Optimization of contrast-enhanced microCT for 3D X-ray-based histology of the kidney

Details

Supervisors

Kerckhofs, Greet

Faculty

Ecole polytechnique de Louvain

Degree label

Master [120] : ingénieur civil biomédical, à finalité spécialisée

Abstract

"The shape makes the function" is a well-known adage in biology. However, even though kidneys are vital organs responsible for essential functions such as blood filtration or blood pressure regulation, the link between the kidney anatomy and its physiology is not always known. Nevertheless, this knowledge is essential in order to understand the mechanisms of disease such as chronic kidney disease, which affects one out of ten people in Belgium. It is thus crucial to have a 3D visualization of the structures of the kidney, as some of them are hihgly convoluted. This can be achieved by using X-rays contrast enhanced microfocus computed tomography. Moreover, the research group of G. Kerckhofs has recently reported that a new approach of micro-CECT, Cryogenic CECT, based on the imaging of frozen samples provides good results for non-destructive 3D visualization of biological tissues. The goal of this master thesis is to obtain images of the kidney (micro)structures that achieve a high contrast enhancement, a micrometer resolution and a non-destructive 3D visualization. To do so, five staining solutions (1:2 Hf-WD POM, Lugol in PBS, Lugol in Sorensen's buffer, CA4+ and PTA) have been used on murine kidney. Their effects on the samples volume, the grey values and the volume of the different kidney layers (i.e. the cortex, the outer stripe of the outer medulla, the inner medulla and the papilla), the shape and size of the glomeruli and the diameter of the cortical tubules were compared. The added value of the cryo-CECT was also evaluated for different staining solutions and at different freezing rates. First, the results showed that acidic staining solutions such as Lugol PBS or PTA lead to extensive tissue shrinkage. Therefore, when in Sorensen's buffer, the Lugol causes almost no shrinkage. However, this change of solvent has a negative impact on the contrast enhancement of the staining solutions compared to Lugol's iodine in PBS. Moreover, we demonstrated that cryo-CECT imaging allows to distinguish more easily the kidney microstructures than classical CECT imaging. Added value of the cryo-CECT was maximized with Hf-WD POM solution. Besides, the results showed that different freezing rates has different impact on the cryo-CECT imaging. When the freezing rate is high (i.e. when the sample is frozen at temperature such as -160°C), vitrification occurs, which reduce the cryo-CECT added value. Furthermore, lower freezing rates (i.e. -20°C and to a lesser extend -45°C), tend to induce shrinkage of the kidney tubuli and glomeruli. Therefore, we propose freezing using isopentane at -78°C as the optimal freezing method for cryo-CECT of Hf-WD POM stained kidney. Finally, preliminary results showed differences in cryo-CECT images between samples frozen only once and samples frozen several times at different freezing rates. Murine kidneys seem to keep in memory their freezing history. This should be further investigated.