When the Utility of Micro-Computed Tomography Collides with Insect Sample Preparation: An Entomologist User Guide to Solve Post-Processing Issues and Achieve Optimal 3D Models

Abstract

Many techniques are used today to study insect morphology, including light and electron microscopy. Most of them require to specifically prepare the sample, precluding its use for further investigation. In contrast, micro-CT allows a sample to be studied in a non-destructive and rapid process, even without specific treatments that might hinder the use of rare and hard-to-find species in nature. We used synchrotron radiation (SR) micro-CT and conventional micro-CT to prepare 3D reconstructions of Diptera, Coleoptera, and Hymenoptera species that had been processed with 4 common preparation procedures: critical-point drying, sputter-coating, resin embedding, and air-drying. Our results showed that it is possible to further utilize insect samples prepared with the aforementioned preparation techniques for the creation of 3D models. Specimens dried at the critical point showed the best results, allowing us to faithfully reconstruct both their external surface and their internal structures, while sputter-coated insects were the most troublesome for the 3D reconstruction procedure. Air-dried specimens were suitable for external morphological analyses, while anatomical investigation of soft internal organs was not possible due to their shrinking and collapsing. The sample included in resin allowed us to reconstruct and appreciate the external cuticle and the internal parts. In this work, we demonstrate that insect samples destined to different analyses can be used for new micro-CT studies, further deepening the possibility of state-of-the-art morphological analyses.

A conceptualisation of computed tomography outputs in entomological research by step by step displaying trough the CT-based visualization of a wood-boring larvae

“…However, it is known, that the μCT allows us to create additional, significantly higherspatial resolution (below 10 μm) images. The μCT permits the mapping of micrometer-sized formulas, which points more towards the study of the morphology of insects (Mensa et al, 2022). Generally, due to the size limitation of micro CT and the significantly longer time required for applying their technique, diagnostic CT can remain the tool of choice for imaging larger organisms.…”

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior

“…This approach applied to chemically-cleared Drosophila adult male individuals allowed to just use the AF of the sample to image both surface (e.g., hexagons of the lens facets, bristles on inter-ommatidial, maxillary palp and distal labellum) and internal (e.g., elements of the compound eye, the optic lobe, cardia) structures, to be made then observable with a highly sharp resolution in a 3D virtual reconstruction [165]. The 3D imaging analysis has been thus used as a technique additional to conventional optical or scanning electron microscopy, and its ability to provide variable views of the target organism has stimulated both new investigations to improve sample preparations, or applications to improve the detection of parasites (e.g., Plasmodium in infected A. stephensi) [36,166,167]. Sample clearing with KOH has been thus recommended as the preferred preparation procedure to obtain specimens suitable to be preserved for long time and thus for reexaminations by confocal microscopy, favoring sample exchange among scientists and comparative and taxonomic studies.…”

130 years from discovery to description: micro‐CT scanning applied to construct the integrative taxonomy of a forgotten moth from Southern Africa (Lepidoptera: Geometridae)

“…Micro-CT imaging has also extensively been used in studies of insect anatomy and physiology (Lowe et al, 2013;Martín-Vega et al, 2021), in forensic entomology (Richards et al, 2012) to time pupal metamorphosis, and to study the functional reproductive morphology (Jandausch et al, 2023;Zlatkov et al, 2023). For imaging soft tissues with synchrotron (e.g., Vommaro et al, 2023) or standard micro-CT, the use of fresh specimens and special preparation methods, such as critical point drying, metal coating and resin embedding (Mensa et al, 2022) or iodine staining (Gignac et al, 2016) are preferable. These sample preparation methods are, however, to a varying degree invasive and not routinely applicable to dried and pinned or mummified specimens.…”