Comparison of directedness and EMR in terms of frequency and polarity and voltage analysis between pDCEFs and bpEFs and comparison of trajectory speed and puzzling circles of the cell in electrically neutral 10 Hz 50% duty cycle bpEF. (a) For directedness, polarity contributed more to statistical variation than frequency, and 10 Hz 80% bpEF induced the highest among all the EFs. Variation on frequency in 2-way ANOVA: ∗ p < 0.05; on polarity: ∗∗∗∗ p < 0.0001; on interaction: ns. For multiple comparisons, 5 Hz pDCEF versus 5 Hz bpEF: ∗ p < 0.05; 5 Hz pDCEF versus 10 Hz bpEF: ∗∗∗∗ p < 0.0001; 10 Hz pDCEF versus 10 Hz bpEF: ∗ p < 0.05. (b) For EMR, 10 Hz 80% bpEF induced the highest among all waveforms. Variation on frequency in 2-way ANOVA: ∗ p < 0.05; on polarity: ∗∗∗∗ p < 0.0001; on interaction: ∗∗∗ p < 0.001. For multiple comparisons, 5 Hz pDCEF versus 10 Hz bpEF: ∗∗∗∗ p < 0.0001; 5 Hz bpEF versus 10 Hz pDCEF: ∗∗ p < 0.01; 5 Hz bpEF versus 10 Hz bpEF: ∗∗∗ p < 0.001; 10 Hz pDCEF versus 10 Hz bpEF: ∗∗∗∗ p < 0.0001. (c) Both the frequency and polarity contributed to statistical variation in voltage transient, and 10 Hz 80% bpEF had the highest voltage transient. Variation on frequency in 2-way ANOVA: ∗ p < 0.05; on polarity: ∗∗∗∗ p < 0.0001; on interaction: ∗ p < 0.05. For multiple comparisons, 5 Hz pDCEF versus 5 Hz bpEF: ∗ p < 0.05; 5 Hz pDCEF versus 10 Hz bpEF: ∗∗∗∗ p < 0.0001; 5 Hz bpEF versus 10 Hz pDCEF: ∗∗ p < 0.01; 5 Hz bpEF versus 10 Hz bpEF: ∗∗∗ p < 0.001; 10 Hz pDCEF versus 10 Hz bpEF: ∗∗∗∗ p < 0.0001. (d) The comparison of trajectory speed of the cells under DCEF, 5 Hz 90% pDCEF, 10 Hz 90% pDCEF, 5 Hz 50% bpEF, and 10 Hz 50% bpEF. ANOVA on different EFs: ∗∗∗∗ p < 0.0001. For multiple comparisons, 5 Hz 50% bpEF versus 10 Hz 50% bpEF: ∗∗∗∗ p < 0.0001; 5 Hz 50% bpEF versus 5 Hz 90% pDCEF: ∗∗∗∗ p < 0.0001; 5 Hz 50% bpEF versus 10 Hz 90% pDCEF: ∗∗ p < 0.01; 5 Hz 50% bpEF versus DCEF: ∗∗∗∗ p < 0.0001; 10 Hz 50% bpEF versus 5 Hz 90% pDCEF: ∗∗∗∗ p < 0.0001; 10 Hz 50% bpEF versus 10 Hz 90% pDCEF: ∗∗∗∗ p < 0.0001; 10 Hz 50% bpEF versus DCEF: ∗∗∗∗ p < 0.0001; 5 Hz 90% pDCEF versus 10 Hz 90% pDCEF: ∗∗∗∗ p < 0.0001; 5 Hz 90% pDCEF versus DCEF: ∗∗∗∗ p < 0.0001; 10 Hz 90% pDCEF versus DCEF: ∗∗∗∗ p < 0.0001. From (e) to (f), cell 1 finished a circular route and cell 2 finished one and a half circles and small curves in 15 mins in the electrically neutral 10 Hz 50% bpEF. From (g) to (h), 3 cells finished acute angle and circle-like routes in 15 mins in electrically neutral 10 Hz 50% bpEF. One-way ANOVA with Tukey's test of multiple comparisons and 2-way ANOVA and Sidak's test of multiple comparisons were used.