Which is the best method to prepare invertebrate shells for SEM analysis? : Testing different techniques on recent and fossil brachiopods

The analysis of invertebrate shell microstructures by Scanning Electron Microscope (SEM) is a method easily available to most palaeontologists and geochemists; sample preparation does not require specialised techniques or instruments but it requires attention to detail in order to capture microstructural information invaluable to researchers in different fields of palaeontology. Herein, several tests were performed on recent and fossil brachiopod shells, excellent archives of ecological/palaeoecological and climatological/palaeoclimatological information, experimenting new and old methodologies in order to identify a general protocol to better highlight and analyse the shell biocomposite. The results of these tests are documented by SEM images. For recent brachiopod shells, we analysed seven specimens belonging to the species Liothyrella uva and Liothyrella neozelanica, respectively collected from Antarctica and New Zealand. We carried out several tests to check the response of the shell fabric to the resin used to embed the valves before cutting (Procedures 1b and 2c) and to different times of exposure to hydrochloric acid (Procedure 10; as the presence of the organic matrix in recent shells represents the main obstacle to obtaining high quality images at the SEM, to remove it we used bleach (Procedure 2a) and hydrogen peroxide (Procedure 2b) with different concentrations and times of exposure. We then analysed two fossil specimens of Terebratula scillae collected from a lower Pleistocene succession in Northern Italy; here, we mainly checked the degree of penetration of the resin into the shell substance, embedding (Procedure 3a) vs not embedding (Procedure 3b) the specimens before cutting them. We show that Procedures 2a and 2b (embedding in the resin, bleach and hydrogen peroxide at the highest time of exposure, hydrochloric acid for 3 seconds) are the best methods to use when preparing recent brachiopods, whereas fossil shells should be subject to Procedure 3b (embedding in the resin, hydrochloric acid for 15 seconds).