Abstract
Knowledge of the three-dimensional (3d) neuronal cytoarchitecture is an important factor in order to understand the connection between tissue structure and function or to visualize pathological changes in neurodegenerative diseases or tumor development. The gold standard in neuropathology is histology, a technique which provides insights into the cellular organization based on sectioning of the sample. Conventional histology, however, misses the complete 3d information as only individual two-dimensional slices through the object are available. In this work, we use propagation-based phase-contrast x-ray tomography to perform 3d virtual histology on cerebellar tissue from mice. This technique enables us to non-invasively visualize the entire 3d density distribution of the examined samples at isotropic (sub-)cellular resolution. One central challenge, however, of the technique is the fact that contrast for important structural features can be easily lost due to small electron density differences, notably between the cells and surrounding tissue. Here, we evaluate the influence of different embedding media, which are intermediate steps in sample preparation for classical histology, on contrast formation and examine the applicability of the different sample preparations both at a synchrotron-based holotomography setup as well as a laboratory source.
Highlights
The gold standard to decipher the cytoarchitecture of neuronal tissue on thecellular scale is histology, based on slicing the sample into thin sections in order to gain insights into the three-dimensional (3d) cellular distribution (Li et al, 2010)
We have demonstrated that drying of tissue according to the evaporation-of-solvent method leads to reconstructions with a high contrast, allowing for the unambiguous identification of single cells within the volume (To€pperwien et al, 2017)
A 1 mm punch was taken from the brain slice in order to reduce absorption of parts of the sample which lie outside the field of view and squeezed into a Kapton tube with 1 mm diameter, which was glued to a sample holder
Summary
The gold standard to decipher the cytoarchitecture of neuronal tissue on the (sub-)cellular scale is histology, based on slicing the sample into thin sections in order to gain insights into the three-dimensional (3d) cellular distribution (Li et al, 2010) It is a time consuming technique which consists of several steps of tissue preparation and processing, including, e.g., fixation, dehydration and embedding of the tissue, followed by the mechanical slicing procedure, staining of features of interest and observation of the individual sections under a light microscope. Computed tomography (CT) offers a high potential for non-destructive 3d virtual histology, without any need for slicing due to the high penetration depth of x-rays through tissue It provides isotropic reconstruction of the 3d cytoarchitecture based on differences in absorption coefficient (absorption contrast) or electron density (phase contrast). This enables imaging of the sample on multiple length scales and fields of view
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