Photon Factory Activity Report 2014 #32 (2015) B%/&*Micro-X-ray imaging of ethanol fixed biological soft tissue
Tohoru Takeda1,* Thet-Thet-Lwin1, Akio Yoneyama2, Kazuyuki Hyodo3
Allied Health Sciences, Kitasato University, Kanagawa 252-0373, Japan
2
Central Research Laboratory, Hitachi Ltd, Saitama 350-0395, Japan
3
High Energy Accelerator Research Organization, Tsukuba 305-0801, Japan
1

1 Introduction
Recently, a phase-contrast X-ray imaging technique fordetecting contrast from differences in refractive index,
was developed. This technique has approximately 1000times greater sensitivity than that of the conventional
absorption technique [1-4]. However, spatial resolution ofphase-contrast X-ray imaging technique is limited by thethickness of half mirror (analyzer). Absorption-contrast
X-ray imaging allows to obtain higher spatial resolution
less than 1 micrometer, however the image-contrast
reduces significantly.
To enhance the image-contrast that enables to obtain
the higher spatial resolution, we changed the fixation
technique of the biological sample from commonly used
formalin to ethanol. Ethanol fixation is popularly used toprepare specimens for biopsy processing and rapid intraoperative pathological diagnosis. It exhibits comparable
histology results and superior immune-staining results toformalin. We surmise that the physical property increases
density differences against back ground and thedehydration by ethanol increases the soft tissue density.
Here, preliminary phase-contrast X-ray imaging withethanol fixation were performed, and the image-contrast
was significantly improved in brain [5, 6], heart [7] andkidney [8, 9] of rat.
2 ExperimentThis study used six male rats (age, 10 weeks). All rats
were anesthetized, and cannulation to the apex of left
ventricle was carried out surgically for perfusion. First,
physiological saline solution was injected from the apex,
and the whole blood in the vessel was replaced toeliminate blood coagulation artifacts. Then, 10% formalin
or 100% ethanol was perfused for fixation, and thesamples were steeped in each fixing solution. The
experimental protocol was approved by the President ofKitasato University through the judgment of the Animal
Care and Use Committee of Kitasato University.
Phase-contrast X-ray CT system with crystal
interferometer was set at the vertical wiggler beam-line
14C of the Photon Factory, Tsukuba, Japan. X-ray CCD
camera with a pixel size of 13 x 13 μm2 was used. The Xray energy was set at 35 keV, and projection number was
250 per 180 degree. CT image was reconstructed FBP.
After phase-contrast X-ray CT imaging, all specimens
fixed by each solution (ethanol and formalin) were sliced
into 3-µm-thick sections and stained with hematoxylin
and eosin (HE).
The density was measurement in phase-contrast X-ray
CT images. And histological image analysis was also
performed by using image-manipulating software (NIHimage 1.63)

3 Results and Discussion
Here, we report the experiment of kidney in detail.
Phase-contrast X-ray imaging using a two-crystal X-ray
interferometer was able to clearly visualize the fine renal
structures such as cortex, medulla and the vessels in bothformalin-fixed and ethanol-fixed kidneys. Especially, theimage-contrast of ethanol-fixed kidneys was much better
than that of formalin-fixed kidneys due to physicallyenhance the density difference between the soft tissue andsurrounded ethanol solution with lower density
33(0.79g/cm ) than that of formalin solution (1.01g/cm ).
Actually in histological image, the shrinkage of soft tissue
was observed in ethanol-fixed kidney.
In quantitative analysis, the ethanol fixation
technique enhances approximately 3.1 times higher image
contrast; the effect of shrinkage is estimatedapproximately 2-times and the physical effect of ethanol
is approximately 28%. Thus, the ethanol-fixation
technique enables to enhance the image contrast in phasecontrast X-ray imaging. Histological conditions ofvarious organs were examined as preliminary studies byusing phase-contrast X-ray CT system.
The ethanol is easily volatilized, and fixed soft tissue
having exact histological structure remains. Here, we areplanning to obtain high spatial resolution image with Xray CT system at room condition.
Acknowledgement
This study was supported by a grant from Kitasato
University School of Allied Health Sciences (Grant-inAid for Research Project, No. 2012-1012), and wasapproved by the KEK (proposal number 2012G044 and2013G584).
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[6] S. Kokubo et al., Med Imag Tech.. 32, 2 (2014).
[7] T Kunii et al., Med Imag Tech.. 31, 132 (2013).
[8] R Sirai et al., Med Imag Tech.. 30, 298 (2012).
[9] R Sirai et al., J. Synchrotron Rad. 21, 795 (2014).
*t.takeda@kitasato-u.ac.jp