nach oben

Erschienen in:

14.03.2019 | Original Article

Ethanol fixation method for heart and lung imaging in micro-CT

verfasst von: Matej Patzelt, Jana Mrzilkova, Jan Dudak, Frantisek Krejci, Jan Zemlicka, Jakub Karch, Vladimir Musil, Jozef Rosina, Viktor Sykora, Barbora Horehledova, Petr Zach

Erschienen in: Japanese Journal of Radiology | Ausgabe 6/2019

Einloggen, um Zugang zu erhalten

Abstract

Purpose

The soft tissue imaging in micro-CT remains challenging due to its low intrinsic contrast. The aim of this study was to create a simple staining method omitting the usage of contrast agents for ex vivo soft tissue imaging in micro-CT.

Materials and methods

Hearts and lungs from 30 mice were used. Twenty-seven organs were either fixed in 97% or 50% ethanol solution or in a series of ascending ethanol concentrations. Images were acquired after 72, 168 and 336 h on a custom-built micro-CT machine and compared to scans of three native samples.

Results

Ethanol provided contrast enhancement in all evaluated fixations. Fixation in 97% ethanol resulted in contrast enhancement after 72 h; however, it caused hardening of the samples. Fixation in 50% ethanol provided contrast enhancement after 336 h, with milder hardening, compared to the 97% ethanol fixation, but the visualization of details was worse. The fixation in a series of ascending ethanol concentrations provided the most satisfactory results; all organs were visualized in great detail without tissue damage.

Conclusions

Simple ethanol fixation improves the tissue contrast enhancement in micro-CT. The best results can be obtained with fixation of the soft tissue samples in a series of ascending ethanol concentrations.

Metscher BD. MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol. 2009;9:11. https://doi.org/10.1186/1472-6793-9-11.CrossRefPubMedPubMedCentral

Vickerton P, Jarvis J, Jeffery N. Concentration-dependent specimen shrinkage in iodine-enhanced microCT. J Anat. 2013;223(2):185–93. https://doi.org/10.1111/joa.12068.CrossRefPubMedPubMedCentral

Silva JMDE, Zanette I, Noel PB, Cardoso MB, Kimm MA, Pfeiffer F. Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography. Sci Rep-UK. 2015;5:Artn 14088. https://doi.org/10.1038/srep14088.CrossRef

Donath T, Pfeiffer F, Bunk O, Grunzweig C, Hempel E, Popescu S, et al. Toward clinical X-ray phase-contrast CT demonstration of enhanced soft-tissue contrast in human specimen. Invest Radiol. 2010;45(7):445–52. https://doi.org/10.1097/RLI.0b013e3181e21866.CrossRefPubMed

Mizutani R, Suzuki Y. X-ray microtomography in biology. Micron. 2012;43(2–3):104–15. https://doi.org/10.1016/j.micron.2011.10.002.CrossRefPubMed

Descamps E, Sochacka A, De Kegel B, Van Loo D, Van Hoorebeke L, Adriaens D. Soft tissue discrimination with contrast agents using micro-CT scanning. Belg J Zool. 2014;144(1):20–40.

Clauss SB, Walker DL, Kirby ML, Schimel D, Lo CW. Patterning of coronary arteries in wildtype and connexin43 knockout mice. Dev Dyn. 2006;235(10):2786–94. https://doi.org/10.1002/dvdy.20887.CrossRefPubMed

Pai VM, Kozlowski M, Donahue D, Miller E, Xiao XH, Chen MY, et al. Coronary artery wall imaging in mice using osmium tetroxide and micro-computed tomography (micro-CT). J Anat. 2012;220(5):514–24. https://doi.org/10.1111/j.1469-7580.2012.01483.x.CrossRefPubMedPubMedCentral

Yamashita T, Kawashima S, Ozaki M, Namiki M, Hirase T, Inoue N, et al. Mouse coronary angiograph using synchrotron radiation microangiography. Circulation. 2002;105(2):E3–4. https://doi.org/10.1161/hc0202.100423.CrossRefPubMed

10.

Degenhardt K, Wright AC, Horng D, Padmanabhan A, Epstein JA. Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ Cardiovasc Imaging. 2010;3(3):314–22. https://doi.org/10.1161/Circimaging.109.918482.CrossRefPubMedPubMedCentral

11.

Wong MD, Spring S, Henkelman RM. Structural stabilization of tissue for embryo phenotyping using micro-CT with iodine staining. PLoS ONE. 2013;8(12):e84321. https://doi.org/10.1371/journal.pone.0084321.CrossRefPubMedPubMedCentral

12.

Gammon ST, Foje N, Brewer EM, Owers E, Downs CA, Budde MD, et al. Preclinical anatomical, molecular, and functional imaging of the lung with multiple modalities. Am J Physiol Lung C. 2014;306(10):L897–914. https://doi.org/10.1152/ajplung.00007.2014.CrossRef

13.

Vande Velde G, Poelmans J, De Langhe E, Hillen A, Vanoirbeek J, Himmelreich U, et al. Longitudinal micro-CT provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume. Dis Model Mech. 2016;9(1):91–8. https://doi.org/10.1242/dmm.020321.CrossRefPubMedPubMedCentral

14.

Ashton JR, Clark DP, Moding EJ, Ghaghada K, Kirsch DG, West JL, et al. Dual-energy micro-CT functional imaging of primary lung cancer in mice using gold and iodine nanoparticle contrast agents: a validation study. PLoS ONE. 2014;9(2):e88129. https://doi.org/10.1371/journal.pone.0088129.CrossRefPubMedPubMedCentral

15.

Rodt T, von Falck C, Dettmer S, Halter R, Maus R, Ask K, et al. Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-beta 1. Resp Res. 2010;11:181. https://doi.org/10.1186/1465-9921-11-181.CrossRef

16.

Thiesse J, Namati E, Sieren JC, Smith AR, Reinhardt JM, Hoffman EA, et al. Lung structure phenotype variation in inbred mouse strains revealed through in vivo micro-CT imaging. J Appl Physiol. 2010;109(6):1960–8. https://doi.org/10.1152/japplphysiol.01322.2009.CrossRefPubMedPubMedCentral

17.

Pauwels E, Van Loo D, Cornillie P, Brabant L, Van Hoorebeke L. An exploratory study of contrast agents for soft tissue visualization by means of high resolution X-ray computed tomography imaging. J Microsc-Oxf. 2013;250(1):21–31. https://doi.org/10.1111/jmi.12013.CrossRef

18.

Takeda T, Thet-Thet-Lwin Kunii T, Sirai R, Ohizumi T, Maruyama H, et al. Ethanol fixed brain imaging by phase-contrast X-ray technique. J Phys: Conf Ser. 2013;425:022004. https://doi.org/10.1088/1742-6596/425/2/022004.CrossRef

19.

Shirai R, Kunii T, Yoneyama A, Ooizumi T, Maruyama H, Lwin TT, et al. Enhanced renal image contrast by ethanol fixation in phase-contrast X-ray computed tomography. J Synchrotron Radiat. 2014;21(Pt 4):795–800. https://doi.org/10.1107/S1600577514010558.CrossRefPubMed

20.

Dudak J, Zemlicka J, Krejci F, Karch J, Patzelt M, Zach P, et al. Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex vivo soft tissue samples. J Instrum. 2016;11:C03005. https://doi.org/10.1088/1748-0221/11/03/C03005.CrossRef

21.

Jakubek J. Data processing and image reconstruction methods for pixel detectors. Nucl Instrum Methods A. 2007;576(1):223–34. https://doi.org/10.1016/j.nima.2007.01.157.CrossRef

22.

Bruker. CTVox: Volume Rendering [computer software] http://bruker-microct.com/products/downloads.htm (2015).

23.

Turecek D, Holy T, Jakubek J, Pospisil S, Vykydal Z. Pixelman: a multi-platform data acquisition and processing software package for Medipix2, Timepix and Medipix3 detectors. J Instrum. 2011;6:C01046. https://doi.org/10.1088/1748-0221/6/01/C01046.CrossRef

24.

Llopart X, Ballabriga R, Campbell M, Tlustos L, Wong W. Timepix, a 65k programmable pixel readout chip for arrival time, energy and/or photon counting measurements. Nucl Instrum Methods A. 2007;581(1–2):485–94. https://doi.org/10.1016/j.nima.2007.08.079.CrossRef

25.

Dudak J, Zemlicka J, Karch J, Patzelt M, Mrzilkova J, Zach P, et al. 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. Sci Rep-UK. 2016;6:30385. https://doi.org/10.1038/Srep30385.CrossRef

26.

Dudak J, Zemlicka J, Krejci F, Polansky S, Jakubek J, Mrzilkova J, et al. X-ray micro-CT scanner for small animal imaging based on Timepix detector technology. Nucl Instrum Methods A. 2015;773:81–6. https://doi.org/10.1016/j.nima.2014.10.076.CrossRef

27.

Jakubek J, Holy T, Jakubek M, Vavrik D, Vykydal Z. Experimental system for high resolution X-ray transmission radiography. Nucl Instrum Methods A. 2006;563(1):278–81. https://doi.org/10.1016/j.nima.2006.01.033.CrossRef

28.

Jakubek J, Jakubek M, Platkevic M, Soukup P, Turecek D, Sykora V, et al. Large area pixel detector WIDEPIX with full area sensitivity composed of 100 Timepix assemblies with edgeless sensors. J Instrum. 2014;9:C04018. https://doi.org/10.1088/1748-0221/9/04/C04018.CrossRef

29.

Howat WJ, Wilson BA. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 2014;70(1):12–9. https://doi.org/10.1016/j.ymeth.2014.01.022.CrossRefPubMedPubMedCentral

Titel: Ethanol fixation method for heart and lung imaging in micro-CT
verfasst von: Matej Patzelt
Jana Mrzilkova
Jan Dudak
Frantisek Krejci
Jan Zemlicka
Jakub Karch
Vladimir Musil
Jozef Rosina
Viktor Sykora
Barbora Horehledova
Petr Zach
Publikationsdatum: 14.03.2019
Verlag: Springer Japan
Erschienen in: Japanese Journal of Radiology / Ausgabe 6/2019
Print ISSN: 1867-1071
Elektronische ISSN: 1867-108X
DOI: https://doi.org/10.1007/s11604-019-00830-6

Update Radiologie

Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.

Newsletter bestellen

Springer Medizin

Ethanol fixation method for heart and lung imaging in micro-CT