Enhanced renal image contrast by ethanol fixation in phase-contrast X-ray computed tomography

Abstract

Phase-contrast X-ray imaging using a crystal X-ray interferometer can depict the fine structures of biological objects without the use of a contrast agent. To obtain higher image contrast, fixation techniques have been examined with 100% ethanol and the commonly used 10% formalin, since ethanol causes increased density differences against background due to its physical properties and greater dehydration of soft tissue. Histological comparison was also performed. A phase-contrast X-ray system was used, fitted with a two-crystal X-ray interferometer at 35 keV X-ray energy. Fine structures, including cortex, tubules in the medulla, and the vessels of ethanol-fixed kidney could be visualized more clearly than that of formalin-fixed tissues. In the optical microscopic images, shrinkage of soft tissue and decreased luminal space were observed in ethanol-fixed kidney; and this change was significantly shown in the cortex and outer stripe of the outer medulla. The ethanol fixation technique enhances image contrast by approximately 2.7-3.2 times in the cortex and the outer stripe of the outer medulla; the effect of shrinkage and the physical effect of ethanol cause an increment of approximately 78% and 22%, respectively. Thus, the ethanol-fixation technique enables the image contrast to be enhanced in phase-contrast X-ray imaging.

X-Ray Phase-Contrast Tomography of Renal Ischemia-Reperfusion Damage

“…Another potential source of error is the influence of formalin fixation on soft-tissue signal characteristics in phase-contrast imaging. This has only been examined for brain tissue [16] and is currently under investigation for other tissue types [19] . Security regulations at the synchrotron radiation facility did not allow for the measurement of fresh kidney samples.…”

“…The majority of PCI-studies on biological tissue have been performed at synchrotron radiation sources [16] which provide monochromatic x-rays with high flux and are used in combination with high-resolution imaging systems. PCI of the kidney without the use of a contrast agent has so far only been performed using crystal-based phase imaging techniques [15] , [17] – [19] . These studies already demonstrate the innovative potential of this technique to depict the smallest anatomical structures and detect parenchymal renal disease such as glomerulosclerosis in hamster kidneys [15] .…”

X-ray phase tomography with near-field speckles for three-dimensional virtual histology

“…Alternative x-ray methods based on extracting the x-ray phase shift induced by the sample [10] can provide significantly better contrast for soft biomedical tissue without the need for staining and have recently been identified as a promising approach for virtual histology [9,[11][12][13][14][15]. However, the x-ray phase-contrast imaging methods employed for this purpose to date, such as propagation-based imaging (PBI) [16][17][18] and x-ray grating interferometry (XGI) [19][20][21], have various limitations that have prevented their practical use and wider uptake for virtual histology.…”

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector

“…Very good soft tissue contrast together with non-destructive volumetric information can be provided also by highly sophisticated methods such as X-ray phase-contrast 23 24 25 26 27 28 , dark-field imaging 29 30 or X-ray microscopy 31 32 . These methods, however, require an advanced setup and very special X-ray beam characteristics, which in most of the cases can be found only at large scale synchrotron facilities.…”

“…In this work we demonstrate performance of this recently developed large area hybrid pixel photon counting detector for high resolution X-ray microradiography and micro-tomography of ex-vivo ethanol-only preserved murine organs. The effect of ex-vivo soft tissue contrast enhancement after ethanol-preservation has been demonstrated in studies related to phase contrast X-ray imaging 23 24 . However, in the case of techniques based on X-ray absorption, ethanol is generally used as a soft tissue fixative and the sample needs to be further stained by a contrast agent (Lugol solution, phosphotungstic acid, osmium tetroxide, etc.)…”