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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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