Abstract
Synchrotron X-ray Phase Contrast micro-Tomography (SXrPCμT) is a powerful tool in the investigation of biological tissues, including the central nervous system (CNS), and it allows to simultaneously detect the vascular and neuronal network avoiding contrast agents or destructive sample preparations. However, specific sample preparation procedures aimed to optimize the achievable contrast- and signal-to-noise ratio (CNR and SNR, respectively) are required. Here we report and discuss the effects of perfusion with two different fixative agents (ethanol and paraformaldehyde) and with a widely used contrast medium (MICROFIL®) on mouse spinal cord. As a main result, we found that ethanol enhances contrast at the grey/white matter interface and increases the contrast in correspondence of vascular features and fibres, thus providing an adequate spatial resolution to visualise the vascular network at the microscale. On the other hand, ethanol is known to induce tissue dehydration, likely reducing cell dimensions below the spatial resolution limit imposed by the experimental technique. Nonetheless, neurons remain well visible using either perfused paraformaldehyde or MICROFIL® compound, as these latter media do not affect tissues with dehydration effects. Paraformaldehyde appears as the best compromise: it is not a contrast agent, like MICROFIL®, but it is less invasive than ethanol and permits to visualise well both cells and blood vessels. However, a quantitative estimation of the relative grey matter volume of each sample has led us to conclude that no significant alterations in the grey matter extension compared to the white matter occur as a consequence of the perfusion procedures tested in this study.
Highlights
Assessment of the effects of different sample perfusion procedures on phase-contrast tomographic images of mouse spinal cord
We found that ethanol enhances contrast at the grey/white matter interface and increases the contrast in correspondence of vascular features and fibres, providing an adequate spatial resolution to visualise the vascular network at the microscale
We performed our analysis on the whole mouse SC, but we report here only results obtained on a selected region. Such an approach ensured a fair comparison between the different sample preparation procedures employing fixative agents, avoiding misleading conclusions due to normal anatomical GM/WM variations occurring across different regions of the SC longitudinal extension
Summary
Assessment of the effects of different sample perfusion procedures on phase-contrast tomographic images of mouse spinal cord. We report and discuss the effects of perfusion with two different fixative agents (ethanol and paraformaldehyde) and with a widely used contrast medium (MICROFIL®) on mouse spinal cord. We found that ethanol enhances contrast at the grey/white matter interface and increases the contrast in correspondence of vascular features and fibres, providing an adequate spatial resolution to visualise the vascular network at the microscale. : Medical-image reconstruction methods and algorithms, computer-aided diagnosis; Medical-image reconstruction methods and algorithms, computer-aided software tomography [16, 17]) do not provide a sufficient spatial resolution and/or an adequate contrast for soft tissues. When higher resolution is achieved by conventional imaging techniques (e.g. scanning electron microscopy, histological sectioning), an invasive or aggressive sample treatment is usually required, such as sectioning, staining, or casting, and only 2D information is derived
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