Open Access, Peer-reviewed
pISSN 2671-5651
eISSN 2671-566X
    Korean J Phys Anthropol. 2018 Dec;31(4):133-142. Korean.
    Published online Dec 31, 2018.
    https://doi.org/10.11637/kjpa.2018.31.4.133
    © 2018 Korean Association of Physical Anthropologists
    Original Article

    3D Histology Using the Synchrotron Radiation Propagation Phase Contrast Cryo-microCT

    Ju-Heon Kim,2 Sung-Mi Han,3 Hyun-Ouk Song,4 Youn-Kyung Seo,5 Young-Suk Moon,1 and Hong-Tae Kim1
      • 1Department of Anatomy, Catholic University of Daegu School of Medicine, Korea.
      • 2Department of Radiology, Yeongju Red Cross Hospital, Korea.
      • 3Optical Convergence Technology Center, Catholic University of Daegu, Korea.
      • 4Department of Parasitology, Catholic University of Daegu School of Medicine, Korea.
      • 5Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Korea.
    • Correspondence to: Hong-Tae Kim (Department of Anatomy, Catholic University of Daegu School of Medicine). Email: htaekim@cu.ac.kr
    Received September 10, 2018; Revised December 02, 2018; Accepted December 03, 2018.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    3D histology is a imaging system for the 3D structural information of cells or tissues. The synchrotron radiation propagation phase contrast micro-CT has been used in 3D imaging methods. However, the simple phase contrast micro-CT did not give sufficient micro-structural information when the specimen contains soft elements, as is the case with many biomedical tissue samples. The purpose of this study is to develop a new technique to enhance the phase contrast effect for soft tissue imaging. Experiments were performed at the imaging beam lines of Pohang Accelerator Laboratory (PAL). The biomedical tissue samples under frozen state was mounted on a computer-controlled precision stage and rotated in 0.18° increments through 180°. An X-ray shadow of a specimen was converted into a visual image on the surface of a CdWO4 scintillator that was magnified using a microscopic objective lens (X5 or X20) before being captured with a digital CCD camera. 3-dimensional volume images of the specimen were obtained by applying a filtered back-projection algorithm to the projection images using a software package OCTOPUS. Surface reconstruction and volume segmentation and rendering were performed were performed using Amira software. In this study, We found that synchrotron phase contrast imaging of frozen tissue samples has higher contrast power for soft tissue than that of non-frozen samples. In conclusion, synchrotron radiation propagation phase contrast cryo-microCT imaging offers a promising tool for non-destructive high resolution 3D histology.

    Keywords
    3D histology; Synchrotron radiation; Cryo-microtomography; X-ray propagation phase contrast; Collagen induced arthritis

    Figures

    Fig. 1
    Schematic drawing of the experimental setup. Unmonochromatic beam arrived from the bending magnet device (B) was reduced to the beam size for matching the scintillator after passing the slit (S). The X-ray irradiated the object (O) positioned 34.8 m away from the source. Visible-light images on the surface of CdWO4 scintillator (St) placed at a distance of 10 cm from object were reflected at 90 degrees by a gold-coated mirror (M) and magnified by a microscope objective lens (X5–X20) (L). Finally, the images reached the CCD camera.

    Fig. 2
    Pictures of the cryo-tomogarphy devices. (a) The sample was directly exposed to liquid nitrogen stream. (b) The sample was embedded in OCT compound.

    Fig. 3
    Typical CT image (a) and three-dimensional micro-structural architecture (b) of the knee joint of collagen induced arthritis (CIA) mouse model. The typical findings of CIA, destructive changes in bones (*), and new bone formation (arrows), can be seen in detail. Scale bar=1 mm

    Fig. 4
    Phase contrast synchrotron micrographic images of mouse liver tissue, hydrated (a) and dried (b and c) samples. The edge enhancement effect was disturbed by water (a), but it was increased after drying tissue samples. Scale bar=500 µm.

    Fig. 5
    Comparison of X-ray phase contrast micro-CT images of the rat metatarsophalangeal joint of the 3rd toe according to the condition of micro-CT, (a) non-frozen agarose embedded sample (b) directly frozen sample (c) and frozen OCT embedded sample. The tomographic slices are on 3 sectional planes, horizontal plane (A and B), sagittal plane (C) and coronal plane(D) and the 3-dimensional volume-rendered images (E). Panel B shows the differences of the thickness of articular cartilage (*), the wideness of joint cavity (Jc) and the lining of synovial membrane (arrows) between each condition of microCT. Scale bar=1 mm.

    Fig. 6
    The tomographic slices of X-ray phase contrast cryo-microCT reconstruction images (A and C) and histologic images (B) of the metatarsophalangeal joints of the 3rd toe of normal control mouse (a) and collagen induced arthritis (CIA) mouse model. The typical findings of CIA, destructive change (arrows) in bone and pannus formation (*), can be seen in detail. Scale bar=1 mm.

    Fig. 7
    A tomographic slice of X-ray phase contrast cryo-microCT reconstruction image (a) and a 3-dimensional volume-rendered microCT image (b) of a mouse eyeball. Ci: ciliary body, Co: cornea, Ir: iris. Scale bar=1 mm.

    References

      1. Park JS, Chung MS, Hwang SB, Lee YS, Har DH, Park HS. Visible Korean human: improved serially sectioned images of the entire body. IEEE Trans Med Imaging 2005;24:352–360.
      1. Hwang SB, Chung MS, Hwang YI, Park HS, Har DH, Shin DS, et al. Improved sectional images of the female pelvis showing detailed urogenital and neighboring structures. Korena J Phys Anthropol 2010;12:187–198.
      1. Fiala JC. Reconstruct: a free editor for serial section microscopy. J Microsc 2005;218:52–61.
      1. Song WC, Hu KS, Kim HJ, Koh KS. A study of the secretion mechanism of the sebaceous gland using three-dimensional reconstruction to examine the morphological relationship between the sebaceous gland and arrector pili muscle in the follicular unit. Br J Dermatol 2007;157:325–330.
      1. Roy D, Steyer GJ, Gargesha M, Stone ME, Wilson DL. 3D cryo-imging: A very high-resolution view of the whole mouse. Anat Rec (Hoboken) 2009;292:342–351.
      1. Le Gros M, McDermott G, Larabell CA. X-ray tomography of whole cells. Curr Opin Struct Biol 2005;15:593–600.
      1. Meuli R, Hwu Y, Je JH, Margaritondo G. Synchrotron radiation in radiology: radiology techniques based on synchrotron sources. Eur Radiol 2004;14:1550–1560.
      1. Zhou SA, Brahme A. Development of phase-contrast X-ray imaging techniques and potential medical applications. Physica Medica 2008;24:129–148.
      1. Momose A. Recent advances in X-ray phase imaging. Jap J Applied Physics 2005;44:6355–6357.
      1. Takeda T, Momose A, Wu J, Yu Q, Zeniya T, Lwin TT, et al. Vessel imaging by interferometric phase-contrast X-ray technique. Circulation 2002;105:1708–1712.
      1. Wilkins SW, Gureyev TE, Gao D, Pogany A, Stevenson AW. Phase-contrast imaging using polychromatic hard X-rays. Nature 1996;384:335–338.
      1. Youn SW, Kim HK, KIm HT, Han SM, Don JK, DO YR, et al. Propagation-based phase-contrast X-ray microtomography of a cerebral protection device retrieved after carotid artery stenting. J Synchrotron Radiat 2014;21:215–222.
      1. Bravin A, Keyriläinen J, Fernández M, Fiedler S, Nemoz C, Karjalainen-Lindsberg KL, et al. High-resolution CT by diffraction-enhanced x-ray imaging: mapping of breast tisssue samples and comparison with their histo-pathology. Phys Med Biol 2007;52:2197–2211.
      1. Sunaguchi N, Yuasa T, Huo Q, Ando M. Convolution reconstruction algorithm for refraction-contrast computed tomography using a Laue-case analyzer for drak field imging. Opt Lett 2011;36:391–393.
      1. Pfeiffer F, Weitkamp T, Bunk O, David C. Phase retrieval and differential phase-contrast imaging with low-brilliance X-ray sources. Nature Physics 2006;2:258–261.
      1. Kim BI, Kim KH, Youn HS, Jheon S, Kim JK, Kim HT. High-resolution X-ray phase contrast synchrotron imaging of normal and ligation damaged rat sciatic nerves. Microsc Res Tech 2008;71:443–447.
      1. Choi CH, Kim HT, Choe JY, Kim SK, Choi GW, Jheon SH, et al. In vivo high-resolution synchrotron radiation imaging of collagen-induced arthritis in a rodent model. J. J Synchrotron Radiat 2010;17:393–399.
      1. Larabell CA, Le Gros MA. X-ray tomography generates 3-D reconstruction of the Yeast, Saccharomyces cerevisiae, at 60-nm resolution. Molecular Biology of the Cell 2004;15:957–962.
      1. Fitzgerald R. Phase-sensitive X-ray imaging. Phys Today 2000;53:23–26.
      1. Berhardt R, van den Dolder J, Bierbaum S, Beutner R, Scharnwebar D, Jansen J, et al. Osteoconductive modification of Ti-implants in a goat defect model: characterization of bone growth with SR μCT and histology. Biomaterials 2005;26:3009–3019.
      1. Kim HT, Han SM, Moon YS, Seo SJ, Kim JK, Kim KH. Comparision of the 3D structures of Hairs from humans and from animals by using synchrotron radiation micro-CT. New Phys: Sae Mulli 2012;62:269–274.
      1. Wu J, Takeda T, Lwin TT, Momose A, Sunaguchi N, Fukami T, et al. Imaging renal structures by X-ray phase-contrast microtomography. Kidney International 2009;75:945–951.
      1. Bravin A, Coan P, Suortti P. X-ray phase-contrast imaging: from pre-clinical applications towards clinics. Phys Med Biol 2013;58:R1–R35.
      1. Tang L, Li G, Sun YS, Li J, Zhang XP. Synchrotron radiation phase-contrast imaging of human stomach and gastric cancer: in vitro studies. J Synchrotron Radiat 2012;19:319–322.
      1. Mizutani R, Takeuchi A, Hara T, Uesugi K, Suzuki Y. Computed tomography imaging of the neuronal structure of Drosophila brain. J Synchrotron Rad 2007;14:282–287.
      1. Studer D, Humbel BM, Matthias C. Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem Cell Biol 2008;130:877–889.
      1. Kuzay TM, Kazmierczak M, Hsieh BJ. X-ray beam/biomaterial thermal interaction in third-generation synchrotron sources. Acta Crystallogr D Biol Crystallogr 2001;57:69–81.
      1. McDermott G, Le Gros MA, Knoechel CG, Uchida M, Larabell CA. Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging. Trends in Cell Biol 2009;19:587–595.
      1. Rosene AL, Roy NJ, Davis BJ. A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact. J Histochem Cytochem 1986;34:1301–1315.
    MeSH Terms
    Gyeongsangbuk-do
    Octopodiformes
    Synchrotrons*
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