## 3 Results and Discussion  
Dissection of the embryo zona pellucida consists of its local destruction as a result of the absorption of energy of the laser pulses following a given repetition rate. The ZP of a mouse embryo consists of three glycoproteins: ZP1, ZP2, and ZP3. Molecules of glycoproteins ZP2 and ZP3 are connected alternately and form chains that link to each other through \"bridges\" of ZP1 molecules, resulting in a network-like structure [28]. Following an approach by [PERSON] et al. [23] for cell nanosurgery, biological objects are considered as water. Such an approach is to some extent fair for the ZP, since the mesh of proteins forming it is permeable to water molecules that can absorb laser photons. Interaction of a femtosecond laser pulse with water then leads to the formation of \"free\" electrons in the conduction band as a result of multiphoton ionization processes and the Zener tunneling effect [23; 29]. Due to the nonlinearity of the absorption process, the electrons are strongly localized in the focal spot of the laser beam. One of the possible mechanisms for  
Figure 2: (**A**) An example of radial cuts of the ZP performed at different energies of laser pulses \(E\) for velocity \(v=0.01\) mm/s and pulse repetition rate \(f=2.5\) kHz. Pulse energies for cuts 1—19 nJ; 2—24 nJ; 3—31 nJ; 4—38.6 nJ; 5—43 nJ; 6—46 nJ; 7—48 nJ; 8—51 nJ. Scale bar is 10 μm. Rectangle shows the area of multiple cross sections. (**B**) Cross section of cut 2.  
the destruction of proteins that form the ZP is a photochemical effect, leading either to the dissociation of water molecules and the creation of reactive oxygen species that destroy proteins, or to the direct rupture of bonds (the mechanisms of femtosecond nanosurgery of cells are described in detail elsewhere [23; 30]). Another distinctive feature of all the aforementioned effects is their high localization within the size of the laser beam's waist, which makes it possible to perform a cut of the zona pellucida narrower than the laser beam's diameter, i.e., \(D<2\omega_{0}\). In this paper, the cuts of the zona pellucida were made for a series of energies \(E\) of femtosecond laser pulses (see Figure 2A) following with a repetition rate \(f=2.5\) kHz and velocity \(v=0.01\) mm/s. The blue circles in Figure 3 demonstrate the obtained dependence of the average cut width \(D_{\mathrm{av}}\) on energy \(E\). The light-blue area indicates the limits (Min-Max) of the values obtained and characterizes the error \(\Delta D\) in determining the width of the cut. In case of millisecond pulse duration, a single pulse is enough to perform a cut. Let us consider the differences in the effects induced by millisecond and femtosecond pulses. When a pulse from a millisecond laser dissector is directed at the ZP, denaturation of the proteins occurs since the temperature in the laser focal spot can reach several hundred degrees. [PERSON] et al. [13] have shown that for laser-pulse parameters considered to be close to optimal (pulse duration \(\tau=0.6\) ms and power \(P=250\) mW for LAH procedure), the temperature at the beam's center reaches 370 \({}^{\circ}\)C. This forms a region of highly superheated water, which is an excellent solvent. Thus, it is not surprising that the region of the ZP nearest to the waist of the laser beam rapidly disappears [12]. The temperature, however, decreases in a radial direction and reaches about 130 \({}^{\circ}\)C at a distance of 5 \(\upmu\)m, corresponding to the radius of a hole drilled in the ZP using the laser-pulse parameters given above [13]. Such a large hole of 10 \(\upmu\)m in size (red star in Figure 3) obtained for a focused beam diameter of \(\sim\)3 \(\upmu\)m is due to the high thermal conductivity of the aqueous medium surrounding the embryo and to the heat transfer to the adjacent area. It is worth mentioning that a perforation of about 1 \(\upmu\)m in diameter can, however, be formed using a millisecond laser pulse.