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Less embryo phototoxicity with spinning disk/EMCCD microscopy setup

Electron multiplying (EM) CCD cameras have helped spinning disk confocal microscopy find more use because the cameras are sensitive enough to detect the low levels of light produced by the microscopy method. For living specimens, this type of microscopy brings the benefits of reducing phototoxicity during the repeated exposures used to acquire time-lapse images or z-axis slices.

Researchers at RIKEN-Kobe in Japan recently used this win-win technology pairing to image mouse embryos. They published a paper in the Journal of Reproduction and Development describing how their methods decreased phototoxicity so much that preimplantation mouse embryos could undergo long-term time-lapse imaging, be implanted, and then survive full-term.

They used an Olympus inverted microscope with a Nipkow disk confocal module and an Andor EMCCD camera. A CVI Melles Griot 70 mW agron-krypton laser provided the confocal light source. The mouse embryos were injected with a mixture of mRNAs encoding for enhanced GFP coupled to alpha-tublin and monomeric RFP fused to histone H2B. At specific time points, the researchers acquired 51 images in the z-axis with 488 and 561 nm excitation. Imaging took place for 70 hours.

Although the setup lessened light exposure, the researchers found that the interval between observations was another important factor -- an interval of 7.5 min. increased embryo viability greatly over 3.75 minutes. They also found that continuous light exposure was worse than repeated intermittent excitation, even when the total exposure was equal.

This microscopy method could aid study of development and assistive reproductive techniques in mammals. For example, it could help reveal why some assistive reproductive techniques (such as in vitro fertilization) work better than others, and it might be used to identify healthy embryos prior to implantation.

In the paper, the researchers pointed out that the mechanisms behind phototoxicity are still not completely understood and that their findings regarding phototoxicity imply that embryos may be able to neutralize toxicity. To find out more about this topic, I am going to compile some of the latest research on photoxicity.

Are you performing research in the area of phototoxicity, or do you know of a good resource?

Image: These time-lapse images show spindle (green) and nuclear (red) dynamics during preimplantation embryo development. Courtesy of Kazuo Yamagata, RIKEN-Kobe.

Note: If you want to know more about EMCCDs, Photometrics is holding webinar covering this topic on Friday April 3:



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