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.

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

“…Thus we conclude that the extent of damage is strongly related to the size of the melanocyte. Therefore, protocols that involve introducing an absorbing dye or marker should account for this dependence on area [25].…”

“…Also notable is the ability of fs laser insults to kill with single cell accuracy. The utility of fs lasers has been demonstrated in chicks, pigs, zebrafish, Drosophila , and C. elegans , [22], [23], [24], [25], [26], [27]. However, despite the importance of Xenopus as an experimental system with an important history of ablation experiments, laser ablation has not been tried on these embryos.…”

Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery

“…Selective disruption of individual mitochondria can be used to study their role in energy production, cell death and subcellular homeostasis [4,14]. Recently, automated ablation of entire metaphase plates in porcine oocytes has been published by our group [15]. This method may serve as an alternative for conventional highly-invasive enucleation techniques of somatic cell cloning.…”

“…As the contrast between subcellular structures is usually very weak in all of these applications, the organelle of interest has to be stained with fluorescent dyes or tagged with fluorescent proteins. Therefore, intracellular nanosurgery is often combined with fluorescence [11], confocal laser scanning [4,13,14] or multiphoton microscopy [12,15]. …”

Continuous enucleation of bovine oocyte by microrobot with local flow distribution control

“…Especially the oocyte enucleation technique is a primary study for cloning process; therefore this topic attracted many studies. The conventional techniques of the enucleation process mainly include manual manipulator operation, chemical treatment methods and femtosecond laser pulses techniques [1][2][3]. However, these methods tend to have problems of low success rate, low repeatability and contamination to the cell, for the chemical treatment may cause an unaware toxicity to the cell.…”