Fig. 1. Schematic illustration of different focusing situations after the laser is tightly focused through the objective lens

Fig. 2. Focal spot intensity distribution for different processing depths

Fig. 3. Spot intensity distribution at focal points of different numerical apertures

Fig. 4. Laser spot image program acquisition section

Fig. 5. Image processing flow for calculating spot centre and radius

Fig. 6. The focal point is located 10 μm below the lower surface of the slide

Fig. 7. The focus is on the lower surface of the slide

Fig. 8. The focus is on the upper surface of the slide

Fig. 9. The focal point is located 10 μm above the upper surface of the slide

Fig. 10. 20× objective simulation-experimental laser spot radius versus relative acquisition position and comparison plot

Fig. 11. 20× objective simulation-experimental laser spot radius versus relative acquisition position and comparison plot（dispersion）

Fig. 12. Proportion coefficient variation with spectral width in 20× objective lens simulation-experiment

Fig. 13. Autofocus flowchart