Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development
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概要
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Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which `out-of focus blur cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research.
- 2009-06-01
著者
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若山 照彦
理化学研究所神戸研究所発生・再生科学総合研究センター
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若山 照彦
理化学研究所発生・再生科学総合研究センターゲノム・リプログラミング研究チーム
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若山 照彦
理化学研究所神戸研究所発生・再生科学総合研究センターゲノム・リプログラミング研究チーム
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若山 照彦
理化学研究所 発生・再生科学総合研究センター
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若山 照彦
神戸大学 大学院・自然科学研究科
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若山 照彦
理化学研究所発生再生科学総合研究センター
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Wakayama Teruhiko
Center For Developmental Biology Riken Kobe
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WAKAYAMA Teruhiko
RIKEN Center for Developmental Biology
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Yamagata Kazuo
Laboratory For Genomic Reprogramming Center For Developmental Biology
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Wakayama Teruhiko
Laboratory For Genomic Reprogramming Center For Developmental Biology
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Wakayama Teruhiko
Laboratory For Genomic Reprogramming Riken Center For Developmental Biology
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Wakayama Teruhiko
Laboratory For Genomic Reprogramming Center For Developmental Biology Riken
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Wakayama Teruhiko
Laboratory For Genomic Reprogramming Center For Developmental Biology Riken Kobe Minatojima-minamima
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Wakayama Teruhiko
Department Of Veterinary Anatomy Faculty Of Agriculture The University Of Tokyo
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SUETSUGU Rinako
Laboratory for Genomic Reprogramming, Center for Developmental Biology
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Suetsugu Rinako
Laboratory For Genomic Reprogramming Center For Developmental Biology
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