To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.

A unit in which two lasers, one with femtosecond and the other continuous radiation, were used was designed and manufactured. Both lasers worked under holographic control conditions provided by two spatial optic modulators. Experiments with creating several polymer structures by several femtosecond optical “traps” simultaneously were performed. The possibility of the production of one polymer article by simultaneous displacement of several femtosecond optical traps was demonstrated. A polymer article was also prepared by several femtosecond traps and manipulated with the use of several continuous traps. The possibility of focusing femtosecond radiation onto an extended object (“thread” 13 μm long) and the preparation of a polymer object by controlling a femtosecond optical trap in three-dimensional space was demonstrated.

SignificancePreparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye.AimThis work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.ApproachWe applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.ResultsWe demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.ConclusionsThis method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.

Near-infrared femtosecond laser is a promising tool for oocyte and embryo manipulation. In nuclear transfer and mitochondrial replacement therapy, this laser can be successfully applied for the enucleation. In addition, it can be used for zygote ploidy normalization by pronuclear destruction. The aim of this work was to develop a new technique for normalizing zygote ploidy. We applied 1033 nm femtosecond laser radiation to eliminate a pronucleus in three-pronuclear human zygotes. The study showed that destruction of pronuclear DNA occurred under the action of the femtosecond laser. At the same time, the zygotes retained their structure and even their ability to cleave. Thus, we suppose that femtosecond laser can be useful for pronuclear destruction and zygote ploidy normalization.