In the fertilization and early embryonic development process, various events are spatiotemporally controlled, and many events are connected in the cause-effect relations toward the final goal of ontogenesis. To understand the mechanism of this process, conventional experimental techniques by fixing and destruction of the cells have limitations. If this process can be observed over time and the development process can be continued after the observation, it will open a new era in the Genetics research. A mammalian developmental biology researcher, Dr. Kazuo Yamagata, established such technique by using the CSU system.
He successfully imaged mouse embryos over a long period of time, from the post-fertilization through to the blastocyst stage, to acquire approximately 60,000 of 3D confocal images. Thereafter, the embryos were transferred to a recipient mouse, and the pups were born all normally, grew healthy, and were capable of reproduction; a firm evidence that this early embryo imaging technique does not adversely affect the process of full-term development. The high speed image acquisition and extremely low excitation light unique for the CSU system enabled greatly reduced phototoxicity and realized intensive but damage-free long time observation. Only by using this technology which does no harm on the embryonic development, it is possible to “utilize the same embryo after intensive analysis by imaging” , and thus to investigate cause- and-effect relationship of various early stage phenomena and their influence on the development.
Figure : The long-time, multi-dimensional live cell imaging on early stage embryos does not affect the process of ontogenesis.
(a) Experimental flow
(b) Movie example: Images were acquired at 7.5-minute intervals over approximately 70 hours.
This figure shows extracted images at 2-hour intervals.
Each image is the maximum intensity projection of a total of 51 images in the Z-axis direction.
Green：Spindle (EGFP-α‒tubulin), Red：Nucleus (H2B-mRFP1)
| Experimental conditions | |
| Total time | 70 hours |
| Interval | 7.5 min/stack |
| Z-axis slices | 51 sections (at 2μm intervals) |
| Channel | 3(DIC, EGFP, mRFP1) |
| Position | 6(Total 72 embryos) |
| Laser power(Measured at objective lens) | 488nm (0.281 mW), 561nm (0.225 mW) |
Data: Kazuo Yamagata, PhD., Laboratory for Genomic Reprogramming,Center for Developmental Biology, Riken
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