Tightly Focused Femtosecond Laser Radiation Induces DNA Double-Strand Breaks in Human Tumor Cells.

We studied the formation of double-strand DNA breaks (DNA DSB) induced by femtosecond laser radiation in A549 human lung adenocarcinoma cells using immunocytochemical staining of the resulting tracks of a specific DSB marker protein phosphorylated ATM kinase (phospho-ATM). Additionally, colocalization of phospho-ATM tracks with γH2AX protein tracks was studied. The results of immunocytochemical analysis showed that 30 min after irradiation of cells with femtosecond pulses with energies of 1 and 2 nJ (radiation power density 2×1011 and 4×1011 W×cm-2, respectively), the formation of tracks consisting of phospho-ATM and γH2AX proteins located in sites where the laser beam passes through the cell nuclei was observed. The presence of phospho-ATM tracks co-localized with γH2AX allows us to conclude that exposure to focused femtosecond infrared laser radiation with a pulse energy of 1-2 nJ leads to the formation of DNA DSB in irradiated cells.

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.