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Confocal Imaging of the Embryonic Heart: How Deep?

Published online by Cambridge University Press:  12 May 2005

Christine E. Miller ,
Robert P. Thompson ,
Michael R. Bigelow ,
George Gittinger ,
Thomas C. Trusk  and
David Sedmera
Christine E. Miller
Affiliation:
Department of Mechanical Engineering, Bucknell University, Lewisburg, PA 17837, USA
Robert P. Thompson
Affiliation:
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
Michael R. Bigelow
Affiliation:
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
George Gittinger
Affiliation:
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
Thomas C. Trusk
Affiliation:
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
David Sedmera
Affiliation:
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
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Abstract

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.

Keywords

chickmouseembryocardiac3D reconstructionwhole-mount immunohistochemistryethanolretinoic acidteratologydevelopment
Type
Research Article
Information
Microscopy and Microanalysis , Volume 11 , Issue 3 , June 2005 , pp. 216 - 223
Copyright
© 2005 Microscopy Society of America

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References

REFERENCES

Germroth, P.G., Gourdie, R.G., & Thompson, R.P. (1995). Confocal microscopy of thick sections from acrylamide gel embedded embryos. Microsc Res Tech 30, 513–520.Google Scholar
Hamburger, V. & Hamilton, H.L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88, 49–92.Google Scholar
Kolker, S.J., Tajchman, U., & Weeks, D.L. (2000). Confocal imaging of early heart development in Xenopus laevis. Dev Biol 218, 64–73.Google Scholar
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, 443–457.Google Scholar
Omens, J.H. & Fung, Y.C. (1990). Residual strain in rat left ventricle. Circ Res 66, 37–45.Google Scholar
Sedmera, D., Pexieder, T., Hu, N., & Clark, E.B. (1997). Developmental changes in the myocardial architecture of the chick. Anat Rec 248, 421–432.Google Scholar
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, 105–119.Google Scholar
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, 238–252.Google Scholar
Sedmera, D., Pexieder, T., Vuillemin, M., Thompson, R.P., & Anderson, R.H. (2000). Developmental patterning of the myocardium. Anat Rec 258, 319–337.Google Scholar
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, 773–777.Google Scholar
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. 157–176. 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, 181–193.Google Scholar
Wong, Y.M., Thompson, R.P., Cobb, L., & Fitzharris, T.P. (1983). Computer reconstruction of serial sections. Comput Biomed Res 16, 580–586.Google Scholar
Zucker, R.M., Hunter, S., & Rogers, J.M. (1998). Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33, 348–354.Google Scholar