Our aim was to evaluate the applicability of micro-CT and diceCT imaging for the detailed visualization of the pediatric hyoid-larynx complex. Traditional forensic imaging modalities face limitations in pediatric cases due to the non-calcified nature of the hyoid-larynx complex, limited resolution, and lack of contrast [3, 4]. In this study, we demonstrate that micro-CT and diceCT overcome these challenges by providing high-resolution and soft tissue contrast.

All pediatric specimens demonstrated a non-fused ossified hyoid bone with either bilateral or unilateral cartilaginous lesser horns (Fig. 3). These findings align with known ossification patterns of the hyoid and laryngeal structures, as hyoid ossification starts within the first two living years and calcification of the larynx does not start before the second decade [2]. In addition, literature reports that the calcification of the larynx starts within the inferior thyroid horns and spreads to the superior thyroid horns [2, 4]. Nevertheless, our results show a sample of a 16-year-old male that already showed one calcified center in the thyroid lamina as the beginning of ossification (Fig. 4). This center was identified as an ossification center as Claassen et al. [20] found that the laryngeal ossification pattern consists of a special mode of endochondral ossification, which includes the mineralization of cartilage and creating islands of cartilage before being covered by the deposition of bone. The hyperdense center could represent the mineralized cartilage, as this cartilage appears denser than bone on X-ray images [20].

Furthermore, hypodense features were identified within the cartilages of the hyoid-larynx complex of this 16-year-old (Fig. 4). These features could present part of the ossification process. Dedivitis et al. [21] studied histological aging changes in the cricoid cartilage and showed that adolescents’ cricoid cartilages only consist of typical hyaline cartilage, while the older cartilages showed central areas of lamellar bone tissue and bone marrow cavities filled with adipose and/or hematopoietic tissue. These cavities could possibly be seen as hypodense irregularities in the larynx cartilages on micro-CT.

In addition, a denser layer was observed around the hypodense cartilages in all pediatric samples (Fig. 4). This layer appeared as a hypodense line surrounding the even more hypodense laryngeal cartilages, and in the younger samples, it also encircled the hypodense cartilaginous greater horns of the hyoid (Fig. 5). This could shed light on the cartilage and bone development, as it was not observed in adults [14]. The dense layer could present the outer layer of hyaline cartilage, the perichondrium. With age, the cartilage is subject to the process of endochondral ossification, resulting in the perichondrium being converted into periosteum [20]. Periosteum is a thin layer of membranous connective tissue, while perichondrium is denser and consists of fibrous tissue [22]. Besides, the perichondrium appears thinner on histological slices of the cartilaginous adult larynx in comparison with the pediatric larynx; therefore, it could show less dense on diceCT as well [19].

In two samples, a uni- or bilateral circular defect in the lamina of the thyroid cartilage was observed on micro-CT (Fig. 4). In the literature, this opening is known as the foramen thyroideum (FT) [23]. The FT can occur in one or both laminae of the thyroid cartilage. We found a FT in two of the hyoid-larynx complexes (40%), in one sample unilateral and in the other one bilateral. Previous research reports an incidence of FT in adults of 24–31%, of which 2–11% were found bilaterally [23,24,25]. The prevalences found in this study showed a slight difference in comparison to current literature, which could be due to our small sample size. According to León et al. [25], the adult FT contains a neurovascular bundle in 73%, a nerve branch in 20%, and in 7% only a vascular branch. In our research, diceCT showed a hyperdense bundle within every FT. Within those hyperdense bundles, two hypodense tubular structures could be visualized in one sample, and the other sample showed one hypodense and one hyperdense tubular structure (Fig. 4). Since blood shows hyperdense on diceCT images, we suspect that the hypodense tubular structures are nerves (branch of the superior laryngeal nerve) and the hyperdense structures are blood-filled vascular branches. As neurovascular bundles are surrounded by connective tissue, the hyperdense staining surrounding the bundles is considered connective tissue. This is in line with the hyperdensely stained tissue we discovered in the cricoid area, paraglottic, and pre-epiglottic spaces (Fig. 5). These spaces contain areolar or loose connective tissue, which is composed of adipose tissue with elastic and collagen fibers [22]. Hence, B-Lugol binds to adipose tissue and/or elastic and collagen fibers as well. Anatomical dissection could provide a definitive answer to the content of the FT.

The observed ossification process, hyperdensely stained perichondrium, and FT provide potential new insights into early hyoid-larynx complex development and anatomy, highlighting the potential of micro-CT and diceCT for developmental anatomy research.

The forensic relevance of these imaging techniques lies in their ability to non-destructively assess the pediatric hyoid-larynx complex in micrometer-level detail, something previously unattainable using standard imaging or autopsy methods. Partial ossifications and transitional zones, which are difficult or impossible to assess macroscopically, become clearly visible. In contrast to traditional histology, diceCT enables three-dimensional mapping of hemorrhages in situ and in anatomical context, without requiring destructive tissue dissection [20]. This allows for more precise, targeted histological sampling and preserves the integrity of delicate pediatric structures. Second, the high resolution of micro-CT and diceCT makes it possible to identify subtle trauma and visualize hematomas with confidence [20]. Given this level of detail, a negative scan may reasonably exclude significant hyoid-larynx trauma. With advancing technology and growing interest, micro-CT and diceCT may in time become valuable tools for routine forensic evaluation. Importantly, current forensic protocols do not recommend separate imaging of the excised pediatric larynx and hyoid, largely due to the insufficient resolution of conventional modalities. Now that micro-CT offers a viable alternative, there is a strong case for systematic research into its forensic value, with the potential to revise and improve existing guidelines. Third, the ability to clearly visualize ossification centers and perichondral layers could support forensic age estimation. Finally, the resulting 3D volume renderings are not only scientifically informative but also provide clear and compelling visualizations for courtroom presentations. Compared to traditional autopsy photos or histological slides, these high-resolution images enhance communication of complex anatomical findings to legal professionals and lay audiences alike.

This study’s small sample size limits the generalizability of the findings. Additionally, incomplete staining in the largest pediatric sample highlights the need for optimized staining protocols. In hindsight, this sample contained a lot of soft tissue and, based on its weight compared to the other samples, required a longer staining time. Based on our experience, we estimate that this particular sample would have required approximately 30 days of staining to achieve complete and uniform contrast enhancement. To strengthen the forensic applicability of diceCT, future research should include trauma cases and incorporate histological validation to confirm the accuracy of imaging findings. Finally, some practical limitations of micro-CT should be acknowledged: the technique requires access to high-end imaging infrastructure, is associated with significant costs, and demands specialized expertise for both image acquisition and interpretation. These factors may currently limit its widespread adoption in routine forensic workflows, particularly outside academic or research-oriented settings.