TY - JOUR
T1 - 3D imaging of the early embryonic chicken heart with focused ion beam scanning electron microscopy
AU - Rennie, Monique Y.
AU - Gahan, Curran G.
AU - López, Claudia S.
AU - Thornburg, Kent L.
AU - Rugonyi, Sandra
PY - 2014/8
Y1 - 2014/8
N2 - Early embryonic heart development is a period of dynamic growth and remodeling, with rapid changes occurring at the tissue, cell, and subcellular levels. A detailed understanding of the events that establish the components of the heart wall has been hampered by a lack of methodologies for three-dimensional (3D), high-resolution imaging. Focused ion beam scanning electron microscopy (FIB-SEM) is a novel technology for imaging 3D tissue volumes at the subcellular level. FIB-SEM alternates between imaging the block face with a scanning electron beam and milling away thin sections of tissue with a FIB, allowing for collection and analysis of 3D data. FIB-SEM was used to image the three layers of the day 4 chicken embryo heart: myocardium, cardiac jelly, and endocardium. Individual images obtained with FIB-SEM were comparable in quality and resolution to those obtained with transmission electron microscopy. Up to 1,100 serial images were obtained in 4 nm increments at 4.88 nm resolution, and image stacks were aligned to create volumes 800-1,500 μm3 in size. Segmentation of organelles revealed their organization and distinct volume fractions between cardiac wall layers. We conclude that FIB-SEM is a powerful modality for 3D subcellular imaging of the embryonic heart wall.
AB - Early embryonic heart development is a period of dynamic growth and remodeling, with rapid changes occurring at the tissue, cell, and subcellular levels. A detailed understanding of the events that establish the components of the heart wall has been hampered by a lack of methodologies for three-dimensional (3D), high-resolution imaging. Focused ion beam scanning electron microscopy (FIB-SEM) is a novel technology for imaging 3D tissue volumes at the subcellular level. FIB-SEM alternates between imaging the block face with a scanning electron beam and milling away thin sections of tissue with a FIB, allowing for collection and analysis of 3D data. FIB-SEM was used to image the three layers of the day 4 chicken embryo heart: myocardium, cardiac jelly, and endocardium. Individual images obtained with FIB-SEM were comparable in quality and resolution to those obtained with transmission electron microscopy. Up to 1,100 serial images were obtained in 4 nm increments at 4.88 nm resolution, and image stacks were aligned to create volumes 800-1,500 μm3 in size. Segmentation of organelles revealed their organization and distinct volume fractions between cardiac wall layers. We conclude that FIB-SEM is a powerful modality for 3D subcellular imaging of the embryonic heart wall.
KW - FIB-SEM
KW - cardiac outflow tract
KW - embryonic heart development
KW - focused ion beam
KW - scanning electron microscopy
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UR - http://www.scopus.com/inward/citedby.url?scp=84905236118&partnerID=8YFLogxK
U2 - 10.1017/S1431927614000828
DO - 10.1017/S1431927614000828
M3 - Article
C2 - 24742339
AN - SCOPUS:84905236118
SN - 1431-9276
VL - 20
SP - 1111
EP - 1119
JO - Microscopy and Microanalysis
JF - Microscopy and Microanalysis
IS - 4
ER -