###### Abstract  
Contrast-enhanced \(\mu\)CT imaging has been used to provide non-destructive 3D images of microdamage, but at a lower quality than found in histology and 2D backscatter electron (BSE) imaging. This study aimed to quantify potential improvements in microdamage characterization by enhancing \(\mu\)CT scanning parameters. Eleven slides from 9 rat vertebrae (healthy = 3, osteolytic metastases = 3, mixed metastases = 3) previously stained for microdamage with \(BaSO_{4}\) and analyzed with BSE imaging (2 um voxel spacing) were used in this study. \(\mu\)CT imaging conducted under varying protocols (\(\kappa\)-ray voltage, tube current, frame averaging demonstrated enhanced scan parameters at 90 kVp, 44 \(\mu\)A, 0.5 mm aluminum filter, 8 times frame averaging, and 4.9 \(\mu\)m voxel spacing. Post-processing with Richardson-Lucy deconvolution further deblurred the \(\mu\)CT images. Labeled microdamage in the baseline, enhanced and deblurred \(\mu\)CT images were segmented and spatially quantified vs. BSE-labeled microdamage using a probability-based correlation metric at six inflation radii. Enhanced \(\mu\)CT scan parameters improved damage visualization and increased spatial correlation probability with BSE images. Deblurring improved the sharpness of stain boundaries but did not significantly improve spatial correlation probabilities in comparison to the enhanced scans. This enhanced \(\mu\)CT protocol facilitates 3D visualization of microdamage, an indicator of bone quality important to bone damage mechanics. b 2023 13 3625  
Distinct from optical microscopes, scanning electron microscopes (SEMs) use electrons (rather than light) to produce images of specimens. BSEs are high-energy electrons that are reflected out of the specimen during SEM imaging that highlight the different elements contained in the specimen. Denser elements (those with a high atomic number) deflect incident electrons more strongly than lighter elements, thus appearing brighter in a BSE image acquired with an SEM [13]. This characteristic of BSE images allows for the study of mineralization in bone tissue, as regions high in hydroxyapatite (a mineral composed of calcium, phosphate, and hydroxide) appear bright. Visualizing high atomic number contrast agents is facilitated by BSE imaging as the bright contrast may be easily located and segmented from the images. SEM imaging acquires images under a vacuum. Thus, specimens must be dehydrated, or a cryo-fixation technique must be employed [13; 14]. Bones' inorganic phase and relatively low water content compared to soft biological tissues allow for the dehydration method without the need for the more complicated cryo-fixation. The dehydration process, however, requires weeks of progressive dehydration to limit artifacts (microcracks) induced by drying [13]. As the surface topology of the samples must be flat, bone specimens require embedding in resin and subsequent surface polishing. This can further induce artifacts and requires additional time for resin polymerization [13]. To avoid static charge buildup on the sample surface, non-conductive samples such as bone must be rendered conductive. Strategies to achieve this include impregnation of the sample with heavy metals, applying a thin conductive coating (such as carbon), or use of ionic liquids [13]. Computed tomography is a non-invasive imaging tool providing three-dimensional (3D) images of biological structures. Micro-computed tomography (\(\mu\)CT) allows for high-resolution images on a smaller scale, which has value for pre-clinical studies of biological tissues. \(\mu\)CT leverages the varying x-ray attenuations of biological tissues to generate images [15]. As the sample is rotated inside the \(\mu\)CT scanner (or the detector is rotated around the specimen), the intensity of x-rays transmitted through the tissues at different angles is measured by the detector [15; 16]. The collection of 2D projections is then reconstructed post-acquisition to create the 3D image using a process called back-projection [15].