High‐resolution micro‐tomographic X‐ray imaging of stained mouse brain
Authors: Martins de Souza e Silva Juliana , Pfeiffer Franz 
Published: December 2016

High‐resolution structural visualization of animal organs at the micro‐ and submicrometer scale is essential for functional, comparative and developmental studies. Histology is the gold standard to examine microstructures in stained thin sections of animal organs using optical and electron microscopies. However, due to limitations intrinsic to these imaging techniques, the sample preparation protocol is usually very time‐consuming, the images obtained are two‐dimensional and they only exhibit structures of small sections of the organs imaged.
Recent works have shown that the chemical staining of animal organs is a good strategy to increase the low inherent contrast of soft tissues in X‐ray absorption micro‐computed tomography (microCT).[1,2] However, it is repeatedly described in several works in this area that the slow and inhomogeneous diffusion of the staining agent into the samples can limit the use of stains in microCT. To overcome this issue, long staining times consisting of one or more weeks are used,[2,3] as well as highly concentrated staining solutions, that can cause sample shrinkage.
We have recently made some significant improvements to a well known staining protocol applied to soft‐tissue samples and non‐distorted three‐dimensional (3D) structural information of entire animal organs were easily accessed.[4] The images obtained with a bench top microCT scanner reveal rich morphological information of the whole organs analysed. Moreover, with our protocol, the chemical agents that increase the contrast were able to reach small structures and we have demonstrated that organs structures and some cells types can be discriminated in the microCT images obtained. As an example, the digital mid‐sagittal section of a mouse brain stained with our protocol (Fig. 1) reveals brain regions including the cerebellum (Cb); the thalamus (Th); the hippocampus (Hp); the striatum (Str), the corpus callosum (cc) and the neocortex (NCx). In a higher‐resolution measurement of the cerebellum (Fig. 2), the stratum moleculare (a); and the stratum granulosum (b) are discriminated. Moreover, we have detected microstructures of tens of mm in diameter, which due to their location, size, shape and density are presumably the Purkinje cells (Fig. 2, arrows). In a higher‐resolution measurement of the brain (Fig. 3), the thalamus; the hypothalamus; the striatum, the corpus callosum and the neocortex are seen in an even more detailed fashion, and the cells organization in the corpus callosum is much more evident.
By selecting the appropriate agent or even by combining different staining agents, we are able to improve the contrast in selected areas and a few distinct structures could then be discriminated in one entire mouse brain using microCT. These images demonstrate that staining allied to microCT is a promising strategy to increase the contrast of features of the same order of magnitude of axons in the corpus callosum and and Purkinje cells in the cerebellum, thus allowing the reconstruction in 3D of the structural organization of some specific cells of interest and therefore being a complementary technique to histology in functional, comparative and developmental studies.
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