title: Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses.
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.
authors.affiliation: Laser Zentrum Hannover eV, Hannover, Germany. k.kuetemeyer@lzh.de
authors.forename: Kai
authors.initials: K
authors.lastname: Kuetemeyer
authors.forename: Andrea
authors.initials: A
authors.lastname: Lucas-Hahn
authors.forename: Bjoern
authors.initials: B
authors.lastname: Petersen
authors.forename: Erika
authors.initials: E
authors.lastname: Lemme
authors.forename: Petra
authors.initials: P
authors.lastname: Hassel
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000295:instrumentation*; D058948:Cell Tracking / Q000295:instrumentation*; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D007834:Lasers; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D053652:Nuclear Transfer Techniques / Q000295:instrumentation*; D009865:Oocytes / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects; D010363:Pattern Recognition, Automated / Q000379:methods; D013552:Swine; D018511:Systems Integration