Skip to main content
    • Aa
    • Aa

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.

Corresponding author
Corresponding author. E-mail:
Hide All


Germroth, P.G., Gourdie, R.G., & Thompson, R.P. (1995). Confocal microscopy of thick sections from acrylamide gel embedded embryos. Microsc Res Tech 30, 513520.
Hamburger, V. & Hamilton, H.L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88, 4992.
Kolker, S.J., Tajchman, U., & Weeks, D.L. (2000). Confocal imaging of early heart development in Xenopus laevis. Dev Biol 218, 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 Rec 224, 443457.
Omens, J.H. & Fung, Y.C. (1990). Residual strain in rat left ventricle. Circ Res 66, 3745.
Sedmera, D., Pexieder, T., Hu, N., & Clark, E.B. (1997). Developmental changes in the myocardial architecture of the chick. Anat Rec 248, 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 Morphol 36, 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 Rec 254, 238252.
Sedmera, D., Pexieder, T., Vuillemin, M., Thompson, R.P., & Anderson, R.H. (2000). Developmental patterning of the myocardium. Anat Rec 258, 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 Rec 274A, 773777.
Thompson, R.P., Reckova, M., DeAlmeida, A., Bigelow, M., Stanley, C.P., Spruill, J.B., Trusk, T., & Sedmera, D. (2003). The oldest, toughest cells in the heart. In Development of the Cardiac Conduction System, Chadwick, D.J. & Goode, J. (Eds), pp. 157176. Chichester, UK: Wiley.
Vuillemin, M., Pexieder, T., Wong, Y.M., & Thompson, R.P. (1992). A two-step alignment method for 3D computer-aided reconstruction based on fiducial markers and applied to mouse embryonic hearts. Eur J Morphol 30, 181193.
Wong, Y.M., Thompson, R.P., Cobb, L., & Fitzharris, T.P. (1983). Computer reconstruction of serial sections. Comput Biomed Res 16, 580586.
Zucker, R.M., Hunter, S., & Rogers, J.M. (1998). Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33, 348354.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 16
Total number of PDF views: 49 *
Loading metrics...

Abstract views

Total abstract views: 224 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 19th October 2017. This data will be updated every 24 hours.