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Investigation of contrast enhancing techniques for the application of Micro-CT in marine biodiversity

studies

Faulwetter, Sarah1, Dailianis, Thanos1, Vasileiadou, Aikaterini1, Arvanitidis, Christos1

1 Institute of Marine Biology and Genetics, Hellenic Centre for Marine Research, Thalassocosmos, P.O. Box 2214, 71003 Heraklion, Crete, Greece, sarifa@hcmr.gr Aims Taxonomy – the science of identifying, describing and naming species – relies on the availability of the so-called type specimens. Those are specimens on which the description of an organism is based and thus are a reference standard. For a new taxon description to be formally recognised by the scientific community, the type material has to be deposited in publicly available museum collections. However, in practice, the process of loaning specimens from museums for study is time-consuming and often unsuccessful. Modern non-destructive 3D-techniques such as MicroCT could overcome the problems of traditional specimen handling through the creation of virtual collections which can be studied instantly and simultaneously by scientists all over the world. Towards this end, the Hellenic Centre of Marine Research is aiming at creating a web-accessible virtual collection of marine invertebrates to facilitate and accelerate taxonomic studies. However, scanning marine invertebrates presents the user with several challenges: a) specimens usually contain both low-density soft tissues and very dense calcified structures (shells, skeletons, mouth parts) which need to be visualised; b) soft-bodied organisms scanned in their preservation medium (ethanol, formalin) result in unsatisfactory low-contrast images and thus need to be stained with electron-dense substances; c) contrast-enhancing methods have to be non-destructive concerning both morphology and DNA to be available for further studies; d) organisms often are very small (<1mm) and need to be stabilised properly during scanning without being squeezed broken or otherwise affected in their anatomy and morphology. Based on techniques for scanning soft-bodied organisms developed by other workers (e.g. Metscher 2009a, 2009b; Tercedor & Sánchez-Tocino 2011), a series of experiments has been conducted to test different methods for sample handling, staining techniques and scanning parameters in order to develop protocols and recommendations for different types of marine organisms. Methods Three different marine invertebrate taxa with distinct size ranges have been used for testing: a) a marine polychaete worm (Eunice sp., 0.3 x 2 mm; fixed and stored in 96% ethanol); b) a bivalve (1 x 3 mm; fixed and stored in 96% ethanol); c) a sponge (Agelas oroides, piece of 4 mm x 10 mm; fixed and stored in formalin, dehydrated to 96% ethanol 3–5 days before scanning). All species contain structures of both high and low density (Fig. 1). The polychaete’s soft body tissues are enclosed within a collagenous cuticle, bearing rigid chitinous chaetae (Figs. 1a–d) . The outer shell of the bivalve, constructed from calcium carbonate, encloses delicate tissues such as gills and muscles (Fig. 1e). Finally, the sponge is characterised by a soft, gelatinous body mass (mesohyl), supported by a skeletal network of anastomosing fibers made of spongin (a protein similar to collagen), as well as hard, siliceous spicules (Fig. 1e).

Figure 1: False-colour volume renderings. Colours indicate relative densities: blue: low density, green: medium density, red: high density. a) Eunice sp., iodine staining in Ethanol, whole animal, ventral view; b) Eunice sp., PTA staining in Ethanol, whole animal, ventral view; c) Eunice sp., cut-away view at midbody, iodine staining in Ethanol; d) Eunice sp., cut-away view at midbody, PTA staining in Ethanol; e) Bivalve, PTA staining in Ethanol, cut-away view; f) Agelas oroides, no staining, HMDS-dried, cut-away view.

Of each organism, three similar-sized individuals or segments were chosen and different stains were applied: a) no staining (control); b) 1% iodine in 100% ethanol (I2E); c) 0.3% phosphotungstic acid (PTA) in 70% ethanol (for protocols of b and c see Metscher 2009a, 2009b). Specimens were stained overnight to 5 days at room temperature, then washed out

in ethanol. For scanning, specimens were placed in 96% ethanol in Eppendorf tubes or polypropylene pipette tips (its conical form allows the specimen to sink into the tube until gently touching the walls of the tip and thus stabilizing itself without being pressed). After scanning in the liquid medium, each organism was dried with Hexamethyldisilazane (HMDS, see Braet et al. 1997) for 2 x 2 hrs in two subsequent baths, dried and scanned again, this time stabilized in styrofoam and scanned in air. To test whether the different chemical substances negatively affect the genetic material, DNA was extracted from three specimens of another polychaete worm (Hermodice carunculata) for which the procedure had previously been standardised. The same staining procedures as for the organisms that were X-rayed were applied to these three specimens: a) drying with HMDS; b) iodine staining; c) PTA staining. For the extraction procedure the DNeasy Blood & Tissue Kit (Qiagen, USA) was used and the protocol provided by the manufacturer was followed. Fragments of 16S rRNA and 18S genes were amplified with Polymerase Chain Reaction (PCR) to verify the quality of the extracted DNA. All results were verified through visualisation on electrophoresis gel. Scans were performed with a SkyScan 1172. Before the actual experiment, a series of test scans was conducted to determine the best scanning and reconstruction parameters: A PTA-stained bivalve was transferred to 96% ethanol in which it was scanned at different settings: a) 40 kV without filter; b) 60 kV without filter; c) 60 kV with 0.5 mm aluminium filter. All settings were tested with both medium (2000 px) and high (4000 px) camera resolution. The reconstructed cross-sections were compared and based on the results the most appropriate parameters were chosen (60kV without filter, camera resolution 4000 px) and subsequently applied during the experiment for all organisms. Reconstruction was always done with the projections from 360° and undersampling was applied to reduce noise and file size. Before reconstruction, projection images were aligned with a reference scan to correct thermal shifts. In cases where strong ring artifacts were present, the thermal correction shift was manually changed to deviate by 1-2 pixels from the calculated values since this was found to significantly reduce ring artifacts. In case of strong density differences in the scanned samples, the “tail” (dense values) of the density histogram was “cut off” for reconstruction to allow a better visualization of soft tissues. SkyScan software (NRecon, CTVox, DataViewer) was used for data processing and viewing. For presentation purposes, images were minimally edited in Adobe Photoshop v.12.0.4 using the standard auto levels correction to enhance contrast and remove noise. Results and Discussion As expected, the unstained control samples in ethanol had very low contrast and the resulting images are not useful for further study (Figs. 2a, 3a, 4a). Drying the specimens with HMDS generally improved the contrast and resulting images showed more detail. However, in organisms that have by nature high contrast differences of tissues (e.g. bivalve shell and soft tissue), HMDS alone does not suffice to adequately visualize the soft tissues. In the current study, of the unstained and dried organisms only the sponge gave excellent results (Fig. 4b). Both the polychaete worm and the bivalve showed lack of detail in the soft tissues (Figs. 2b, 3b).

Figure 2: Micro-CT cross-sections of the polychaete worm Eunice sp. a) unstained, scanned in 96% ethanol; b) unstained, dried with HMDS, embedded in styrofoam and scanned in air; c) I2E staining, scanned in 96% ethanol; d) I2E staining, dried with HMDS, embedded in styrofoam and scanned in air; e) PTA staining, scanned in 96% ethanol; f) PTA staining, dried with HMDS, embedded in styrofoam and scanned in air. Scale bar 150µm.

Figure 3: Micro-CT cross-sections of a bivalve. a) unstained, scanned in 96% ethanol; b) unstained, dried with HMDS, embedded in styrofoam and scanned in air; c) I2E staining, scanned in 96% ethanol; d) I2E staining, dried with HMDS, embedded in styrofoam and scanned in air; e) PTA staining, scanned in 96% ethanol; f) PTA staining, dried with HMDS, embedded in styrofoam and scanned in air. Scale bar 500 µm.

Figure 4: Micro-CT cross-sections of the sponge Agelas oroides. a) unstained, scanned in 96% ethanol; b) unstained, dried with HMDS, embedded in styrofoam and scanned in air; c) I2E staining, scanned in 96% ethanol; d) I2E staining, dried with HMDS, embedded in styrofoam and scanned in air; e) PTA staining, scanned in 96% ethanol; f) PTA staining, dried with HMDS, embedded in styrofoam and scanned in air. Scale bar 1mm.

Both stains (I2E and PTA) in ethanol gave excellent results for the polychaete worm, displaying fine details of soft tissues (Figs. 2c, e). In the bivalve, iodine did not stain the tissues successfully, even after prolonging the staining to over a week, however, the PTA worked very well (Figs. 3c, e). In the sponge, the two stains did not significantly improve the results compared to the unstained and dried sample. The contrast was slightly stronger in the I2E-stained images, but additional details were not discernible (Figs. 4b, 4d). Concerning the contrast, the PTA stain gave similar results to iodine but did not penetrate further than 0.5–1 mm into the tissue, giving the specimen a hollow appearance (Fig. 4e). Additional attempts to stain the sponge with a 2% PTA solution in 96% ethanol both at room temperature and for up to 18 hrs at 60°C (Locke & Krishnan 1971) and with 0.3% PTA solution in 70% ethanol for up to 18 hrs at 60°C did not facilitate the penetration of the stain into the tissue. The combination of a stain with HMDS drying did not notably improve the results in any of the organisms. There was no further increase in contrast or additional visualization of details. Overall, in the dried samples the signal-to-noise ratio is better since the noise from the liquid medium is not present. However, slight shrinkage of tissues might be observed when drying samples; this plays a role especially in small organisms and might be negligible in larger organisms or low-resolution scans. The combination of I2E with HMDS increased the contrast even further and resulted in slight “streaking” artifacts – to remove these and obtain a higher quality of the images, re-scanning these samples at higher voltages might be desirable. The used methodologies all enhance the contrast of the scanned images, however, the underlying principles are different. Whereas drying a specimen (either with HMDS or through other techniques, such as critical point drying) increases the contrast because the liquid medium is removed both from the cells and the surrounding area, the two stains bind to tissues and thus make them more electron-dense. The used stains generally bind to all structures but exhibited stronger affinities to certain anatomical components of the scanned organisms (Fig. 1), staining them more intensely. Iodine seems to bind strongly to calcified structures and polysaccharides and stained in the polychaete worm especially epidermic tissues (cuticle – containing polysaccharides), the jaw apparatus (mineralized with aragonite) and certain types of bristles (possibly also calcified) (Figs. 1a, c). In the bivalve, Iodine stained all soft tissues very weakly and without differentiation. Phosphotungstic acid is known to bind to certain proteins (fibrin, collagen) and stained the epidermis of the polychaete even stronger than iodine, especially in the anterior end of the animal (binding to collagen fibres), as well as muscular fibers of the jaw apparatus and of the basis of the bristles (Figs. 1b, d). Contrarily to the iodine stain, the jaw apparatus itself was not stained noticeably, neither were the bristles. In the bivalve, PTA was the most successful stain, strongly binding to all tissues and especially to muscle fibers (Fig. 1e). There was no differentiation in staining performance in the sponge tissues. The different size of scanned specimens has an apparent effect in the results. In the largest scanned specimen, the sponge fragment, structures sized at several microns were clearly visible; however, the resolution was not sufficient to distinguish tinier dense structures, such as the siliceous spicules, requiring scanning of smaller fragments. Contrastingly, most anatomical structures were visible in the Eunice specimen, its dimensions approaching the lower limit of the micro-CT. Furthermore, an improved signal-to-noise ratio with decreasing specimen size was evidenced for scans performed in ethanol, suggesting that scanning in this medium may be preferable for small organisms, especially as the latter are rendered especially susceptible to damage by handling after desiccation. Finally, none of the used substances seem to be detrimental to the DNA quality, since DNA could be successfully extracted both from the specimens dried with HDMS, as well as from the stained specimens (PTA and iodine).

Conclusion All tested methods (PTA and iodine staining, drying with HMDS) are useful techniques to enhance the contrast of soft tissues in marine invertebrates. However, all methods have dissimilar effects on different organisms, making general recommendations difficult. Marine organisms exhibit – even among closely related taxa – a large diversity of tissue types, chemical components and material combinations, making it almost impossible to predict the effect of a certain contrast-enhancing technique. For each new organism, the best method has thus to be experimentally determined. However, a few general statements can be made: Drying samples with HMDS increases the fragility of specimens, especially for small

specimens or animals with thin appendages (such as arthropod legs or antennae). Specimen handling in these cases becomes very difficult and almost always results in damaging the specimens. The technique is therefore only recommended for larger and more compact samples.

HMDS might result in a slight shrinkage of tissues, which is usually negligible in larger organisms, but might considerably influence the results in smaller species.

Combinations of a stain with HMDS do not significantly improve the results; however, in air-scanned samples there are less ring artifacts and the signal-to-noise ratio is better. This approach might therefore be useful if very clear details are needed.

Iodine seems to have a strong affinity to calcified tissues and polysaccharides, whereas PTA binds strongly to certain proteins (collagen, fibrin).

PTA is not suitable for staining large organisms since its penetration depth is limited. Staining at higher temperatures and for longer periods might increase the penetration depth to a certain amount.

None of the tested chemicals seems to degrade the quality of the extracted DNA, making all stains suitable for biodiversity studies for which molecular analyses of the scanned organisms are to be performed after scanning. To what extent the nucleotide sequence might be modified, either through the chemical treatment or through the exposition to x-rays, has to be tested in future studies.

The present study is only a preliminary study in order to establish some guidelines for scanning marine invertebrates. Other methods of contrast enhancement (critical point drying, freeze-drying, other staining methods) remain to be tested in the future in order to optimise the results. Acknowledgements Many thanks go to Jeroen Hostens (SkyScan, Belgium) for a thorough training in using the SkyScan 1172 and an introduction to the possibilities of Hexamethyldisilazane, and to Dr. Brian Metscher (Department of Theoretical Biology, University of Vienna, Austria) for training in application of the various staining methods and for valuable discussions. This study was funded by the EU FP7 project MARBIGEN (FP7-REGPOT-2010-1). References

1. Braet F, De Zanger R, Wisse E, Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells, Journal of Microscopy, 186, 84-87, 1997.

2. Locke M, Krishnan N, Hot alcoholic phosphotungstic acid and uranyl acetate as routine stains for thick and thin sections, The Journal of Cell Biology, 50, 550–557, 1971.

3. Metscher BD, MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions, Developmental dynamics : an official publication of the American Association of Anatomists, 238, 632–640, 2009a.

4. Metscher BD, MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues, BMC physiology, 9, 11, 2009b.

5. Tercedor JA, Sánchez-Tocino L, The use of the SkyScan 1172 high-resolution micro-CT to elucidate if the spicules of the “sea slugs” (Mollusca: Nudibranchia, Opisthobranchia) have a structural or a defensive function, Proceedings of the 2001 SkyScan MicroCT User meeting, 113-121, 2011.