advantages of this method are the perfect alignment of sections with identical orientation of structures, the completeness of series, and the significant saving of time. This facilitates tracing of stained and labeled structures, yielding quick 3-D reconstruction. Semithin sections can be cut from 0.5 to 2 micro m and several ribbons can be mounted side by side onto the slide. Two examples are presented to illustrate the advantages of the method.

Microanatomy and ultrastructure of the protonephridial system in the larva of the limpet, Patella vulgata L. (Mollusca, Patellogastropoda).

The microanatomy and ultrastructure of the larval excretory system of Patella vulgata L., 1758 has been examined by means of semithin and ultrathin serial sections, reconstructions, and transmission electron microscopy. The protonephridial system appears after torsion and consists of two terminal flame bulbs with narrow, ciliated ducts. Whereas the polyciliary terminal cells (cyrtocytes) are only slightly asymmetrically placed below the mantle cavity, the distal excretory ducts and their openings show remarkable asymmetry due to torsion. Further larval ultrafiltration sites with identical fine-structure (meandering slits with diaphragms covered by extracellular matrix) are present in the solitary rhogocytes (pore cells). The presence of larval protonephridia is regarded as plesiomorphic for Mollusca and the Trochozoa (Spiralia) as a whole and the specific conditions (asymmetry, simplicity) in Patella are probably plesiomorphic for the Gastropoda.

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda).

Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered.