Method for adjusting optical and mechanical properties of cornea tissue through photosensitizer-assisted femtosecond laser linkage

Abstract

The invention belongs to the technical field of laser treatment of corneal tissues, and particularly relates to a method for adjusting optical and mechanical properties of corneal tissues through photosensitizer-assisted femtosecond laser linkage. The method comprises the following steps: introducing an exogenous photosensitizer into cornea tissues, and pretreating the cornea tissues; a femtosecond laser direct writing system is adopted to focus a laser beam in the cornea tissue; the femtosecond laser is subjected to three-dimensional fine customized crosslinking in the cornea tissue by adjusting various parameters of a laser beam of a light path of the femtosecond laser direct writing system; the femtosecond pulse laser generates a two-photon effect at a light focus, low-density plasma is induced under the assistance of a photosensitizer, collagen tissue in cornea tissue generates oxygen free radicals, then the oxygen free radicals react with surrounding protein to form crosslinking, and synergistic change of the refractive index and the mechanical property of the cornea tissue is initiated. The method can effectively inhibit generation of bubbles in the laser treatment process, and enables the optical refractive index of the treated cornea tissue to be increased; the optical light transmittance is kept in a normal interval; and the mechanical strength is improved in linkage.

Description

Technical Field

[0001] The present invention belongs to the technical field of corneal tissue laser processing, and particularly relates to a method for photosensitizer-assisted femtosecond laser linkage regulation of corneal tissue optical and mechanical properties. Background Art

[0002] Currently, corneal cross-linking (CXL) is an effective technology for treating ectatic corneal diseases such as keratoconus. This technology primarily uses ultraviolet light (UVA) and riboflavin (Rf) to induce molecular cross-linking of collagen fibers in corneal tissue, causing a photochemical reaction, thereby increasing the biomechanical strength of the corneal tissue and preventing or delaying the progression of the disease. This technology uses UVA light to irradiate the corneal tissue for a long time, which can easily cause certain damage to the corneal epithelium, corneal endothelium, and even the lens. Therefore, it requires a corneal thickness of 380μm at its thinnest point, which limits its scope of application. In addition, due to the severe absorption and low penetration depth of UVA light, it is difficult to precisely control the cross-linking range and structure when cross-linking corneal tissue. Traditional UV LED light sources cannot be focused on the key lesion area, and only produce a uniform cross-linking effect across the entire cornea, failing to improve the optical properties of the cornea. This is a major drawback of current traditional technologies.

[0003] In recent years, femtosecond laser micro-nanoprocessing technology has been widely used in the medical field due to its ultrashort pulse duration (femtosecond level), high energy density, and extremely high processing precision. When a femtosecond laser is used to act on the target tissue, the impact on surrounding tissue is extremely low, and it can precisely act on the target tissue without damaging surrounding tissue, especially in ophthalmic corneal refractive surgery. The femtosecond laser corneal tissue processing technologies currently used mainly include small incision lenticule extraction (SMILE) and femtosecond laser assisted laser-assisted in situ keratomileusis (FS-LASIK). These procedures mainly achieve the desired optical effect by invasively cutting or ablating the cornea with a femtosecond laser or excimer laser. Postoperatively, there are risks such as dry eye caused by corneal nerve damage, changes in corneal biomechanical properties, and even corneal ectasia.

[0004] The current problem is that when using existing femtosecond laser ophthalmology technology, it will produce an ablation and resection effect on the corneal tissue, and it is impossible to change the refractive index in situ without damage; when using UVA light for cross-linking, cross-linking can only be achieved on a two-dimensional plane. The cross-linking effect and accuracy are low, and it is impossible to control the fine cross-linking position and depth, or even the three-dimensional fine adjustment of optical and mechanical properties. Summary of the Invention

[0005] To solve the above problems, the present invention provides a safe and efficient method for photosensitizer-assisted femtosecond laser linkage regulation of the optical and mechanical properties of corneal tissue.

[0006] The present invention introduces photosensitizers and uses femtosecond laser amplifiers and femtosecond laser oscillators to treat corneal tissue, thereby achieving in situ and simultaneous linkage regulation of the optical and mechanical properties of corneal tissue under non-damaging or low-damaging conditions, providing a new minimally invasive or even non-invasive solution for corneal tissue repair.

[0007] The present invention provides a method for regulating the optical and mechanical properties of corneal tissue by combining photosensitizer with femtosecond laser, which introduces exogenous photosensitizer into corneal tissue to achieve pretreatment of corneal tissue; a femtosecond laser direct writing system (specifically, Figure 1 As shown in the figure, the laser beam is focused on the corneal tissue; by adjusting the optical path components of the femtosecond laser direct writing system and the various parameters of the laser beam, the femtosecond laser performs three-dimensional fine-tuned cross-linking in the corneal tissue; at this time, the femtosecond pulsed laser produces a two-photon effect at the light focus, and with the assistance of a photosensitizer, it induces low-density plasma (LDP), causing the collagen tissue inside the corneal tissue to produce reactive oxygen species (ROS), which then react with surrounding proteins to form cross-links, triggering synergistic changes in the refractive index and mechanical properties of the corneal tissue.

[0008] The method of the present invention is a novel non-invasive laser-corneal tissue interaction mode, which avoids severe thermal ablation and ablation cutting and does not cause significant damage to the corneal tissue.

[0009] The method provided by the present invention for regulating the optical and mechanical properties of corneal tissue by femtosecond laser linkage comprises the following specific steps:

[0010] Step (1), introduction of exogenous photosensitizer;

[0011] The corneal tissue is placed in a photosensitizer and immersed;

[0012] Step (2), preparation and debugging of the femtosecond laser direct writing system;

[0013] By rotating 1 / 2 of the glass slide, the polarization state of the laser beam entering the polarization beam splitter is adjusted, thereby controlling the energy of the laser beam entering the objective lens, and the power adjustment range is 3mw to 8mw. According to the processing requirements, objective lenses and Z-axis translation stages with different magnifications and numerical apertures are selected to adjust the focus and depth of focus of the laser focus. The femtosecond laser direct writing system is debugged to complete the setting of the laser parameters: the scanning speed, point spacing and other parameters of the laser beam are set through the femtosecond laser direct writing control system. The preset three-dimensional cross-linking pattern is loaded into the system's computer program, and the laser focus is controlled to move according to the specified program and path by processing the text file instructions.

[0014] Step (3), femtosecond laser cross-linking treatment of corneal tissue;

[0015] The corneal tissue infiltrated with photosensitizer is placed under the laser emission window. The laser beam is perpendicular to the processing plane of the corneal tissue, and the system accurately positions the laser focus at the specified depth of the corneal tissue. Three-dimensional fine processing is performed using a femtosecond laser direct writing system. The laser beam triggers a two-photon effect inside the corneal tissue, generating low-density plasma to induce the production of oxygen free radicals (ROS). The oxygen free radicals react with the corneal tissue collagen to form a cross-linked structure. This process does not cause damage to the surface of the corneal tissue. By adjusting the laser power density, scanning speed, point spacing and other parameters, the mechanical strength, refractive index and transmittance of the cross-linked area can be controlled.

[0016] Step (4), preservation and detection of the corneal tissue after treatment.

[0017] Preservation of corneal tissue: For ex vivo corneas, after laser processing, the corneal tissue is placed in 0.01M phosphate buffered saline (PBS) and continued to be stored at 2-8°C to maintain the activity of the processed corneal tissue and the stability of the processing effect.

[0018] Testing of processing effects: For in vitro corneas, the mechanical strength of the cross-linked area is tested using a mechanical strength tester; the femtosecond laser parameters are adjusted to regulate the refractive index growth change.

[0019] Further:

[0020] In step (1), the exogenous photosensitizer can be riboflavin, with a concentration of 0.1% to 1.2%, and the infiltration time is 15 to 45 minutes.

[0021] In step (2), the preparation and debugging of the femtosecond laser direct writing system specifically include: adjusting the laser power, scanning speed, and point spacing; using different long working distance objective lenses; writing a specified text file to move the laser focus according to the specified program.

[0022] In the present invention, the photosensitizer exemplified is riboflavin, but it also includes other photosensitizers that can be excited by femtosecond lasers of different wavelengths to produce a two-photon effect.

[0023] In the present invention, the femtosecond laser types used are near-infrared band femtosecond laser amplifiers and femtosecond laser oscillators. However, femtosecond laser amplifiers and femtosecond laser oscillators of other wavelengths are also included.

[0024] In the present invention, a 1 / 2 glass slide and a polarization beam splitter prism are used to achieve regulation of laser power. The femtosecond laser power regulation range is 3mw to 8mw, and also includes powers outside this range.

[0025] The objective lens used in the present invention has adjustable focusing magnifications of 5x, 10x, 20x, and 50x; the corresponding effective numerical apertures are 0.15, 0.30, 0.45, and 0.65, respectively; the corresponding working distances are 45 cm, 34 cm, 31 cm, 20.2 cm, and 20.16 cm, respectively; and the Z-axis translation stage can be linked to adjust the height range from 0 to 38 mm. (Modifications to the above values are not considered innovations of this patent.)

[0026] In the present invention, the refractive index increase Δn of corneal tissue femtosecond cross-linking with the introduction of photosensitizer (riboflavin) in multiple cases was significant, ranging from 0.08 to 0.19, and was stable and controllable. When no photosensitizer was introduced, the increase in refractive index Δn was between 0 and 0.10 (not controllable and easily disturbed to form a removal effect).

[0027] In the present invention, multiple cases of introducing photosensitizer (riboflavin) for corneal tissue femtosecond amplifier cross-linking showed that the lowest transmittance was 90.65%, which met the normal imaging requirements. The highest transmittance without the introduction of photosensitizer was 85.85%, which was lower than the normal corneal tissue imaging level.

[0028] In the present invention, multiple cases of corneal tissue femtosecond amplifier cross-linking using a photosensitizer (riboflavin) resulted in an average change in corneal tissue transmittance of 1.09%, a maximum change of 2.65%, and a minimum of 90.65%, without affecting normal corneal imaging. In the absence of a photosensitizer, the average change in corneal tissue transmittance due to femtosecond amplifier-induced cross-linking was approximately 5.45%, with a maximum change of 7.40% and a minimum of 85.85%, which is lower than normal corneal tissue imaging levels.

[0029] In the present invention, the introduction of photosensitizer (riboflavin) for corneal tissue femtosecond amplifier cross-linking in many cases increased the mechanical strength by up to 10%, and the mechanical properties were improved. Without the introduction of riboflavin, the mechanical properties of corneal tissue induced by femtosecond amplifier-induced cross-linking decreased.

[0030] After introducing an exogenous photosensitizer, this method uses a femtosecond laser amplifier and a femtosecond laser oscillator with different parameters to treat corneal tissue. This method modulates the optical and mechanical properties of the corneal tissue in situ at the laser focal point. With the aid of an exogenous photosensitizer (such as riboflavin), the generation of bubbles during laser treatment is effectively suppressed, and the optical refractive index Δn of the treated corneal tissue increases. The optical transmittance remains within the normal range, and the mechanical strength is also enhanced. This method is beneficial for the treatment of corneal tissue diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a structural diagram of the femtosecond laser direct writing system of the present invention.

[0032] Figure 2 This is a rendering of the refractive index change microscope of Example 1 of the present invention.

[0033] Figure 3 This is a rendering of the refractive index change microscope of Example 2 of the present invention.

[0034] Figure 4 This is a diagram of the refractive index phase change after riboflavin-assisted corneal tissue femtosecond laser amplifier treatment according to an embodiment of the present invention.

[0035] Figure 5 This is a graph showing the grayscale change in refractive index after riboflavin-assisted corneal tissue femtosecond laser amplifier treatment according to an embodiment of the present invention.

[0036] Figure 6 It is a curve diagram of light transmittance change of corneal tissue treated with riboflavin-assisted femtosecond laser amplifier, treated with simple laser amplifier and a blank control group implemented by the present invention.

[0037] Figure 7 This is a diagram showing the maximum change in light transmittance between corneal tissue treated with riboflavin-assisted femtosecond laser amplifier and corneal tissue treated with laser amplifier alone, as implemented in the present invention.

[0038] Figure 8 A curve diagram showing the relative transmittance variation between riboflavin-assisted corneal tissue femtosecond laser amplifier treatment and simple laser amplifier treatment implemented in the present invention.

[0039] Figure 9 This is a graph showing the mechanical strength changes of various parameters between the riboflavin-assisted corneal tissue femtosecond laser amplifier treatment and the simple laser amplifier treatment of the present invention.

[0040] Figure 10 Schematic diagram of riboflavin-assisted femtosecond laser treatment of corneal tissue for the coordinated regulation of optical and mechanical properties.

[0041] Figure 11 This is a comparison chart of the microscopic effects of riboflavin-assisted corneal tissue femtosecond laser oscillator treatment and simple laser oscillator treatment. DETAILED DESCRIPTION

[0042] The present invention is further described below through embodiments in conjunction with the accompanying drawings.

[0043] like Figure 1 The optical path structure of the femtosecond laser direct writing system shown in the figure includes components such as an adjustable attenuation plate, a 1 / 2 glass slide, a polarization beam splitter prism, a convex lens, a concave lens, a CCD, a beam splitter prism, three reflective mirrors, an objective lens, an XY translation stage, and a Z-axis translation stage.

[0044] The figure shows the relationship between the components of the femtosecond laser direct writing system. After being emitted by the laser, the femtosecond laser first passes through an adjustable attenuator for energy regulation. It then enters a laser polarization control module consisting of a half-wave plate and a polarization beam splitter prism, which outputs the desired polarized beam. The laser beam then passes through a concave and convex lens, reducing the laser's divergence angle during transmission and adjusting its spatiotemporal energy density. Finally, the laser beam passes through a series of reflectors and is guided to an objective lens suspended on a Z-axis translation stage for focusing. The upper portion of the translation stage houses the imaging module of the processing system, consisting of a beam splitter prism, a visible-wavelength convex lens, and a CCD. An illumination source is placed at the incident end on the other side of the beam splitter prism. The illumination light is refracted downward by the beam splitter prism onto the corneal tissue surface, forming the reflective illumination module of the system. The corneal tissue is fixed to the translation stage, which has high-speed XY motion and hundred-nanometer positioning capabilities. After the optical gate is opened, the laser beam is transmitted through the optomechanical system, ultimately forming a tight focus within the corneal tissue, where it produces a two-photon effect. The XYZ three-axis linkage translation stage moves according to computer instructions, such as Figure 1 As shown in the dotted box in the upper right corner,

[0045] Example 1, a method for regulating the optical and mechanical properties of corneal tissue by femtosecond laser linkage, comprising the following steps:

[0046] (1) Pretreatment of in vitro corneal tissue and introduction of riboflavin photosensitizer.

[0047] Corneal tissue pretreatment: After removal from a human eye donor, corneal tissue was immediately placed in a 20% T-500 dextran solution in 0.01M phosphate buffered saline (PBS) to maintain bioactivity. The corneal tissue was then stored at 4°C. The preserved corneal tissue was then removed and placed flat in a 0.1% riboflavin solution for 30 minutes. After immersion, the corneal tissue was rinsed with PBS to remove any residual riboflavin solution.

[0048] (2) Preparation and debugging of femtosecond laser system.

[0049] Femtosecond laser system setup: A femtosecond laser amplifier direct writing system was used for corneal tissue processing. The system adjusts the polarization state of the laser beam entering the polarizing beam splitter by rotating 1 / 2 of the glass slide, thereby controlling the laser beam energy entering the objective lens, and the power range is adjusted to 5.5 mw. The objective lens parameters used are 5X-0.15NA-40WD, and the Z-axis translation stage can be adjusted in a linkage within the height range of 0 to 38 mm. The laser parameters are set to 5.5 mW laser power and 2500 μm / s scanning speed. The system loads the preset rectangular pattern into the computer program and controls the movement of the laser focus according to the specified program through text file instructions.

[0050] (3) Femtosecond laser cross-linking care of corneal tissue.

[0051] Corneal tissue fixation and laser processing: Riboflavin-infiltrated corneal tissue is placed under the laser emission window. The laser beam is perpendicular to the processing plane of the corneal tissue, and the system precisely positions the laser focus at a specified depth in the corneal tissue. Three-dimensional fine processing is performed using a femtosecond laser direct writing system. The laser beam induces a two-photon effect within the corneal tissue, forming a cross-linked structure.

[0052] (4) Preservation and detection of corneal tissue after laser processing.

[0053] Preservation of corneal tissue: After laser processing of the ex vivo cornea, the corneal tissue was placed in 0.01M phosphate buffered saline (PBS) and stored at 4°C to maintain the activity of the processed corneal tissue and the stability of the processing effect. Testing of processing effect: Changes in the grayscale value of the corneal tissue were measured using a phase microscope to deduce changes in the refractive index; the transmittance of the corneal tissue was measured using a UV-visible spectrophotometer; and the mechanical strength of the cross-linked area was tested using a mechanical strength tester.

[0054] The femtosecond laser processing equipment and processing trajectory of this embodiment are shown in FIG. Figure 1 As shown; the microscope effect after riboflavin-assisted femtosecond laser amplifier cross-linking is shown Figure 2 As shown, the surface of the cross-linked area is relatively uniform, with a different refractive index from the surrounding environment and a clear boundary.

[0055] In Example 2, steps (2), (3), and (4) are the same as those in Example 1, and step (1) is different from that in Example 1. In this example, after the corneal tissue is removed from the human eye donor in step (1), it is immediately placed in 0.01M phosphate buffered saline (PBS), a 20% T-500 dextran solution is prepared to maintain the biological activity of the corneal tissue, and the corneal tissue is stored at 4°C. The preserved corneal tissue is removed, the surface is washed with PBS, and then it can be used for processing. The microscope effect after riboflavin-free photosensitizer femtosecond laser cross-linking in this example is shown in FIG. Figure 3As shown, the left image is a low-power microscopic image of the cross-linked region, and the right image is a high-power microscopic image of the cross-linked region. The cross-linked region has a striped surface, with a different refractive index from the surrounding area and a clear boundary.

[0056] In Example 3, steps (1), (3), and (4) are the same as those in Example 1, and step (2) is different from that in Example 1. In step (2), this example uses the same laser scanning parameters as in Example 1 (5X-0.15NA-40WD objective lens, 5.5mW laser power, 2500μm / s scanning speed), but the scanning structures are circular ring structures and rectangular structures. The refractive index phase change diagram of the lens tissue with riboflavin photosensitizer film after femtosecond laser cross-linking in this example is shown in FIG. Figure 4 As shown, the large picture is a phase microscope image. The phase difference of the cross-linked structure in the picture can be used to preliminarily observe the change in refractive index. The small picture in the lower right corner is the corresponding microscope image. The grayscale change of refractive index after femtosecond laser cross-linking of corneal tissue assisted by riboflavin is shown in the figure. Figure 5 As shown, the large image is a grayscale image converted from the phase image. The grayscale differences in the cross-linked structure in this image can be used to analyze refractive index changes. The small image in the lower right corner is the grayscale change curve corresponding to the marked area. In summary, this example demonstrates a significant refractive index change structure at the corneal tissue cross-linking process using a femtosecond laser.

[0057] In Example 4, steps (3) and (4) are the same as in Example 1, and steps (1) and (2) are different from those in Example 1. In this example, two groups of operations are performed on the same piece of corneal tissue in step (1), which are the same as in Example 1 and the same as in Example 2, and blank control groups are set up, which are named laser + riboflavin group, simple laser processing group, and blank control group, respectively. In this example, the same laser scanning parameters (5X-0.15NA-40WD objective lens, 5.5mW laser power, 2500μm / s scanning speed) as in Example 1 are used in step (2), but the scanning structure is changed to a full coverage scanning mode. The transmittance change curves of riboflavin-assisted corneal tissue femtosecond laser treatment, simple laser treatment, and blank control group in this example are shown in FIG. Figure 6 As shown in Figure 1, the transmittance change curves of three groups of corneal tissues in the 400-800 nm band are shown. The maximum transmittance change of corneal tissue treated with riboflavin-assisted femtosecond laser and that treated with laser alone is shown in Figure 1. Figure 7 The figure shows the maximum value of the transmittance change of corneal tissue in the 400-800 nm band after these two different treatments. The relative transmittance change curve of corneal tissue treated with riboflavin-assisted femtosecond laser and treated with laser alone is shown in the figure. Figure 8 , which shows the relative change curve of the transmittance of corneal tissue in the 400-800 nm band after these two different treatments.

[0058] Example 5, steps (1), (3), and (4) are the same as Example 4, and step (2) is different from Example 4. In this example, some of the same laser scanning parameters as those in Example 4 are set in step (2) (5X-0.15NA-40WD objective lens 2500μm / s scanning speed), but the laser power is changed to three cases of 3.5, 4.5, and 5.5mW. The laser + riboflavin group, the simple laser processing group, and the blank control group were processed in the same manner of full coverage scanning. The transmittance and refractive index detection effects of this example are the same as those of Examples 1, 2, 3, and 4, but they are subjected to mechanical strength tests respectively. The mechanical strength change diagram of each parameter of riboflavin-assisted corneal tissue femtosecond laser treatment and simple laser treatment in this example is shown in the figure below. Figure 9 The figure shows the change in mechanical strength of corneal tissue under three laser powers in two different treatment methods. The schematic diagram of the riboflavin-assisted corneal tissue femtosecond laser treatment in this embodiment to regulate the optical and mechanical properties is shown in FIG. Figure 10 As shown, this embodiment uses the processing scheme of the present invention to obtain the linked regulation of the optical refractive index, optical transmittance and mechanical property Young's modulus as shown in the four figures on the right.

[0059] In Example 6, steps (1), (3), and (4) are the same as those in Example 1, and step (2) is different from that in Example 1. In this example, in step (2), the femtosecond laser light source is a femtosecond laser oscillator, and the laser scanning parameters are set as follows: 1KHz repetition frequency, 360mW power, 150μs point dwell time, and 20X objective lens. With such laser scanning parameters and riboflavin, the corneal tissue is treated in the range of 100μm*50μm, and the microscope effect is obtained as shown in the figure below. Figure 11 The left picture shows the low-power microscope observation of the rectangular treatment area, and the right picture shows the high-power microscope observation of the rectangular treatment area.

Claims

1. A method for regulating the optical and mechanical properties of corneal tissue by photosensitizer-assisted femtosecond laser linkage, characterized in that: An exogenous photosensitizer is introduced into the corneal tissue to achieve pretreatment of the corneal tissue; a femtosecond laser direct writing system is used to focus the laser beam into the corneal tissue; by adjusting the optical path components of the femtosecond laser direct writing system and various parameters of the laser beam, the femtosecond laser performs three-dimensional, finely customized cross-linking within the corneal tissue; the femtosecond pulsed laser produces a two-photon effect at the light focus, and with the assistance of the photosensitizer, it induces low-density plasma, causing the collagen tissue within the corneal tissue to produce oxygen free radicals, which then react with surrounding proteins to form cross-links, triggering synergistic changes in the refractive index and mechanical properties of the corneal tissue; the specific steps are as follows: Step (1), introduction of exogenous photosensitizer; The corneal tissue is placed in a photosensitizer to be infiltrated; Step (2), preparation and debugging of the femtosecond laser direct writing system; By rotating 1 / 2 of the glass slide, the polarization state of the laser beam entering the polarizing beam splitter is adjusted, thereby controlling the laser beam energy entering the objective lens, with a power adjustment range of 3mw to 8mw. According to processing requirements, objective lenses and Z-axis translation stages with different magnifications and numerical apertures are selected to adjust the focus and depth of the laser focus. The femtosecond laser direct writing system is debugged to set laser parameters such as laser beam scanning speed and point spacing. The preset three-dimensional cross-linking pattern is loaded into the system's computer program, and the laser focus is controlled to move according to the specified program and path by processing text file instructions. Step (3), femtosecond laser cross-linking treatment of corneal tissue; The corneal tissue infiltrated with a photosensitizer is placed under the laser emission window of a femtosecond laser direct writing system. The laser beam is perpendicular to the processing plane of the corneal tissue, and the laser focus is precisely positioned at a specified depth in the corneal tissue. Three-dimensional fine processing is performed using the femtosecond laser direct writing system. The laser beam triggers a two-photon effect inside the corneal tissue, generating low-density plasma that induces the production of oxygen free radicals. The oxygen free radicals react with corneal tissue collagen to form a cross-linked structure. The mechanical strength, refractive index, and transmittance of the cross-linked area can be controlled by adjusting the laser power density, scanning speed, and point spacing parameters. Step (4), preservation and testing of the corneal tissue after treatment; Preservation of corneal tissue: After laser processing, the corneal tissue is placed in 0.01M phosphate buffer and stored at 2-8°C to maintain the activity of the processed corneal tissue and the stability of the processing effect; Detection of processing effect: Use a mechanical strength tester to detect the mechanical strength of the cross-linked area; adjust the femtosecond laser parameters and adjust the refractive index growth change.

2. The method according to claim 1, characterized in that The exogenous photosensitizer in step (1) is riboflavin, the concentration of which is 0.1% to 1.2%, and the infiltration time is 15 to 45 minutes.

3. The method according to claim 1, characterized in that The preparation and debugging of the femtosecond laser direct writing system described in step (2) specifically include: adjusting the laser power, scanning speed, and point spacing; using different long working distance objective lenses; writing a specified text file to move the laser focus according to the specified program.

4. The method according to claim 1, wherein The femtosecond laser types used are near-infrared band femtosecond laser amplifiers and femtosecond laser oscillators.

5. The method according to claim 1, wherein The objective lens used has adjustable focusing magnifications of 5x, 10x, 20x, and 50x; the corresponding effective numerical apertures are 0.15, 0.30, 0.45, and 0.65 respectively; the corresponding working distances are 45 cm, 34 cm, 31 cm, 20.2 cm, and 20.16 cm respectively; the Z-axis translation stage can be linked to adjust the height range from 0 to 38 mm.

6. The method according to claim 1, characterized in that The refractive index growth change Δn of corneal tissue femtosecond cross-linking is obvious, ranging from 0.08 to 0.19, and is stable and controllable.

7. The method according to claim 1, characterized in that The average change in corneal tissue transmittance was 1.09%, the highest transmittance change was 2.65%, and the lowest transmittance was 90.65%, which met the requirements for normal corneal tissue imaging.

8. The method according to claim 1, characterized in that The mechanical strength of corneal tissue cross-linked by femtosecond amplifiers increased, with the maximum increase being 10%.

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