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A multi-sample thin section preparation method: interest for the study of fossil eggshells

Une méthode de préparation de lames minces multi-échantillons : intérêt pour l'étude des coquilles d'œufs fossiles
Lionel Marié, Thibaut Guiragossian et François Fournier
Traduction(s) :
Une méthode de préparation de lames minces multi-échantillons : intérêt pour l'étude des coquilles d'œufs fossiles [fr]

Résumés

La découverte de coquilles d'œufs dans des contextes paléontologiques et archéologiques est fréquente. Les coquilles d'œufs de dinosaures du Mésozoïque sont retrouvées en très grand nombre et font l'objet d'études diverses, rejoignant parfois même le domaine de l'archéologie. De même que pour les dinosaures non-aviens, les œufs d'autruche présentent un intérêt taxonomique pour l'identification de l'espèce qui les a pondus, mais aussi archéologique pour l'utilisation qu'en ont fait les humains au fil du temps. Ce type d'artefacts nécessite parfois la préparation de lames minces pour comprendre leur affinité taxonomique ou d'éventuelles modifications diagénétiques. La production de lames minces de coquilles d'œufs fossiles est un processus complexe impliquant des matériaux coûteux, des consommables et des compétences précises. Ainsi, la multiplication des échantillons par lame présente l'avantage de limiter les consommables utilisés, de réduire le temps de préparation et de faciliter l'étude des coquilles. En effet, ce type de méthode permet la visualisation simultanée de toutes les coquilles d'œufs appartenant à une couvée. Enfin, la présence de plusieurs échantillons de coquilles d'œufs sur une même lame permet un gain de temps significatif lors d’analyses nécessitant l’utilisation d’une chambre à vide (catholuminescence, microscope électronique à balayage).

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Notes de la rédaction

This article was initially submitted to the Journal of Paleontological Techniques (03/02/2024). It was peer-reviewed and accepted by the journal (23/08/2024), but never progressed beyond this editorial stage. Having received no response from the journal, the authors decided to exercise their right of withdrawal (email sent on: 28/04/2025). As a result, this article is now published in Préhistoires méditerranéennes. In accordance with the journal's submission policy, the article underwent a new evaluation process.

Texte intégral

We would like to thank Guy André from the Laboratoire Méditerranéen de Préhistoire Europe Afrique (LAMPEA), and Alain Tonetto from the Technological Analytical and Imaging Research Platform (PRATIM) for their help in setting up this protocol for the preparation of thin section. We also thank Anaïs Coccaro-Turet for the illustrations and English correction corrections, Jean-Philip Brugal for his help and the reviewers for their useful corrections that improved this work. Finally, we would like to thank Mike Mathieu and Christelle, who provided us with the ostrich eggshells used in this study.

Introduction

1Non-avian dinosaur and ostrich eggshells are the subject of numerous studies linking methods used in paleontology and archaeology. The study of eggshells is based on microscopic and macroscopic morphological criteria. The criteria for identifying different oofamilies are primarily based on the shape of the shell units observed in radial sections seen in transparency with transmitted polarized light and parallel and cross nicols. These criteria were described by K.E. Mikailov (Mikailov 1994). This parataxonomy permit a better understanding of the evolution of the geographical and chronological distribution through biostratigraphic and ootaxonomic correlations. Since then, the discovery of eggs containing fossilized embryos has allowed scientists to link certain oofamilies to known taxa.

2The study of eggshells requires a wide range of methods, from surface analysis to the preparation of thin sections. In addition to classical approaches such as optical microscopy, SEM and cathodoluminescence, new methods using tomography have emerged to study the internal structures of the shell in 3D. Each method provides different information, allowing for a better understanding of the biological, geographical, ethological, and climatic phenomena related to the animals that laid eggs.

3Ostrich eggshells from the Pleistocene were shown to have characteristics that allow their identification to the specific level (Mikailov & Zelenkov 2020). These shells are well documented in Pleistocene and Holocene deposits (Segalen & Person 2017, Texier et al. 2013) and the study of their thin-section would allow to refine the information on their taxonomic distribution and their state of preservation. Similarly, other authors have shown that some dinosaur eggshells from the Mesozoïc were worked by humans and used as beads during prehistoric periods (Andrew 1932, Pauc & Buffetaut 1998). The study of fossils eggshells is therefore a field of research using different disciplines and employing various methods and techniques.

4The paleontological preparation of dinosaur eggshell samples must allow both the study and the conservation of the material. Preparing thin sections of eggshells uses the shell individually, thus separating it from some of its context when it comes from a clutch containing multiple eggs. Preparing multiple thin sections makes it easier to preserve and study the relationships that certain shells might have with each other.

5The oldest thin section of dinosaur eggshells from South-Eastern France appeared in the work of Paul Gervais in 1877 (Gervais 1877). Since then, preparation methods have considerably evolved and a significant diversity of embedding products, machinery-tools and supports are now being used by paleontologist preparers all over the world. Several authors have described protocols allowing to produce thin sections of fossilized eggshells (Lamm 2013, May et al. 1994, Quinn 1994) and these methods are well mastered and reproduced in many paleontology laboratories (May et al. 1994). “Preparation and Sectioning of Specimens” by Ellen-Thérèse Lamm (Lamm 2013), is an important work in paleontological preparation. This work gives a very complete vision of the steps to follow in the preparation of thin sections for different fossils. In addition, the listing of the different consumables, machines and protective equipment allows to develop a protocol adapted to the needs of the study.

6Although consumables and machines are modernized, the overall principles of preparation of thin sections analyzed in these publications are similar, with adaptations depending on the type of equipment and methods of analysis (Quinn 1994). Methods for preparing eggshells for macroscopic surface study have been experimented (Val et al. 2013) and many scientific studies typically use eggshells radials thin section in addition to surface condition observations. These studies relate to various fields such as parataxonomy (Fernández & Khosla 2014, Garcia & Vianey-Liaud 2001, Hirsch 1996, Mikhailov 1991, 1997, Mohabey 1996, 1998, Sellés et al. 2014a, Vianey-Liaud et al. 1994), paleobiology (Jackson et al. 2004, Jackson & Varricchio 2003, Sellés et al. 2017, Zelenitsky & Hills 1997) or taphonomy (Dauphin 1990, Fiorelli et al. 2013, Grellet-Tinner et al. 2010, Moreno-Azanza et al. 2016).

7The preparation of eggshells must therefore adapt to a diversity of analyses for specialized fields of research. However, in most of these studies, the thin section preparation protocol is poorly described, and only the final thickness of the prepared samples is indicated (Coria et al. 2010, Deeming 2006, Fernández & Khosla 2014, Jackson et al. 2013, Jackson & Varricchio 2016, Salgado et al. 2009, Sellés et al. 2013, 2014b).

8This paper provides a simple and reproducible preparation method of multiple thin sections for modern and fossil eggshell that can be used in classical microscopic study (i.e., transmitted polarized light and parallel and cross nicols), cathodoluminescence, and fluorescence analysis.

Material and methods

9The material used in this study comes from Megaloolithid dinosaur eggshells collected in the Upper Cretaceous deposits (Campanian and Maastrichtian) of the Arc Basin and ostrich eggshells (Struthio camelus). Eggshells from different fossilization contexts were used and sometimes mounted on the same slide. The eggshells are composed mainly of calcium carbonate (CaCO3) and have a thickness between 0.7 mm and 3 mm. The specimens study were preserved in calcareous rocks and in clay sediments.

10Some eggshells were washed beforehand in an ultrasonic tank to test if the presence of microcracks has an impact on the production of thin sections. The samples were washed 15 minutes in a mixture of water and detergent at the Mediterranean Laboratory of Prehistory Europe Africa (LAMPEA). The conclusion was that potential microcracks caused by the ultrasonic tank all not have a negative impact on the success of preparation of the thin sections.

11The creation of a protocol and its implementation were carried out at the European Center for Research and Education in Environmental Geosciences (CEREGE). All samples were prepared using the same consumables and the same machines. The number of samples placed on each slide varied between 2 and 10.

12The preparation of consumables, as well as the machines use, was carried out in adequation with safety instructions and proper equipment (extractor hoods, gloves, masks and protective glasses).

13This protocol must serve as a basis and be adapted by each paleontologist preparer according to the different types of samples and consumables used.

Protocol

14The first step in the production of thin sections is the choice of the samples. The order of each sample on the slide can be important if they share common points. This choice belongs to both the paleontologist preparer and the person who will study the samples, depending on their nature and the different possibilities of placing them on the slide.

15The samples are impregnated a first time individually in resin Araldite 2020 to maintain structural consistency during the first cutting steps (fig. 1.a). The samples are dipped in the resin using forceps, then placed under a vacuum to remove air bubbles. This step has to be done on fragile or badly preserved eggshells but is not necessary for eggshells in good condition. After drying for 24 h, the eggshells are cut and polished on their transverse radial section in the direction of their natural curvature (fig. 1.a). The cutting surface of the sample must be coherent with the highlighting of the characteristics of the eggshell sought for the study. This step allows to obtain a flat section to place all the samples vertically on a cover slip 150 m thick. The order of the samples and their references are written on an adhesive label placed next to each cover slip.

16This cover slip allows the samples to be held vertically during the second impregnation phase and will not be kept during the final preparation stages. The samples are laid out and fixed with strong glue (fig. 1.b). For the tangential sections, the eggshell is glued with the external face in contact with the cover slip (the protocol does not change for the following steps).

17After drying for 2 h, the samples are set, and the cover slip is placed in silicone mold with the slide facing down and the samples facing up. The sample references are written on an adhesive label stuck to the mold. Araldite 2020 impregnating epoxy resin is then poured into the mold until all the eggshells are completely covered (fig. 1.c). The mold is placed under a vacuum in an impregnation stand to bring out any air bubbles and at a negative pressure of -0.9 bar (fig. 1.d).

18Once the air bubbles have been expelled, the mold is placed in an oven to dry for 24 hours.

19Once the resin is dry, a mark is made on the resin block to indicate the order of distribution of the samples. Then, the side of the resin block that received the cover slip is polished on a diamond disc (fig. 1.e). The polished resin block is then dehydrated and then polished again on a cast iron steel plate with a mixture of silicon carbide (8 µm grain) and water. The resin block is then cleaned with water and placed on a hot plate set at 40° for two hours.

20The resin block is then aligned to the slide and the references are annotated on it, according to the distribution of the samples in the resin block.

21Bonding Hillquist epoxy resin is used to bond the resin block to the slide with the samples facing their respective references (fig. 1.f). The resin block is placed over the slide and held down by a bonding press (fig. 2.a). Drying is carried out cold because the epoxy resin shrinkage phenomenon is amplified by heat. Given the large resin surface in contact with the slide, the glass is not strong enough and cracks when the resin shrinks rapidly. The resin block is left to dry cold over a period of at least 48 hours. In addition, a constant temperature and weight allow an even distribution of the bonding resin between the block and the slide.

1.Steps for preparing multiple thin sections (part 1)

1.Steps for preparing multiple thin sections (part 1)

a. Preparation of the section of the eggshells, b. gluing of the eggshells to a coverslip, c. impregnation of the eggshells, d. removal of air bubbles under a vacuum, e. polishing of the underside and removal of the coverslip, f. gluing of the resin block of the slide.

Credit: Anaïs Coccaro-Turet

2. Steps for preparing multiple thin sections (part 2)

2. Steps for preparing multiple thin sections (part 2)

a. Cold drying under a press, b. cutting of the resin block with a leveling saw, c. thinning of the sample with a precision grinder, d. polishing of the sample, e. thickness control with a micrometer.

Credit: Anaïs Coccaro-Turet

22Once the resin block is set on the slide, the cut is made with a circular blade (fig. 2.b). The sample is then brought to a thickness of approximately 50 μm with a precision grinding machine (fig. 2.c). Once this step has been completed, the thickness of the sample is checked with a micrometer.

23The slide is placed on a sample holder with diamond studs set to 30 μm thick. The sample support is placed on a polishing machine with a steel cast iron plate (fig. 2.d). The polishing is carried out by adding a first mixture with a 6 μm grain, then a second with a 3 μm grain. At the end of each cycle, the slide is checked with a micrometer and the operation is repeated until a 30 μm thickness is reached (fig. 2.e). Once the desired thickness has been reached, the slide undergoes a final treatment with a 1 μm grain polishing mixture for cathodoluminescence analyses.

Discussion

24The interest of studying many samples to overcome diagenetic modifications problems has been described both in methodological (Quinn 1994) and research (Dauphin 1990) articles.

25The protocol proposed by Betty Quinn in 1994 is very complete and gives all the steps necessary for the preparation of thin sections of eggshells. However, it lacks figures and does not consider the possibility of placing multiple samples on a single thin section. The strength of the multi-sample thin section preparation method compared to a conventional preparation mode is that it increases the number of samples per slide (fig. 3), reduces the quantities of consumables used and improves study time. However, this method requires a few additional steps increasing the preparation time. Thus, the method to be used must be defined in advance, taking into account both the material to be prepared and the type of analysis to be performed. The study of eggs is carried out by parataxonomic, taphonomic and paleobiological approaches. The eggs are often found in clusters that often correspond to nests and mark an intentionality of the nesting animals. A comparative study of the different eggshells present within the same cluster is, therefore, a necessary step in understanding the nesting behavior of dinosaurs. The presence of several specimens per slide allows convenient comparison of taxonomic criteria and diagenetic modifications (fig. 4).

26

3. View of the use of multiple thin sections

a. view of the processing of thin sections by Hirox digital microscopy, b. view of the distribution of dinosaur eggshell samples in radial section (30 mm width), c. view of the distribution of eggshells of Ostrich eggs in tangential (top) and radial (bottom) section (30 mm width).

Crédits : Thibaut Guiragossian

27To achieve this result, several important modifications have been made. This differences between classical thin preparation methods and multi-sample preparation are in the sample placement before embedding and cold polymerization of the resin when bonding the resin block to the slide, were carried out. The main difficulty was the slides cracking after sticking the resin block on them. Cracking of the slides could appear spontaneously during drying and retractation of the resin, or during the cutting step (fig. 2.b).

28The polishing stage can also be a time when part of the sample is lost if it has not been well prepared beforehand. Indeed, if the resin block has a convex surface and is not perfectly flat before being glued to the slide, the outer edge of the samples disappears during the final polishing steps. Several successive polishings are therefore carried out after unmolding the resin blocks before sticking them on the slides.

29The study of eggshells in cathodoluminescence or scanning electron microscope involves the placement of thin sections under a vacuum. This preparatory phase involves an incompressible time that is renewed with each change of sample. Thus, the presence of several samples on the same slide saves considerable amount of time.

30

4. Example of the different views possible on a thin section of an ostrich eggshell with multiple samples

a. morphology of the pores in tangential section, b. morphology of the crystalline units in tangentiall section, c. morphology of the eggshell and of the pores in radial section, d. morphology of the mammillary layer in radial section.

Crédits : Thibaut Guiragossian

31This method of preparing multiple thin sections has some experimental limitations that still need to be explored. The thinnest shells included in the multiple thin sections were 0.8 mm thick. This method has not been tested on the eggs of crocodiles, turtles, or modern birds other than ostriches. Similarly, this technique has not been tested on recently discovered soft-shelled eggs of non-avian dinosaurs (Norell et al. 2020).

Conclusion

32Studies on dinosaur eggshells are numerous and employ a wide range of protocols depending on the subject. The bibliographic references used in this work were selected to illustrate the value of preparing multiple thin-sections in comparison with the traditional method. The production of thin sections requires the implementation of skills in paleontological preparation, specialized machines, as well as varied and expensive consumables. Thus, the grouping of several samples with contextual consistency on the same slide is an advantage in terms of costs, preparation time and analysis time. In addition, the presence of several samples on the same thin section has the advantage of being able to overcome the problems of diagenetic modification by multiplying the observations.

33Conservation conditions of certain fossils, such as holotypes, are also improved with this type of protocol. It allows to bring together several eggshells belonging to a new oospecies, for example, on the same thin section. This protocol can be adapted to any type of eggshell whatever its preservation and allows to obtain radial and tangential views on a single slide. The machines and consumables, widespread in the field of archaeological and paleontological preparation make this method of thin sections preparation reproducible and advantageous.

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Table des illustrations

Titre 1.Steps for preparing multiple thin sections (part 1)
Légende a. Preparation of the section of the eggshells, b. gluing of the eggshells to a coverslip, c. impregnation of the eggshells, d. removal of air bubbles under a vacuum, e. polishing of the underside and removal of the coverslip, f. gluing of the resin block of the slide.
URL http://journals.openedition.org/pm/docannexe/image/5265/img-1.jpg
Fichier image/jpeg, 385k
Titre 2. Steps for preparing multiple thin sections (part 2)
Légende a. Cold drying under a press, b. cutting of the resin block with a leveling saw, c. thinning of the sample with a precision grinder, d. polishing of the sample, e. thickness control with a micrometer.
URL http://journals.openedition.org/pm/docannexe/image/5265/img-2.jpg
Fichier image/jpeg, 231k
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Lionel Marié, Thibaut Guiragossian et François Fournier, « A multi-sample thin section preparation method: interest for the study of fossil eggshells »Préhistoires Méditerranéennes [En ligne], 13 | 2025, mis en ligne le 29 janvier 2026, consulté le 13 avril 2026. URL : http://journals.openedition.org/pm/5265 ; DOI : https://doi.org/10.4000/15lcw

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Auteurs

Lionel Marié

Aix Marseille Univ, CNRS, IRD, Coll France - CEREGE, Technopôle de l'Arbois-Méditerranée, BP80, 13545 Aix-en-Provence, France - lionel.marie@univ-amu.fr

Thibaut Guiragossian

Aix Marseille Univ, CNRS, Minist Culture, Inrap – LAMPEA, MMSH, 5 rue du Château de l’Horloge, 13094 Aix-en-Provence, France, thibaut.guiragossian@univ-amu.fr

François Fournier

Aix Marseille Univ, CNRS, IRD, Coll France - CEREGE, Technopôle de l'Arbois-Méditerranée, BP80, 13545 Aix-en-Provence, France - fournier@cerege.fr

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