## 4 Micro-CT Bone Structure Analysis in OP-Related Studies
### 3D Morphometric Analyses
#### 4.1.1 Influence of Acquisition Resolution and Voxel Size  
Clinical CT usually works at resolutions around several hundreds of micrometres, in order to have acceptable scanning durations and radiation exposure for the patient, but this hinders the assessment of the trabecular feature modifications deriving from OP [9]. For this reason, micro- and nano-CT have been adopted in bone characterisation with the aim to obtain a more reliable assessment of trabecular architectural parameters thanks to their higher resolutions. According to the literature, in fact, the image resolution and consequently the voxel size play a key role in the correct quantification of trabecular feature values. [PERSON] and collaborators compared the results of trabecular morphometric analyses performed on cadaveric radii through micro-CT using a voxel size of 18 \(\upmu\)m, with those obtained through HR-pQCT working at 41, 82 and 123 \(\upmu\)m [67]. With bigger voxel sizes, a reduction in the trabecular thickness and separation as well as in the bone volume fraction was observed due to the partial volume effect. According to this effect, when the trabecular edge is encompassed in avoxel, the grey intensity of the resulting voxel is generated by averaging the adsorption contribution of the trabecula and that of the pore. Consequently, during the following segmentation phase, the voxel can be attributed to the background, leading to a thinning of the trabecula and even to the loss of the thinner structures. Therefore, it is evident that the partial volume effect can strongly hinder the accurate evaluation of the differences in the morphometric parameters between healthy and osteoporotic bone, particularly considering the variation in the trabecular thickness and in the trabecular interconnectivity.  
However, the partial volume effect and the consequent drawbacks can be reduced using smaller voxel sizes. As visible in Figure 12, [PERSON] and collaborators observed that the use of a 6 \(\upmu\)m voxel size led to a more reliable measure of connectivity density in murine vertebral bones compared to greater voxel sizes (10, 15, 20, 30 \(\upmu\)m), thanks to the preservation of the thinner trabeculae responsible for the interconnection of the bone structure [68]. In general, the partial volume effect can be considered negligible when the voxel size is inferior to one-tenth of the mean dimension of the structure of interest [69]. Therefore, in the case of human trabecular bone, which has a mean thickness of 100-200 \(\upmu\)m depending to the anatomical site and the presence of OP [26,70], the maximum voxel size allowing a correct evaluation of trabecular features is in the range of 10-20 \(\upmu\)m.  
To increase the resolution of the reconstructed images in the case of low-resolution scans, [PERSON] and collaborators proposed the \"discrete algebraic reconstruction technique\". This alternative iterative reconstruction technique allows for enhancing the reconstructed image resolution by imposing compositional assumptions on the material constituting the sample scanned. In particular, the grey level of bone and other materials present in the bone biopsy (e.g., air and bone marrow) are defined _a priori_. However, due to the strong assumptions considered and the high computational costs, the best  
Figure 12: Effect of scanning voxel size on the trabecular interconnectivity of the same vertebral body. Image obtained from the work of [PERSON] et al. [68] and licensed under a Creative Commons Attribution 4.0 International Licence.  
approach to perform reliable morphometric analyses is to scan the sample at the highest resolution available [71].