Traditional light microscopy used to study plant tissues use destructive methods and protocols that do not allow for 3D visualization and are labor-intensive. We investigated the use of micro-CT in exploring the anatomy of oat plants as a higher throughput tool to visualize stem structures. However, low-density plant tissues with limited differences in micro-CT grey values create a challenge to differentiating the cellular structures. Therefore, we developed a method to address these challenges. Initial scans of fresh tissues indicated the need to fix, dry, and improve the contrast of the oat tissues in order to minimize internal movements for image quality and to widen the grey scale to better visualize the cellular structures. We tested three types of fixatives and found that formalin–acetic acid–alcohol (FAA) allowed for the best absorption of the chemicals into the tissue. In place of the lengthy critical point drying (CPD), we found liquid evaporation drying with hexamethyldisilazane (HMDS) resulted in a clearer image. Contrast agent allows low-density tissues like oat stems to widen the grey scale for better image analysis. We evaluated the length of time needed for absorption of two contrast agents, osmium tetroxide and phosphotungstate. The stems were scanned and reconstructed into 3D images. Grey value measurements were made across sections of the images. Samples soaked in contrast agents were compared against fresh samples. Both phosphotungstate and osmium tetroxide were absorbed into the tissues, but phosphotungstate was distributed more evenly through the nodes. The combination of fixing, drying and using a contrast agent greatly improved our ability to use micro-CT as a viable option to study the internal structures and morphology of oats. However, denser plant tissues may require different sample preparation protocols due to the difference in tissue chemical makeup.