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Journal of Biomedical Optics • Open Access

Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses
Author(s): Kai Kuetemeyer; Andrea Lucas-Hahn; Björn Petersen; Erika Lemme; Petra Hassel; Heiner Niemann; Alexander Heisterkamp

Paper Abstract

Since the birth of "Dolly" as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

Paper Details

Date Published: 1 July 2010
PDF: 8 pages
J. Biomed. Opt. 15(4) 046006 doi: 10.1117/1.3463012
Published in: Journal of Biomedical Optics Volume 15, Issue 4
Show Author Affiliations
Kai Kuetemeyer, Laser Zentrum Hannover e.V. (Germany)
Andrea Lucas-Hahn, Friedrich-Loeffler-Institut (Germany)
Björn Petersen, Friedrich-Loeffler-Institut (Germany)
Erika Lemme, Friedrich-Loeffler-Institut (Germany)
Petra Hassel, Friedrich-Loeffler-Institut (Germany)
Heiner Niemann, Friedrich-Loeffler-Institut (Germany)
Alexander Heisterkamp, Laser Zentrum Hannover e.V. (Germany)

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