Published online by Cambridge University Press: 17 April 2014
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.
Monique Y. Rennie: Mouse Imaging Centre, Toronto Centre for Phenogenomics, 25 Orde Street, Toronto, ON, Canada, M5T 3H7.Kent L. Thornburg: Center for Health and Healing, Oregon Health and Science University, 3303 SW Bond Avenue, Mail Code CH15H, Portland, OR 97239, USA.Claudia S. López: Oregon Health & Sciences University, 3181 SW Sam Jackson Park Road, R. T Jones Hall for Basic Sciences, Room 6369, Mail Code L220, Portland, OR 97239, USA.Curran G. Gahan: Oregon State University, SW Jefferson Way, Corvallis, OR 97331, USA.Sandra Rugonyi: 3303 SW Bond Avenue M/C: CH13B, Portland, OR 97239, USA.
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