I. V. Ilina

Laser microsurgery of cells by femtosecond laser scalpel and optical tweezers

This paper provides a brief overview of the current state of research in the field of laser microsurgery of preimplantation embryos. The combined system femtosecond laser tweezers-scalpel developed in JIHT RAS is described. A scheme of device and peculiarities of functioning its nodes as well as the ground for the choice of their parameters are presented. The results of the development of methodology for noncontact polar body biopsy of the preimplantation embryo are also presented to demonstrate the potentials of the combined multi-purpose system femtosecond laser tweezers-scalpel.

Application of femtosecond laser scalpel and optical tweezers for noncontact biopsy of late preimplantation embryos

We demonstrate that one of the key steps of preimplantation genetic diagnosis called embryo biopsy can be successfully performed in noncontact mode by means of femtosecond laser scalpel and optical tweezers. Embryo biopsy was carried out in late-stage mouse preimplantation embryos. Femtosecond laser pulses were applied to detach the desired amount of trophectoderm cells from the blastocyst, while the optical tweezers trapped the cells and moved them out of the embryo. The parameters of laser radiation were optimized so as to efficiently perform embryo biopsy and preserve the viability of the treated embryos.

Application of femtosecond laser pulses in biomedical cell technologies

The results are presented of the works in the field of development of equipment, investigation techniques, and technologies for biology and medicine performed in the Joint Institute for High Temperatures of the Russian Academy of Scienses (JIHT RAS). On the base of the new generation infrared femtosecond lasers, the experimental models are developed and manufactured of laser tweezers, scalpel, and the “tweezers-scalpel” combined system. The results are presented of the experimental studies on the noncontact mammalian cell fusion (blastomeres of mouse embryos on day 1.5 of development) by means of the femtosecond laser pulses.

Biomedical and biotechnology applications of noncontact femtosecond laser microsurgery of living cells

We employed femtosecond (fs) laser pulses to solve important biomedical and biotechnology problems, embryo biopsy and cell fusion respectively. We report on the results of fully contactless laser-mediated polar body (PB) and trophectoderm (TE) biopsy of early mammalian embryos. In the former case the fs laser scalpel (Cr:Forsterite seed oscillator and a regenerative amplifier, 100 fs, 10 Hz) was initially used to drill an opening in the outer covering of the embryo, and then the PB was extracted out of the zygote by means of optical tweezers (cw fiber laser, 1064 nm). In the latter case the laser scalpel was employed to dissect 5-7 TE cells that had just left the zona pellucida (ZP) during the hatching. The energy of laser pulses was thoroughly optimized to prevent cell damage and provide high viability of treated cells. Morphological and fluorescent analysis showed that femtosecond laser-based embryo biopsy did not compromise further in vitro embryo development. We also demonstrated the possibilities of ...

Femtosecond Laser Assisted Hatching: Dependence of Zona Pellucida Drilling Efficiency and Embryo Development on Laser Wavelength and Pulse Energy

Ultrashort laser pulses have enabled highly precise and delicate processing of biological specimens. We present the results of using femtosecond laser pulses for dissection of zona pellucida (ZP) in mouse embryos during assisted hatching procedure. We studied the effects of application of femtosecond laser radiation in the infrared (1028 nm) and visible (514 nm) wavelength ranges. Laser irradiation parameters were optimized so as not to compromise the viability of the treated embryos. We have demonstrated that application of femtosecond laser pulses with the energies in the range of 250–320 nJ (for the wavelength of 1028 nm) and 47–112 nJ (for 514 nm) resulted in efficient ZP dissection. Femtosecond laser-assisted ZP drilling does not slow down the development of pre-implantation embryos and leads to 90–95% frequency of complete hatching. The thermal effects can be significantly lower when femtosecond lasers are used as compared to continuous wave or long-pulse lasers. It is crucial when dealing with living cells or organisms. By optimizing femtosecond laser radiation parameters assisted hatching as well as a wide range of embryo-surgical procedures can be efficiently performed, thus creating a great potential of using femtosecond lasers as a multi-purpose “tool of choice” for specialists in the fields of embryology and developmental biology.

Noncontact microsurgery and micromanipulation of living cells with combined system "Femtosecond laser scalpel-optical tweezers"

We report on the results of using self-developed combined laser system consisting of a femtosecond laser scalpel (Cr:Forsterite seed oscillator and a regenerative amplifier, 620 nm, 100 fs, 10 Hz) and optical tweezers (cw laser, 1064 nm) for performing noncontact laser-mediated polar body (PB) and trophectoderm (TE) biopsy of early mammalian embryos. To perform PB biopsy the femtosecond laser scalpel was initially used to drill an opening in the zona pellucida, and then the PB was extracted out of the zygote with the optical tweezers. Unlike PB biopsy, TE biopsy allows diagnosing maternally-derived as well as paternally-derived defects. Moreover, as multiple TE cells can be taken from the embryo, more reliable diagnosis can be done. TE biopsy was performed by applying laser pulses to dissect the desired amount of TE cells that had just left the zona pellucida during the hatching. Optical tweezers were then used to trap and move the dissected TE cells in a prescribed way. Laser power in optical tweezers and energy of femtosecond laser pulses were thoroughly optimized to prevent cell damage and obtain high viability rates. In conclusion, the proposed techniques of laser-based embryo biopsy enable accurate, contamination-free, simple and quick microprocessing of living cells.

Measurement of ablation threshold of oxide-film-coated aluminium nanoparticles irradiated by femtosecond laser pulses

We report the results of experiments on estimation of femtosecond laser threshold intensity at which nanoparticles are removed from the substrate surface. The studies are performed with nanoparticles obtained by femtosecond laser ablation of pure aluminium in distilled water. The attenuation (or extinction, i.e. absorption and scattering) spectra of nanoparticles are measured at room temperature in the UV and optical wavelength ranges. The size of nanoparticles is determined using atomic force microscopy. A new method of scanning photoluminescence is proposed to evaluate the threshold of nanoparticle removal from the surface of a glass substrate exposed to IR femtosecond laser pulses with intensities 1011 – 1013 W cm-2.

Photoluminescence of silicon nanoparticles under the action of infrared femtosecond laser pulses

We present the experimental results for the absorption spectra and photoluminescence of silicon nanoparticles synthesized by femtosecond laser ablation in distilled water under action of femtosecond laser pulses at a wavelength of 1048 nm with intensities in the range from 1010 to 1012 W/cm2.

Noncontact laser microsurgery of three-dimensional living objects for use in reproductive and regenerative medicine

Laser microsurgery has enabled us to make highly precise and delicate processing of living biological specimens. We present the results of using femtosecond (fs) laser pulses in assisted reproductive technologies. Femtosecond laser dissection of outer shells of embryos (so-called laser-assisted hatching) as well as laser-mediated detachment of the desired amount of trophectoderm cells (so-called embryo biopsy) required for preimplantaion genetic diagnosis were successfully performed. The parameters of laser radiation were optimized so as to efficiently perform embryo biopsy and preserve the viability of the treated embryos. Effects of application of fs-laser radiation in the infrared (1028 nm) and visible (514 nm) wavelength ranges were studied. We also applied laser microsurgery to develop a new simple reproducible model for studying repair and regeneration in vitro. Nanosecond laser pulses were applied to perform localized microdissection of cell spheroids. After microdissection, the edges of the wound surface opened, the destruction of the initial spheroid structure was observed in the wound area, with surviving cells changing their shape into a round one. It was shown that the spheroid form partially restored in the first six hours with subsequent complete restoration within seven days due to remodeling of surviving cells.

Femtosecond scalpel-optical tweezers: efficient tool for assisted hatching and trophectoderm biopsy

Ultrashort laser pulses have enabled highly precise and delicate processing of biological specimens. We present the results of using femtosecond (fs) laser pulses for dissection of zona pellucida (ZP) in mouse embryos during assisted hatching procedure and for trophectoderm biopsy as well. We studied the effects of application of fs laser radiation in the infrared (1028 nm) and visible (514 nm) wavelength ranges. Laser irradiation parameters were optimized so as not to compromise the viability of the treated embryos. Embryo biopsy was carried out in late-stage mouse preimplantation embryos. Femtosecond laser pulses were applied to detach the desired amount of trophectoderm cells from the blastocyst, while the optical tweezers trapped the cells and moved them out of the embryo. The parameters of laser radiation were optimized so as to efficiently perform embryo biopsy and preserve the viability of the treated embryos. The thermal effects can be significantly lower when fs lasers are used as compared to CW or long-pulse lasers. It is crucial when dealing with living cells or organisms.