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Volume Shrinkage of Bone, Brain and Muscle Tissue in Sample Preparation for Micro-CT and Light Sheet Fluorescence Microscopy (LSFM)

Published online by Cambridge University Press:  25 June 2014

Jan Buytaert ,
Jana Goyens ,
Daniel De Greef ,
Peter Aerts  and
Joris Dirckx
Jan Buytaert*
Affiliation:
Laboratory of Biomedical Physics, Groenenborgerlaan 171, 2020 Antwerpen, Belgium
Jana Goyens
Affiliation:
Laboratory of Biomedical Physics, Groenenborgerlaan 171, 2020 Antwerpen, Belgium Laboratory of Functional Morphology, Universiteitsplein 1, 2610 Antwerp, Belgium
Daniel De Greef
Affiliation:
Laboratory of Biomedical Physics, Groenenborgerlaan 171, 2020 Antwerpen, Belgium
Peter Aerts
Affiliation:
Laboratory of Functional Morphology, Universiteitsplein 1, 2610 Antwerp, Belgium
Joris Dirckx
Affiliation:
Laboratory of Biomedical Physics, Groenenborgerlaan 171, 2020 Antwerpen, Belgium
*
*Corresponding author. jan.buytaert@uantwerpen.be
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Abstract

Two methods are especially suited for tomographic imaging with histological detail of macroscopic samples that consist of multiple tissue types (bone, muscle, nerve or fat): Light sheet (based) fluorescence microscopy (LSFM) and micro-computed tomography (micro-CT). Micro-CT requires staining with heavy chemical elements (and thus fixation and sometimes dehydration) in order to make soft tissue imageable when measured alongside denser structures. LSMF requires fixation, decalcification, dehydration, clearing and staining with a fluorescent dye. The specimen preparation of both imaging methods is prone to shrinkage, which is often not mentioned, let alone quantified. In this paper the presence and degree of shrinkage are quantitatively identified for the selected preparation methods/stains. LSFM delivers a volume shrinkage of 17% for bone, 56% for muscle and 62% for brain tissue. The three most popular micro-CT stains (phosphotungstic acid, iodine with potassium iodide, and iodine in absolute ethanol) deliver a volume shrinkage ranging from 10 to 56% for muscle and 27–66% for brain, while bone does not shrink in micro-CT preparation.

Keywords

stainingshrinkagevolumesoft tissuebonespecimen preparationlight-sheet microscopyX-ray tomography
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 4 , August 2014 , pp. 1208 - 1217
Copyright
© Microscopy Society of America 2014 

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References

Aja-Fernandez, S., Vegas-Sanchez-Ferrero, G. & Martin Fernandez, M.a. (2010). Soft thresholding for medical image segmentation. IEEE Eng Med Biol Soc Conf 2010, 4752–4755.Google ScholarPubMed
Bussolati, G. (1978). A fixation-decalcification procedure for bone biopsies. Histopathology 2(5), 329–334.CrossRefGoogle ScholarPubMed
Buytaert, J.A.N., Descamps, E., Adriaens, D. & Dirckx, J.J.J. (2012). The OPFOS microscopy family: High-resolution optical sectioning of biomedical specimens. Anat Res Int 2012, 1–9.CrossRefGoogle ScholarPubMed
Buytaert, J.A.N. & Dirckx, J.J.J. (2007). Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution. J Biomed Opt 12(1), 014039.CrossRefGoogle ScholarPubMed
Buytaert, J.A.N. & Dirckx, J.J.J. (2009). Tomographic imaging of macroscopic biomedical objects in high resolution and three dimensions using orthogonal-plane fluorescence optical sectioning. Appl Opt 48(5), 941–948.CrossRefGoogle ScholarPubMed
Buytaert, J.A.N., Johnson, S.B., Dierick, M., Salih, W.H.M. & Santi, P.A. (2013). MicroCT versus sTSLIM 3D imaging of the mouse cochlea. J Histochem Cytochem 61(5), 372–385.CrossRefGoogle ScholarPubMed
Buytaert, J.A.N., Salih, W.H.M., Dierick, M., Jacobs, P., Dirckx, J.J.J. & Structures, T. (2011). Realistic 3D computer model of the gerbil middle ear, featuring accurate morphology of bone and soft tissue structures. J Assoc Res Otolaryngol 12(6), 681–696.CrossRefGoogle ScholarPubMed
Christie, W. (1993). Preparation of lipid extracts from tissues. In Advances in Lipid Methodology , Christie, W. (Ed.), pp. 195–213. Dundee, Schotland: Oily Press.Google Scholar
Cunningham, M. (2000). Shrinkage of inland silverside larvae preserved in ethanol and formalin. North Am J Fish Manage 20, 816–818.2.3.CO;2>CrossRefGoogle Scholar
D’Arceuil, H. & de Crespigny, A. (2007). The effects of brain tissue decomposition on diffusion tensor imaging and tractography. NeuroImage 36(1), 64–68.CrossRefGoogle ScholarPubMed
Degenhardt, K. & Wright, A. (2010). Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ Cardiovasc Imaging 3(3), 314–322.CrossRefGoogle ScholarPubMed
Descamps, E., Buytaert, J., Kegel, B.D., Dirckx, J., Adriaens, D. & De Kegel, B. (2012). A qualitative comparison of 3D visualization in Xenopus laevis using a traditional method and a non-destructive method. Belgian J Zool 142(2), 99–111.Google Scholar
Dodt, H.-U., Leischner, U., Schierloh, A., Jahrling, N., Mauch, C.P., Deininger, K. & Becker, K. (2007). Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4(4), 331–336.CrossRefGoogle ScholarPubMed
Faulwetter, S., Dailianis, T., Vasileiadou, A. & Arvanitidis, C. (2013). Investigation of contrast enhancing techniques for the application of Micro-CT in marine biodiversity studies. In Bruker-microCT Annual User Meeting, pp. 12–20.Google Scholar
Ferguson, S.J., Bryant, J.T. & Ito, K. (1999). Three-dimensional computational reconstruction of mixed anatomical tissues following histological preparation. Med Eng Phys 21(2), 111–117.CrossRefGoogle ScholarPubMed
Fox, C.H., Johnson, F.B., Whiting, J. Roller, P.P. (1985). Formaldehyde fixation. J Histochem Cytochem 33(8), 845–853.CrossRefGoogle ScholarPubMed
Gea, S.L.R., Decraemer, W.F. & Dirckx, J.J.J. (2005). Region of interest micro-CT of the middle ear: A practical approach. J X-Ray Sci Technol 13(3), 137–147.Google Scholar
Gignac, P. & Kley, N. (2013). Methodological refinements to using Lugol’s iodine as a contrast agent in X-ray micro-CT imaging. In Society for Integrative and Compartive Biology Annual Meeting, p. 133.Google Scholar
Goyens, J., Dirckx, J., Dierick, M., Van Hoorebeke, L. & Aerts, P. (2014 a). Biomechanical determinants of bite force dimorphism in Cyclommatus metallifer stag beetles J Exp Biol 217, 1065–1071.CrossRefGoogle ScholarPubMed
Goyens, J., Soons, J., Aerts, P., Dierick, M. & Dirckx, J. (2014 b). Unraveling the biomechanics of stag beetle armature using microcomputed X-ray tomography. Microscopy and Analysis, March, 19–22.Google Scholar
Huisken, J., Swoger, J., Bene, D., Wittbrodt, J. & Stelzer, E.H. (2004). Live embryos by selective plane illumination microscopy. Science 305(August), 1007–1009.CrossRefGoogle ScholarPubMed
Jonmarker, S., Valdman, A., Lindberg, A., Hellström, M. & Egevad, L. (2006). Tissue shrinkage after fixation with formalin injection of prostatectomy specimens. Virchows Archiv 449(3), 297–301.CrossRefGoogle ScholarPubMed
Keller, C. & Bahadur, A. (2009). Process and apparatus for microCT imaging of ex vivo specimens. US patent 2009/0080600 A1.Google Scholar
Kushida, H. (1962). A study of cellular swelling and shrinkage during fixation, dehydration and embedding in various standard media. J Electron Microsc (Tokyo) 11(3), 135–138.Google Scholar
Lane, J., Rális, Z.A. & Ráliš, Z. (1983). Changes in dimensions of large cancellous bone specimens during histological preparation as measured on slabs from human femoral heads. Calcif Tissue Int 35(1), 1–4.CrossRefGoogle ScholarPubMed
Lessells, C. & Boag, P. (1987). Unrepeatable repeatabilities: A common mistake. The Auk 2(January), 116–121.CrossRefGoogle Scholar
Maciá-Botejara, E., Morán-Penco, J.M., Espín-Jaime, M.T., Botello-Martínez, F., Salas-Martínez, J., Caballero-Loscos, M.J. & Molina-Fernández, M. (2013). Brain lipid composition in rabbits after total parenteral nutrition with two different lipid emulsions. Nutrition 29(1), 313–317.CrossRefGoogle ScholarPubMed
Martinez, P.a., Berbel-Filho, W.M. & Jacobina, U.P. (2013). Is formalin fixation and ethanol preservation able to influence in geometric morphometric analysis? Fishes as a case study. Zoomorphology 132, 87–93.CrossRefGoogle Scholar
Metscher, B.D. (2009 a). MicroCT for comparative morphology: Simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol 9(11), 1–14.CrossRefGoogle ScholarPubMed
Metscher, B.D. (2009 b). MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions. Dev Dyn 238(3), 632–640.CrossRefGoogle ScholarPubMed
Moore, J., Aros, M., Steudel, K. & Cheng, K. (2002). Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis. Biotechniques 32(2), 2–4.CrossRefGoogle ScholarPubMed
Nadeau, J. (2009). Preservation causes shrinkage in seahorses: Implications for biological studies and for managing sustainable trade with minimum size limits. Aquatic Conserv Mar Freshw Ecosyst 19(2009), 428–438.CrossRefGoogle Scholar
Pauwels, E., Van Loo, D., Cornillie, P., Brabant, L. & Van Hoorebeke, L. (2013). An exploratory study of contrast agents for soft tissue visualization by means of high resolution X-ray computed tomography imaging. J Microsc 250, 21–31.CrossRefGoogle ScholarPubMed
Rau, T.S., Würfel, W., Lenarz, T. & Majdani, O. (2013). Three-dimensional histological specimen preparation for accurate imaging and spatial reconstruction of the middle and inner ear. Int J Comp Assist Radiol Surg 8(4), 481–509.CrossRefGoogle ScholarPubMed
Rostgaard, J. & Tranum-Jensen, J. (1980). A procedure for minimizing cellular shrinkage in electron microscope preparation: A quantitative study on frog gall bladder. J Microsc 119(2), 213–232.CrossRefGoogle Scholar
Rown, M.A.B., Eed, R.B.R. & Enry, R.W.H. (2002). Effects of dehydration mediums and temperature on total dehydration time and tissue shrinkage. J Int Soc Plastination 17, 28–33.Google Scholar
Salih, W., Buytaert, J.A.N., Aerts, J.R.M., Vanderniepen, P., Dierick, M. & Dirckx, J.J.J. (2012). Open access high-resolution 3D morphology models of cat, gerbil, rabbit, rat and human ossicular chains. Hearing Res 284, 1–5.CrossRefGoogle ScholarPubMed
Santi, P.A. (2011). Light sheet fluorescence microscopy: a review. J Histochem Cytochem 59(2), 129–138.CrossRefGoogle ScholarPubMed
Sonka, M., Boyle, R. & Hlavac, V. (1999). Image Processing, Analysis, and Machine Vision (p. 866). Stamford, CT, USA: Cengage Learning.Google Scholar
Spalteholz, W. (1911). Über das Durchsichtigmachen von menschlichen und tierischen Präparaten (p. 48). Leipzig, Germany: Verlag Hirzel.Google Scholar
Van der Jeught, S. & Dirckx, J.J.J. (2013). Full-field thickness distribution of human tympanic membrane obtained with optical coherence tomography. J Assoc Res Otolaryngol 14(4), 483–494.CrossRefGoogle ScholarPubMed
Voie, A.H. (2002). Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy. Hearing Res 171(1–2), 119–128.CrossRefGoogle ScholarPubMed
Voie, A.H., Burns, D.H. & Spelman, F.A. (1993). Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens. J Microsc 170(3), 229–236.CrossRefGoogle ScholarPubMed
Vourazeris, J.D., Lawless, M.W., Markert, R.J., Stills, H.F. & Boivin, G.P. (2013). Semitendinosus muscle fatty infiltration following tendon harvest in rabbits. J Orthop Res 31(8), 1234–1239.CrossRefGoogle ScholarPubMed