Microscopy and Microanalysis


Special Issue: Cardiovascular Development and Disease

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 c1
a1 Department of Mechanical Engineering, Bucknell University, Lewisburg, PA 17837, USA
a2 Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA

Article author query
miller ce   [PubMed][Google Scholar] 
thompson rp   [PubMed][Google Scholar] 
bigelow mr   [PubMed][Google Scholar] 
gittinger g   [PubMed][Google Scholar] 
trusk tc   [PubMed][Google Scholar] 
sedmera d   [PubMed][Google Scholar] 

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 [mu]m. 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 [mu]m 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.

(Received March 9 2004)
(Accepted October 28 2004)


Key Words: chick; mouse; embryo; cardiac; 3D reconstruction; whole-mount immunohistochemistry; ethanol; retinoic acid; teratology; development.

Correspondence:
c1 Corresponding author. E-mail: sedmerad@musc.edu