Research Papers: Imaging

Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses

[+] Author Affiliations
Kai Kuetemeyer

Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany

Andrea Lucas-Hahn, Bjoern Petersen, Erika Lemme, Petra Hassel, Heiner Niemann

Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics, Hoeltystrasse 10, 31535 Neustadt, Germany

Alexander Heisterkamp

Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany

J. Biomed. Opt. 15(4), 046006 (July 21, 2010). doi:10.1117/1.3463012
History: Received December 03, 2009; Revised April 09, 2010; Accepted May 19, 2010; Published July 21, 2010; Online July 21, 2010
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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 micro-manipulation 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.

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© 2010 Society of Photo-Optical Instrumentation Engineers

Citation

Kai Kuetemeyer ; Andrea Lucas-Hahn ; Bjoern Petersen ; Erika Lemme ; Petra Hassel, et al.
"Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses", J. Biomed. Opt. 15(4), 046006 (July 21, 2010). ; http://dx.doi.org/10.1117/1.3463012


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