Objective Corneal laser refractive surgery is a method for correcting vision using lasers to reshape the cornea and change its curvature and thickness. Femtosecond laser corneal cutting is widely used in ophthalmic refractive surgery as a precise, minimally invasive, and controllable surgical technique. In femtosecond laser refractive surgery, the numerical aperture of the optical system determines the focal spot size and required single- pulse energy, which are critical parameters that influence the corneal cutting quality. In this study, we built a femtosecond laser surgery system with an adjustable numerical aperture. We investigated the effect of numerical aperture on cutting quality in the corneal stroma by analyzing the differences in bubble morphology, smoothness of flap separation, and proportion of damaged stromal cells. This study aimed to assist clinicians in selecting the appropriate surgical parameters more effectively. Methods Freshly enucleated pig eyeballs and New Zealand white rabbits were selected as experimental subjects. By adjusting the diameter of the incident beam, corneal flaps were formed on the pig eyeballs using a femtosecond laser with numerical aperture values of 0.16, 0.30, and 0.80. The morphology of the bubbles after cutting was recorded, and the smoothness of the separation was observed when the corneal flaps were lifted. Cell damage experiments were conducted by cutting New Zealand white rabbit eyeballs with a femtosecond laser at numerical aperture values of 0.16, 0.30, and 0.80. After creating the flap with the femtosecond laser, the rabbit eyeballs were placed in corneal active medium (DX solution) and incubated at 4. for 6 h to induce apoptosis in the damaged corneal stromal cells. Subsequently, the rabbit eyeballs were removed and prefixed in a 4% paraformaldehyde (PFA) solution for 2 h. After dewaxing and rehydration, the corneal sections were double-stained with DAPI (4,6-diamidino- 2- phenylindole, D9542, Sigma-Aldrich) and TUNEL (TdT-mediated dUTP nick end labeling, C1088, Beyotime). Finally, the apoptotic cell counts were determined by imaging the sections under a fluorescence microscope. Results and Discussions Under the three different numerical aperture values (0.16, 0.30, and 0.80), as the numerical aperture increases, the volume of the bubbles decreases gradually, and the density of the bubble layer increases (Figs. 5 and 6). This is mainly attributed to the decreasing volume of the focal spot with an increasing numerical aperture, which decreases cavitation bubbles. Corneal flaps formed at a higher numerical aperture are easier to separate. This is primarily because smaller cavitation bubbles result in a denser bubble layers, which facilitates the separation of the interlamellar space with less adhesions between the tissue layers. In the cell damage experiment, as numerical aperture increases, the number of apoptotic cells decreases significantly, as shown in Fig. 7. This is attributed to the decreased single-pulse energy and decreased focal spot size associated with an increase in numerical aperture, which results in smaller photodisruption zones and reduced damage to the surrounding tissues. Therefore, increasing the numerical aperture is beneficial for reducing the extent of stromal cell damage. Conclusions The effects of femtosecond laser corneal cutting for different numerical aperture values were investigated experimentally. The morphological differences in cavitation bubbles induced by a femtosecond laser at different numerical aperture values were analyzed, and the ease of separation of the lamellar layers and the extent of cell damage were compared. The results of the experiment show that during femtosecond laser corneal cutting, a higher numerical aperture yields smaller bubbles, denser bubble layers, easier separation of corneal flaps, and lower levels of damage to stromal cells. Therefore, a higher numerical aperture is beneficial in femtosecond laser refractive surgery. Overall, this study provides valuable insights into the effects of numerical aperture on femtosecond laser corneal cutting and highlights the importance of optimizing the numerical aperture to achieve improved treatment outcomes in corneal procedures.
