## 4 Discussion  
Enhancements to \(\mu\)CT scanning parameters and deblurring post-acquisition both qualitatively and quantitatively improved visualization of damage deposition. Enhanced parameter voxel spacing of 4.9 \(\upmu\)m represents higher resolution imaging than historically used for 3D microdamage analysis with \(BaSO_{4}\) labeling [10,25]. Visualization of microdamage with this \(\upmu\)CT protocol is dependent on the efficacy of the \(BaSO_{4}\) staining (ability to precipitate into all microdamage sites). This is an important consideration, as contrary to BSE imaging, the enhanced \(\upmu\)CT parameters are not able to distinguish microdamage that is not labeled by the \(BaSO_{4}\) contrast. \(BaSO_{4}\) labels all damage types (microfractures, diffuse damage, linear microcracks), limiting the specificity of damage characterization by intensity alone; the enhanced techniques presented in this investigation potentially allow the examination of morphology to determine the damage type. Effective assessment of damage distribution in 3D allows for the study of how damage interacts with remodeling, prediction of failure, and treatment responses. This investigation studied trabeculae with thicknesses of 75-95 \(\upmu\)m and spacing of 90-126 \(\upmu\)m [25], which may explain the lack of difference between the enhanced techniques and baseline \(\upmu\)CT imaging at larger inflation radii (Figure 5a). Our tuning of \(\upmu\)CT acquisition parameters was limited by pre-set voltage and current combinations available on the scanner, preventing any determination if higher voltage could further increase the contrast between bone and \(BaSO_{4}\). Spatial correlation accuracy was impacted by registration errors between BSE and \(\upmu\)CT image pairs, limiting the strength of findings at the smaller inflation  
Figure 5: Probability of spatial correlation of \(BaSO_{4}\)-labeled pixels in paired \(\upmu\)CT (baseline, enhanced, and deblurred) and BSE images at six inflation radii. (**a**) the probability that \(BaSO_{4}\)-labeled pixels in a \(\upmu\)CT image (baseline, enhanced, or deblurred) fall within some radius of \(BaSO_{4}\)-labeled pixels in a BSE image; (**b**) the probability that \(BaSO_{4}\)-labeled pixels in a BSE image fall within some radius of \(BaSO_{4}\)-labeled pixels in a \(\mu\)CT image (baseline, enhanced, or deblurred). The data presented are from eleven sagittal BSE slides from rat L1 vertebrae (healthy = 5, osteolytic = 3, mixed = 3). * \(p\) < 0.05, **_p_ < 0.01, *** \(p\) < 0.001, *** \(p\) < 0.0001, Tukey pair-wise comparison. radii. Frame averaging and small voxel spacing in the enhanced scan parameters resulted in long scan times and large file sizes for whole rat vertebrae (~11 h, ~8 GB for ~1500 slices), which is not suitable for samples without fixation. The deblurring algorithm requires extensive computational resources that may be unavailable for total volume deblurring using the enhanced parameters. Eight times frame averaging is a major contributor to increased scan acquisition time in this protocol. However, the reduced noise in these images facilitates the segmentation of small \(BaSO_{4}\)-labeled regions. Increasing integration time may have further reduced noise, however, at the expense of greater scan acquisition times when combined with eight times averaging. Thus, this study did not investigate the magnitude of noise reduction when increasing integration time and frame averaging concurrently. While the acquisition time is long, the process for obtaining BSE images is extensive, requiring weeks for sample dehydration and hard embedding (5 weeks total for the BSE slides used in this study). The enhanced \(\upmu\)CT protocol allows results to be obtained by the next day, is non-destructive to the sample, and provides 3D spatial location and distribution of damage. Scan acquisition time and file size prevents the enhanced \(\upmu\)CT parameters outlined in this protocol from being used for the microdamage analysis of whole bones from large mammals or humans.