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Proceedings Paper

Functional enucleation of porcine oocytes for somatic cell nuclear transfer using femtosecond laser pulses
Author(s): K. Kuetemeyer; A. Lucas-Hahn; B. Petersen; P. Hassel; E. Lemme; H. Niemann; A. Heisterkamp
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Paper Abstract

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer over the last decade. However, this method still results in very low efficiencies originating from biological and technical aspects. The highly-invasive mechanical enucleation belongs to the technical aspects and requires considerable micromanipulation skill. In this paper, we present a novel non-invasive 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 determined the metaphase plate position and shape. Subsequent irradiation of this volume with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation. We show that functional fs laser-based enucleation of porcine oocytes completely inhibited further embryonic development while maintaining intact oocyte morphology. In contrast, non-irradiated oocytes were able to develop to the blastocyst stage without significant differences to control oocytes. Our results indicate that fs laser systems offer great potential for oocyte imaging and enucleation as a fast, easy to use and reliable tool which may improve the efficiency of somatic cell clone production.

Paper Details

Date Published: 25 February 2010
PDF: 6 pages
Proc. SPIE 7589, Frontiers in Ultrafast Optics: Biomedical, Scientific, and Industrial Applications X, 75890A (25 February 2010); doi: 10.1117/12.840045
Show Author Affiliations
K. Kuetemeyer, Laser Zentrum Hannover e.V. (Germany)
A. Lucas-Hahn, Friedrich-Loeffler-Institute (Germany)
B. Petersen, Friedrich-Loeffler-Institute (Germany)
P. Hassel, Friedrich-Loeffler-Institute (Germany)
E. Lemme, Friedrich-Loeffler-Institute (Germany)
H. Niemann, Friedrich-Loeffler-Institute (Germany)
A. Heisterkamp, Laser Zentrum Hannover e.V. (Germany)


Published in SPIE Proceedings Vol. 7589:
Frontiers in Ultrafast Optics: Biomedical, Scientific, and Industrial Applications X
Alexander Heisterkamp; Joseph Neev; Stefan Nolte; Rick P. Trebino, Editor(s)

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