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

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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.

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References

  1. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  3. 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.

    Article  CAS  Google Scholar 

  4. 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.

    Article  PubMed  Google Scholar 

  5. 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.

    Article  PubMed  Google Scholar 

  6. 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.

    Google Scholar 

  7. 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.

    Article  PubMed  Google Scholar 

  8. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

The work was supported by the Charles University Grant Agency [GAUK 130 717] and from European Regional Development Fund-Project “Engineering applications of microworld physics” [No. CZ.02.1.01/0.0/0.0/16_019/0000766].

Author information

Authors and Affiliations

  1. Specialized Laboratory of Experimental Imaging, Third Faculty of Medicine, Charles University, Prague, Czech Republic

    Matej Patzelt, Jana Mrzilkova, Jan Dudak, Frantisek Krejci, Jan Zemlicka, Jakub Karch, Vladimir Musil, Jozef Rosina & Petr Zach

  2. Institute of Experimental and Applied Physics and Faculty of Biomedical Engineering, Czech Technical University in Prague, Prague, Czech Republic

    Matej Patzelt, Jana Mrzilkova, Jan Dudak, Frantisek Krejci, Jan Zemlicka, Jakub Karch, Vladimir Musil, Jozef Rosina & Petr Zach

  3. Department of Anatomy, Third Faculty of Medicine, Charles University, Ruska 87, 100 34, Prague, Czech Republic

    Matej Patzelt, Jana Mrzilkova, Vladimir Musil & Petr Zach

  4. Institute of Experimental and Applied Physics, Czech Technical University in Prague, Prague, Czech Republic

    Jan Dudak, Frantisek Krejci, Jan Zemlicka & Jakub Karch

  5. Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic

    Jan Dudak, Jakub Karch & Jozef Rosina

  6. Centre of Scientific Information, Third Faculty of Medicine, Charles University, Prague, Czech Republic

    Vladimir Musil

  7. Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Charles University, Prague, Czech Republic

    Jozef Rosina

  8. Center for Experimental Biomodels, First Faculty of Medicine, Charles University, Prague, Czech Republic

    Viktor Sykora

  9. Department of Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, The Netherlands

    Barbora Horehledova

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  1. Matej Patzelt
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  2. Jana Mrzilkova
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  3. Jan Dudak
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  11. Petr Zach
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Corresponding author

Correspondence to Jana Mrzilkova.

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All applicable institutional and national guidelines for the care and use of animals were followed.

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Patzelt, M., Mrzilkova, J., Dudak, J. et al. Ethanol fixation method for heart and lung imaging in micro-CT. Jpn J Radiol 37, 500–510 (2019). https://doi.org/10.1007/s11604-019-00830-6

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  • DOI: https://doi.org/10.1007/s11604-019-00830-6

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