TY - JOUR

T1 - Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses

AU - Kuetemeyer, Kai

AU - Lucas-Hahn, Andrea

AU - Petersen, Bjoern

AU - Lemme, Erika

AU - Hassel, Petra

AU - Niemann, Heiner

AU - Heisterkamp, Alexander

N1 - Funding information: We thank APE GmbH for providing us with the autocorrelator CARPE. This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Cluster of Excellence “REBIRTH” (From Regenerative Biology to Reconstructive Therapy).

PY - 2010/7/1

Y1 - 2010/7/1

N2 - 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.

AB - 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.

KW - Cell surgery

KW - Femtosecond laser

KW - Multiphoton microscopy

KW - Oocyte enucleation

KW - SCNT

KW - Somatic cell nucleartransfer

UR - http://www.scopus.com/inward/record.url?scp=79952201066&partnerID=8YFLogxK

U2 - 10.1117/1.3463012

DO - 10.1117/1.3463012

M3 - Article

C2 - 20799808

AN - SCOPUS:79952201066

VL - 15

JO - Journal of biomedical optics

JF - Journal of biomedical optics

SN - 1083-3668

IS - 4

M1 - 046006

ER -