## 1 Introduction  
Micro-computed tomography (micro-CT) is one of the most recent imaging techniques of interest for diagnosis and research in several disciplines. It uses the same technology as a CT scanner, namely an X-ray source and a two-dimensional (2D) array detector to obtain multiple images from different angles across an area of interest. Subsequently, these images are used to reconstruct a three-dimensional (3D) volume representation, which increases the precision when observing elements and making measurements compared with an X-ray [1]. The main difference is that micro-CT works at a smaller scale, from a few millimetres to a few centimeters, and with higher resolution in the micron range [2]. Micro-CT is a non-invasive technique that allows one to preserve and standardise the sample. Hence, numerous measurements and calculations can be made regarding the analysed structures, and these measurements can be replicated [3, 4]. These qualities have favoured the use of micro-CT imaging for tooth analysis because this approach allows one to reconstruct the tooth in its entirety. Moreover, one can even work with tooth fragments. This imaging tool has been preferred over traditional CT scanners because medical measurements based on conventional CT images can overestimate the values obtained from sectioned teeth [5]. Moreover, micro-CT imaging has proved to be a reliable method for the external observation of internal structures; so, it has been considered a gold standard in comparison with the degree of accuracy of the measurements made with analogous techniques, such as cone beam computed tomography (CBCT) [6; 7; 8; 9]. The precision of this imaging technique in the study of hard tissues allows one to observe all dental structures, given the mineral density differences between dentine and enamel, both of which are mineralised [10; 11]. As a result, micro-CT is applicable to various fields in dental research [11]. Enamel, dentine, and pulp cavity thickness, area, volume, and morphology have been used in palaeontology [12], bioarcheology [13; 14; 15], and clinical odontology [16; 17; 18] for different purposes related to each discipline. For example, in clinical odontology, micro-CT can help clinicians to evaluate osseointegration and thus treatment success [19; 20]. However, the application of micro-CT is limited by the availability of scanners and the limited space available to place the sample inside the equipment (from an isolated tooth to small laboratory animals) [21]. Many authors have pointed out that micro-CT is useful for estimating mineral density in dental tissues, as a means to evaluate treatment effectiveness, tissue development, or the possible effects of an intervention [22; 23; 24; 25]. When viewing micro-CT images in specialised software, they are made up of black, white, and grey pixels due to the differences in the attenuation of the radiation when passing through the different tissues. Hounsfield units (HUs) are used to quantify X-rays that pass through or are absorbed by tissues. Because these units are standardised, the attenuation of X-rays in micro-CT images is correlated to mineral density [26; 27]. Hence, HUs can be compared and analysed. However, possibly due to the recent development of the technique, there is a lack of standardised and validated protocols for measuring dental mineral density with micro-CT. It is important to develop standardised and validated protocols because detailed information and documentation regarding the context of image acquisition ensures reproducibility of the methods, validation of the results, and consistency and facilitates peer review [28; 29]. In this context, it is relevant to mention that evidence from the specialised literature indicates that there are similarities between bovine and human teeth in terms of the hardness, morphology, and chemical composition of enamel and dentine [27; 30; 31; 32; 33]. Although there are some differences, they do not limit the use of bovine teeth as an alternative for human teeth.