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Background  Micro-CT holds promising potential for phenotyping and histological purposes. However, few have clarified the difference in the neuroimaging quality between ex vivo and in vivo micro-CT scanners. In addition, no direct comparison has been made between micro-CT scans and standard microscopy. Furthermore, while the efficacy of various stains for yielding soft-tissue contrast in CT scans have been compared in other studies for embryos, staining protocols for larger samples have yet to be clarified. Lastly, post-acquisition processing for image enhancements have not been addressed.    Methods  Comparisons of postnatal rat brain micro-CT scans obtained through custom-built ex vivo and commercially available in vivo micro-CT scanners were made. Subsequently, the scanned rat brains were then H&E stained for microscopy. Neuroanatomy on micro-CT scanning and 4× microscopy of rat brain were compared.  Diffusion and perfusion staining using iodine or PTA were trialled on adult and neonatal encapsulated rat brains. Different combinations of stain concentration and staining time were trialled.  Post-acquisition denoising with NLM filter was completed using a modern General-Purpose Graphic Processing Unit (GPGPU) and custom code for prompt processing.    Results  Ex vivo micro-CT scans of iodine-stained postnatal rat brains yields 3D images with details comparable to 4× H&E light micrographs. Neural features shown on ex vivo micro-CT scans were significantly more distinctive than those on in vivo micro-CT scans.  Both ex vivo and in vivo micro-CT scans required diffusion staining through small craniotomy. Perfusion staining is ineffective. Iodine staining was more efficient than PTA in terms of time.  Consistently, enhancement made by NLM denoising on in vivo micro-CT images were more pronounced than that on ex vivo micro-CT scans due to their difference in image signal-to-noise indexes.    Conclusions  Micro-CT scanning is a powerful and versatile visualization tool available for qualitative and potential quantitative anatomical analysis. Simple diffusion staining via craniotomy with 1.5% iodine is an effective and minimal structural-invasive method for both in vivo and ex vivo micro-CT scanning for studying the microscopic morphology of neonatal and adult rat brains. Post-acquisition NLM filtering is an effective enhancement technique for in vivo micro-CT brain scans.    Electronic supplementary material  The online version of this article (10.1186/s12880-018-0280-6) contains supplementary material, which is available to authorized users.

High-definition neural visualization of rodent brain using micro-CT scanning and non-local-means processing