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Confocal Imaging of the Embryonic Heart: How Deep?

  • Christine E. Miller (a1), Robert P. Thompson (a2), Michael R. Bigelow (a2), George Gittinger (a2), Thomas C. Trusk (a2) and David Sedmera (a2)...

Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 μm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 μm was achieved with a 10× dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5× objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.

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Germroth, P.G., Gourdie, R.G., & Thompson, R.P. (1995). Confocal microscopy of thick sections from acrylamide gel embedded embryos. Microsc Res Tech30, 513520.

Hamburger, V. & Hamilton, H.L. (1951). A series of normal stages in the development of the chick embryo. J Morphol88, 4992.

Kolker, S.J., Tajchman, U., & Weeks, D.L. (2000). Confocal imaging of early heart development in Xenopus laevis. Dev Biol218, 6473.

Laan, A.C., Lamers, W.H., Huijsmans, D.P., Te Kortschot, A., Smith, J., Strackee, J., & Los, J.A. (1989). Deformation-corrected computer-aided three-dimensional reconstruction of immunohistochemically stained organs: Application to the rat heart during early organogenesis. Anat Rec224, 443457.

Omens, J.H. & Fung, Y.C. (1990). Residual strain in rat left ventricle. Circ Res66, 3745.

Sedmera, D., Pexieder, T., Hu, N., & Clark, E.B. (1997). Developmental changes in the myocardial architecture of the chick. Anat Rec248, 421432.

Sedmera, D., Pexieder, T., Hu, N., & Clark, E.B. (1998). A quantitative study of the ventricular myoarchitecture in the stage 21–29 chick embryo following decreased loading. Eur J Morphol36, 105119.

Sedmera, D., Pexieder, T., Rychterova, V., Hu, N., & Clark, E.B. (1999). Remodeling of chick embryonic ventricular myoarchitecture under experimentally changed loading conditions. Anat Rec254, 238252.

Sedmera, D., Pexieder, T., Vuillemin, M., Thompson, R.P., & Anderson, R.H. (2000). Developmental patterning of the myocardium. Anat Rec258, 319337.

Sedmera, D., Reckova, M., DeAlmeida, A., Coppen, S.R., Kubalak, S.W., Gourdie, R.G., & Thompson, R.P. (2003). Spatiotemporal pattern of commitment to slowed proliferation in the embryonic mouse heart indicates progressive differentiation of the cardiac conduction system. Anat Rec274A, 773777.

Wong, Y.M., Thompson, R.P., Cobb, L., & Fitzharris, T.P. (1983). Computer reconstruction of serial sections. Comput Biomed Res16, 580586.

Zucker, R.M., Hunter, S., & Rogers, J.M. (1998). Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry33, 348354.

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Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
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