# The Development of Piezo-Driven Tools for Cellular Piercing
## 3 Results and Discussion  
In experiments, the temperature was maintained at 25 \({}^{\circ}\)C. To demonstrate the validity of our two modified injectors, a series of experiments were performed. Three measures were defined. (1) Lateral vibration: This measure is defined as the difference between the entire lateral oscillation of the injection pipette tip and the outer diameter of the injection pipette tip. Less lateral vibration causes less damage to mouse oocytes. (2) Cleavage rate: This measure is defined as the ratio between the number of oocytes pierced successfully and the total number of oocytes. (3) Stability: This measure is defined as the statistical analysis on different cleavage rates gained from a series of experiments with optimal parameters, which represents the reproducibility and stability of the modified injectors. Since structures of the membrane and zona pellucida are different, the voltages applied to pierce the membrane and zona pellucida was 30 V and 75 V, respectively. For both modified injectors, the lateral vibration strongly stirred the cytoplasm of the cells after puncturing the mouse oocytes when the frequencies were higher than 120 Hz. For the first modified injector where only the distance between the front of the piezoelectric actuator and the back of the pulled section of the injection pipette was shortened, we found  
Figure 6: Cellular piercing flow chart. that 60 Hz was effective in reducing the lateral vibration to 6 \(\upmu\)m. The parameters of cellular piercing were \(U_{1}=75\) V and \(f_{1}=60\) Hz for the rupture of the zona pellucida and \(U_{2}=30\) V and \(f_{2}=60\) Hz for the rupture of the oocyte membrane, shown in Figure 7. Table 1 shows that the achieved cleavage rate of oocytes varied from 76.9% to 84.2%. On average, 80.1% of the injected oocytes were cleaved. Standard deviation was calculated to be 2.73%, indicating a high cleavage rate was achieved. For the second modified injector where the injection pipette and piezoelectric actuator was combined by friction force instead of a screw joint, the reduction of the deflection angle \(\theta\) is ensured. Experimentally, we found that the frequencies of 30 Hz and 45 Hz both led to the lateral vibration below 1 \(\upmu\)m, which is invisible at 40\(\times\) (objective) and 10\(\times\) (eye-piece), shown in Figure 8. Each of the two frequencies was applied to pierce the membrane and zona pellucida on ten mouse oocytes with the same voltages mentioned above. As shown in Table 2, the frequency of 45 Hz was more effective for the rupture of the membrane and zona pellucida. To testify the validity of this frequency, \begin{table}
\begin{tabular}{c c c c} \hline
**Replicate** & **Number of Oocytes Prepared** & **Number of Oocytes Cleaved** & **Cleavage (\%)** \\ \hline
1 & 13 & 10 & 76.9 \\
2 & 21 & 17 & 81.0 \\
3 & 19 & 16 & 84.2 \\
4 & 25 & 20 & 80.0 \\
5 & 27 & 22 & 81.5 \\ Total & 105 & 85 & 80.1 \\ \hline \end{tabular}
\end{table} 
Table 1: Set of experimental results on mouse cells with the final parameters for first injector. Figure 7: Directions of cellular piercing using the first modified injector. (**A**) Modulate the injection pipette close to the zona pellucida and ensure that vibration of pipette is small, about 6 \(\upmu\)m. (**B**) Touch the zona pellucida. (**C**) Pierce through the zona pellucida. (**D**) Push the tip to the opposite side of the oocytes. (**E**) Pierce through the membrane of the oocytes. (**F**) Apply a gentle positive pressure to prevent the intensive suction of cytoplasm. Scale bars = 15 \(\upmu\)m throughout. more experiments were performed, and the results are summarized in Table 3. The cleavage rate varied from 88.0% to 94.7% with the average cleavage rate of 91.5%. Two modified piezo-driven injectors were developed for the piercing of mouse oocytes. The novelty of the first modified injector is the more compact structure. The novelty of the second modified injector is in the connection of the injection pipette and the piezoelectric actuator.