## 4 Discussion  
Here, we presented our results using micro-CT and synchrotron micro-CT on 84 samples that belonged to 3 orders of insects: Coleoptera, Diptera and Hymenoptera. The samples had been stored and treated in different ways: air-dried, critical point dried, metallized and resin embedded. This allowed us to evaluate what could be the advantages and disadvantages of reconstructing models in 3D by using samples not specifically stained for micro-CT analyses [25], determining if it was possible to utilize specimens that had previously been used for other purposes (e.g., electron microscopy) with regard to 3D reconstruction. Out of 84 total specimens, procedures for 3D image analysis and visualization could be performed on almost all of them. This is very important, as many samples used were coming from collections where they were the only specimens, preserved for many years without any treatment. Moreover, in the case of species belonging to the genus _Paussus_, the extreme rarity of these insects meant that no other specimens were available and that it was not possible to perform destructive procedures, such as dissections [32]. The latter, in fact, irreparably damage the specimen, which is not always possible in the case of holdtypes or extremely rare species and is difficult to obtain in the field or from museums and other private collections [18; 33; 34]. Thanks to 3D scanning and reconstruction techniques, today it is possible to study in detail many animal species without damaging their morphology [19; 27; 35]. In addition, scans by micro-CT do not appear to damage DNA or hinder further molecular analysis, although additional studies would need to be conducted as museum specimens vary widely in preservation methods and organism types [36]. In our case, the samples that allowed to obtain the best results were those that had been first fixed in absolute alcohol and then prepared by critical point drying. This, in fact, allowed for the almost perfect preservation of internal organs and musculature, also making the 3D volume creation procedure less troublesome. The gray value of these specimens was often well differentiated from the background, and this allowed to easily segment all parts that were of interest, such as the cephalic musculature. In the case of ants from the  
Figure 9: Sputter coated Coleoptera. (**a**) 3D render of _Paussus_ antenna. Notice the surrounding noise, probably due to the artifacts caused by the gold-coating; (**b**) The same antenna, after being manually and semi-automatically cleaned from the external noise. Scalebars: (**a**)—1 mm; (**b**)—1 mm. genus _Messor_, for example, it was sufficient to remove (virtually) the outer cuticle to get an overview of all the body musculature without having to perform other operations. In the Coleoptera, a very important taxonomic character is the aedeagus. With the samples preserved by critical point drying, it was possible to segment and isolate this important organ without having to extract it manually (thus ruining the sample). However, even with air-dried samples, it was possible to extrapolate this type of information. Insects coming from museum collections, prepared by gluing them on cards, presented a few more problems than described so far. In some cases, the amount of glue covering some parts of the animals (such as the mouthparts, the thorax, the legs, and part of the abdomen) proved difficult to remove digitally, without also removing some parts of the animal itself. In fact, the glue had a gray value like that of the external cuticle of the beetle, and this, therefore, forced a meticulous manual removal of the glue. The tag, on the other hand, proved to be easier to remove without having to resort to complex workflows both in Avizo and Geomagic software. Obviously in this type of specimen, unlike those described above, the internal organs and apparatus were collapsed and therefore could only be partially used for further analyses.