Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm2 (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm2 (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.
Although femtosecond laser cell surgery is widely used for fundamental research in cell biology, the mechanisms in
the so-called low-density plasma regime are largely unknown. To date, it is still unclear on which time scales free
electron and free radical-induced chemical effects take place leading to intracellular ablation. In this paper, we
present our experimental study on the influence of laser parameters and staining on the ablation threshold. We
found that the ablation effect resulted from the accumulation of single-shot multiphoton-induced photochemical
effects finished within a few nanoseconds. In addition, fluorescence staining of subcellular structures significantly
decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for
providing seed electrons for the ionization cascade.
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.
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.
Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.
Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years.
However, this method still results in very low efficiencies around 1% which originate from suboptimal culture
conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell using micromanipulators.
In this paper, we present a new minimal invasive method for oocyte imaging and enucleation based
on the application of femtosecond (fs) laser pulses. After imaging of the oocyte with multiphoton microscopy,
ultrashort pulses are focused onto the metaphase plate of MII-oocytes in order to ablate the DNA molecules.
We show that fs laser based enucleation of porcine oocytes completely inhibits the first mitotic cleavage after
parthenogenetic activation while maintaining intact oocyte morphology in most cases. In contrast, control
groups without previous irradiation of the metaphase plate are able to develop to the blastocyst stage. Further
experiments have to clarify the suitability of fs laser based enucleated oocytes for SCNT.
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