Quantitative fs-laser crystalline lens softening surgery towards presbyopia treatment based on optical coherence elastography

Abstract

Presbyopia, a prevalent age-related vision disorder, is primarily characterized by the progressive loss of accommodative ability due to crystalline lens hardening, as described by the Helmholtz theory of accommodation. Among emerging presbyopia correction techniques, femtosecond laser lentotomy (fs-lento) has gained attention for its potential to restore accommodative function through the creation of intracapsular gliding planes via laser-induced optical breakdown (LIOB). This approach offers distinct advantages, including potential accommodative recovery, a non-invasive nature, minimal tissue disruption, and negligible bleeding risk. However, despite two decades of technical development, the clinical translation of fs-lento remains hindered by challenges in quality control and outcome heterogeneity, primarily due to the absence of precise quantitative methods for assessing the surgical effects on tissue mechanical properties. To address these limitations, we developed a femtosecond laser ocular research apparatus (FLORA) system for surgical investigations. Furthermore, we implemented a novel PZT-contact optical coherence elastography (OCE) system, modified from SD-OCT and validated through phantom experiments, for quantitative tissue mechanical characterization. Our experimental results using ex vivo porcine lenses demonstrated that the sequential creation of 16 flower-patterned gliding planes (2 × 3 mm²) with pulse energy of 4.1 μJ, generating cavitation bubbles <50 μm in diameter, effectively reduced the stiffness from 36.88 ± 1.93 kPa to 31.39 ± 2.11 kPa in the anterior lens OCE measurement region. This study provides the quantitative method of the relationship between fs-lento surgery patterns and resultant biomechanical modifications in lens tissue.

1. Introduction

Presbyopia is a common age-related loss of the ability to dynamically focus [1], typically occurs around the age of 40 and progresses to a loss of accommodation ability within 15 years [2]. At least 800 million people are suffering from presbyopia that has not been effectively corrected, which has a significant impact on productivity [3–5]. This condition significantly impacts the quality of life, particularly in tasks requiring near vision, such as reading, computer work, or checking the dashboard while driving. The primary physiological cause of presbyopia, as proposed by the von Helmholtz theory, is the progressive hardening of the crystalline lens [2,6]. This hardening process reduces the lens's ability to change shape, thereby impairing its capacity to focus on near objects. Interestingly, while the crystalline lens hardens with age, other components of the accommodative elements, such as the ciliary muscle, remain relatively active and elastic throughout life [7].

At present, there is still no satisfactory solution for presbyopia treatment [1]. Conventional presbyopia correction method is wearing progressive glasses, but patients still have vertigo and the risk of falling when seeing objects. And the drug treatment of presbyopia is limited by its efficiency and is still in the early stage of research. The laser surgery of presbyopia currently approved by the FDA only includes corneal excimer laser surgery, lens replacement and corneal implant, and there still have shortcomings like invasive and non-dynamic. Other methods such as intrastromal fs-laser cuts [8], laser scleral microporation [9], fs-laser photodisruption of the crystalline lens [10–12] etc., are still in the preclinical research stage.

According to the researches in the past two decades, significant efforts have been directed toward developing surgical interventions to restore the accommodative ability of the crystalline lens by using pulsed laser [13–17,12]. Compared to other types of ultrafast laser (ns or ps laser), fs laser has higher peak power densities with shorter pulse width, and produce finer cutting results based on nonlinear laser induced optical breakdown (LIOB) [18–21]. Due to the smaller size of laser induced cavitation bubbles, fs-laser is suitable for minimally treatment in deep tissues for eye surgery. The fs-laser lentotomy (fs-lento) surgery, a softening strategy to regain the deformation ability of the crystalline lens by creating precise microincisions within the cortex and nucleus tissue [12,15,17]. These microincisions are designed to construct gliding planes inside the lens, which theoretically allow the lens to regain some of its flexibility [22–24].

The mechanical measurements for the intact lens include spinning test [25], Brillouin microscopy [26], ultrasound elastography [27], bubble acoustics [28,29], and optical coherence elastography [30]. Additionally, the shear wave elastography has previously been applied to assess changes in lens elasticity under various influencing factors, utilizing both OCT and ultrasound [31–36]. However, the only mechanical characterization method for fs-lento surgery [14] is still the Fisher spinning test [37] or mechanical stretcher [17], which could not easily obtain specific quantitative mechanical parameters, but only the relative ability of deformation. Thus, the lack of fine characterization methods of elasticity might be the reason why the fs-lento surgery only has ∼40% of patients for recovering amplitude of accommodation (AoA) from 0 to 2 diopter in clinical trials [12]. Although there are many studies for the aging crystalline lens, including structural [38] and elasticity imaging [39,40], there remains an unknown gap in quantifying fs-lento surgery softening effects in mechanical properties. The laser surgery-induced changes in the structural-mechanical properties of the lens [41], might affect the dynamic range of human eye accommodation [42]. Thus, it is meaningful to measuring surgically-derived changes in lens stiffness for clinical applications.

Therefore, in this study, we aims to address this gap by focusing on system setup for fs-laser surgery demonstration and optical coherence elastography measurement. The extracted porcine eyeballs are chosen as a model due to their anatomical and biomechanical similarities to human eyes. By quantifying the stiffness of the lens before and after fs-laser treatment under different cutting patterns, this research seeks to provide valuable insights into the mechanical changes induced by the fs-lento surgery.

2. Materials and methods

2.1. Design of surgery pattern and sample position

Here, we used the extracted porcine lenses that placed within a 3D-printed hemispherical sample chamber to keep upright position. The upper surface of the 3D-printed sample chamber is sealed with a cover glass, and the anterior capsule of the lens is almost in contact with the cover glass. The isolated crystalline lens is surrounded by Ringer's solution to match the refractive index (RI) and maintain osmotic pressure. In this initial system, we used low pulse energy to observe plasmonic spark on the cover glass surface, to determine the relative position of focal volume in Z-Axis. The sample holder was placed on a 3-Axis XYZ translational stage for position control. With the scanning and focusing system, it is possible that the desired nonlinear optical breakdown could be positioned precisely in transparent ocular tissue within the accuracy less than 10 μm.

Based on the FLORA system, the cutting pattern is shown in the Fig. 1(C) with the LIOB distance from both the anterior and posterior poles being at least >700 μm to minimize the risk of photic cataract or capsular rupture near the optical axis. Considering the size of the average daytime pupil, we set a central optical zone of 2 mm in diameter where the fs-laser does not emit, and only performs layer-by-layer scanning and sculpting outside the optical zone, controlling the galvanometer scanning with a sinusoidal electrical signal. Each set of etchings in the XY plane is approximately 2 mm in radial length, composed of multiple cavitation bubbles spaced 50 μm apart, with each layer of bubbles spaced 50 μm apart in the Z direction.

Fig. 1.

Illustration of experiment on crystalline lenses within 3D-printed chamber. (A) The immersed lens sample undergoes fs-lentotomy surgery with corverslip beneath anterior capsule. (B) The upright lens is used for OCE elastic wave speed and stiffness quantification. (C) The fs-laser generates varying numbers of gliding planes inside the lens tissue (group of 0/control, 4, 8 and 16 cuts).

2.2. Femtosecond lentotomy surgery system design

In order to conduct engineering research on fs-laser surgery for the whole eye, we set up a experimental system with large FOV, which we refer to as the Femtosecond Laser Ocular Research Apparatus (FLORA). The FLORA platform was constructed on an optical table to demonstrate simplified fs-laser lentotomy surgery, according to the design seen in Fig. 2. In this system, the femtosecond laser beam is delivered from a commercialized 1035 nm fiber-based fs source (FemtoYL-40, YSL Photonics), with measured pulse width of 500 fs, and repetition rate of 500 kHz. After beam alignment and energy adjustment, the fs-laser is delivered through beam expander, and then directed into XY galvo scanner (PMXA024A, Scanner Optics) and focused on sample using a F-theta field lens with effective focal length 63 mm (NA ≈ 0.1). The theoretical spot focal diameter is about 12.6 μm. The beam expansion device is programmable, achieving remote focusing through the relative movement of the 4-f Galilean lens pair, enabling fine-tuning of the Z-axis focal position. To achieve real-time investigation during the procedure, we used a dichroic mirror (DM) to reflect the surgery beam, and the sample was illuminated by white light source while recorded through the tube lens and CMOS camera (MV-SUF401GM, MindVision).

Fig. 2.

Schematic diagram of Femtosecond Laser Ocular Research Apparatus (FLORA). (A) System setup. PM: power monitor, HWP: half-wave plate, PBS: polarized beam splitter, BE: beam expander, DM: dichroic mirror, SPF: short pass filter, TL: tube lens. (B) Scheme of the flower-like cutting patterns inside the lens.

2.3. OCE system set-up

The OCE system setup was based on a custom-designed spectral domain OCT (SD-OCT) system with contact PZT excitation system, as shown in Fig. 3. The SD-OCT system constructed from a superluminescent diode light source with a central wavelength of 840 nm (SLD840, Thorlabs), 3 dB bandwidth of 70 nm, and measured 2.3 mW output power on sample. The light was split 50:50 to the sample and reference arms. The light in sample arm was scanned by a 2D galvo scanner (S-8107, Sunny Technology) and then focused by an objective lens (MAD405-B, LBTEK) with a focal length of 2.8 mm and scanning FOV of 10 × 10 mm². The spectrometer is consisted of 3000 line transmitted grating and 2048 pixels CMOS linear array detector with 80 kHz A-line rate (Octoplus, Teledyne e2 V). The interference signals were acquisted by a frame grabber (PCIe-9310, Art-control) into computer and converted to depth-resolved A-line signals via FFT through LabVIEW code. The SD-OCT had a 2.8 mm imaging depth with an axial resolution of 10.90 μm, a lateral resolution of 19.69 μm, a 84.43 dB maximum sensitivity, and a phase sensitivity of 1.42 nm (all calibrated and measured in air).

Fig. 3.

OCE system setup. (A) Schematic of the PZT contact excitation elastography system modified from an SD-OCT. (B) Diagram of the repeated A-line along the same direction in XZ cross-section. (C) The sequence diagram of signal control in M-B scan mode. (D) By acquiring 3D data through M-B scan and slicing it along the XZ plane, the temporal signal variations at each point within the tissue can be obtained.

In this study, we used a probe combined with piezoelectric transducer (PK4DLP1, Thorlabs) and a hemispherical tip with 2.5 mm diameter to achieve a ± 2 μm vibrational amplitude. For homogeneous agar phantom, the PZT excitation signal was a 1500 Hz sinusoidal waveform in 1 ms pulse duration (1.5 sine cycles) for each lateral position. For the crystalline lens surface, the excitation signal pulse duration was set as 3 ms (4.5 sine cycles) to enhance the effective numbers of elastive wave in the spatiotemporal displacement map.

The M-B scan mode is used to detect and reconstruct the propagation process of elastic waves, as shown in Fig. 1(b). Specifically, the M-scan is performed by repeating the A-line 500 times at each sampling point. Then, a two-dimensional (2D) galvanometer moves the detection beam to the next sampling point, and the M-scan is repeated. By comparing the phase change values between two adjacent frames before and after, the deformation displacement of the sample reflective interface during that time can be obtained by phase sensitive detection [43].

$$\mathrm{\Delta }d=\frac{\phi (z_i,t_{j+1})-\phi (z_i,t_j)}{2n{k}_0}$$ (1)

where $\mathrm{\Delta }d$ is the displacement of the reflective sample surface between the adjacent frames, n is the serial number of the reflecting surface $z_n$ between discrete depth $z_i$ and $z_{i+1}$ , k₀ is the wavenumber of central wavelength from SLD, and $\phi (z_i,t_j)$ is the phase of depth $z_i$ as the peak intensity in A line signal at j moment.

Based on Eq. (1), the spatial and temporal distribution map of the sample surface displacement can be obtained. The group velocity of elastic wave propagation can be obtained by further linear fitting based on the peak slope in the plot. Here, it was assumed the crystalline lens as homogenous medium without multilayer structure. And the stiffness was assumed as the Young’s modulus. We selected the ∼ 2.5 mm central part of the lens for the group velocity estimation. The group velocity of the elastic wave was converted to the Young’s modulus value (E) by the surface wave equation [44],

$$E=\frac{2\rho {(1+\nu )}^3}{{(0.87+1.12\nu )}^2}{c}_g^2$$ (2)

where ρ = 1183 kg/m³ is the crystalline lens density, ν = 0.5 is the Poisson’s ratio [44], while c_g is the group velocity of elastic wave measured by the OCE system.

2.4. Phantom preparation and uniaxial mechanical validation protocol

Here, we utilized agar phantoms to verify the consistency of Young’s modulus measurements between our OCE system and standard uniaxial mechanical compression test. Agar powder with a gel strength of ≥1300 g/cm² (CAS: 9002-18-0) was dissolved, heated, and cooled to solidify into phantoms of varying hardness, with concentrations of 1.0%, 1.5%, and 2.0%. Three samples were prepared for each concentration, resulting in a total of nine cylindrical phantom samples. Each phantom was subjected to a standard uniaxial compression test using a universal testing machine (TH-8203S, TOPHUNG) to obtain the Young's modulus. Subsequently, the OCE system was used to measure the elastic wave group velocity and calculate the modulus, thereby verifying the consistency of the custom-built OCE system. In the uniaxial mechanical compression test, a preload of 2 N was first applied to ensure contact between the compression plate and the sample, followed by 8 “loading-unloading” cycles (2∼80 N, 5 mm/min) to obtain the force-displacement curve, from which the Young's modulus was derived.

2.5. Extracted crystalline lens preparation

The crystalline lenses were surgically extracted from fresh intact eyeballs of 6-month-old pigs, which were obtained from a local supplier. For the ocular tissue experiments, freshly enucleated porcine eyeballs were delivered to the laboratory within 12 hours post-slaughter. The intact porcine eyes were consistently stored in a low-temperature environment at approximately 4 ℃. The extracted crystalline lenses were fully immersed in Ringer's solution at room temperature (21 ℃) for 1 h to eliminate temperature and humidity interferences, after which subsequent surgical procedures and measurement experiments were conducted. After extraction, all lenses were ensured to have intact capsules without any rupture. Lenses with relatively consistent dimensions (equatorial diameter of ∼ 10 mm, thickness of ∼ 6 mm) were selected as experimental samples (n = 10 for control group and n = 10 for fs-laser treatment group). Before the OCE measurements, the control and experimental samples were kept at the same temperature and solution. Each lens sample was placed upright into a custom 3D-printed sample chamber, which featured a hemispherical inner surface with a curvature radius of 5 mm and a sloped transition around the edges (as shown in Fig. 1). The samples were fully immersed in a Ringer's solution within the 3-D printed chamber to match the refractive index (RI) and maintain osmotic pressure as closely as possible. While a coverslip was placed on top of the lens as an applantation, in the chamber to flatten it during surgery. All experiments were completed within 6 hours of lens extraction.

3. Results

3.1. Optical coherence elastography results in the agar phantom

The agar phantom sample and PZT stimulus probe are shown in Fig. 4(A). And the raw original B-Scan image is shown in Fig. 4(B). The spatio-temporal displacement map of elastic wave propagation can be mapped onto the 2D B-Scan, and the elastic wave propagation process can be reconstructed by visualising the slices. The slice interval is 0.25 ms as shown in Fig. 4(C). In a scanning area of nearly 10 mm, the direction of wave propagation in the spatial–temporal displacement map of agar is almost linear without obvious dispersion, and its slope fitting value can be regarded as the elastic wave group velocity.

Fig. 4.

OCE measurement on agar phantom. (A) Photography of an agar phantom in measurement. (B) Cross-sectional image of the phantom in OCT B-Scan. (C) The propagation process of OCE wave displacement at different times. (D) Spatial-temporal displacement map of the PZT induced shear wave.

As shown in Fig. 5(A), the elastic wave propagated in approximately same velocity along the vertical depth direction in the samples under the same concentrations, that indicates the homogeneity of the agar phantom. The response of the OCE measurements to differences in mechanical properties is also demonstrated by the fact that the wave velocities of the different samples increase as the concentration increases. The OCE-based Young's modulus was calculated by Eq. (2), and compared to standard unaxial mechanical test data, as shown in Fig. 5(B). The OCE measured Young’s modulus data keeps less than 3% in deviation for same concentration (n = 5 in each group). It verifies the repeatability of the proposed OCE system built in this paper. Consistency with compression mechanical testing also validates the reliability of the elastography system.

Fig. 5.

OCE System Performance Test Results. (A) Group velocity of elastic waves along the depth direction in 1%, 1.5%, and 2% agar phantoms; (B) Stiffness of 1%, 1.5%, and 2% agar phantoms assessed by OCE and uniaxial mechanical testing.

3.2. Fs-lentotomy cuts in transparent medium

In the FLORA system, the fs-lento surgery was performed with a sagittal floral cut as shown in Fig. 6(A). The central optical zone was preserved approximately 2 mm in diameter. The number of sliding planes cut was 0, 4, 8 and 16, referring to the protocol as Holger Lubatschowski et al. reported [14,15,12]. The fs-laser pulse energy was 4.1 μJ and the theoretical diameter of the spot was 12.6 μm, and the final radius of the cavitation bubble was less than 50 μm, with no overlap in the depth direction, as shown in Fig. 6(C). Since the XZ cross-section of crystalline lens is not easy to observe under a microscope, the PMMA samples was used instead as shown in Fig. 6(D), which showed spot overlay in Z-direction. This study primarily discusses the influence of the number of slip surfaces generated by cutting on the softening effect.

Fig. 6.

Photograph of fs-laser induced gliding planes. (A) Capsuled lens with inner incision of 4 cuts pattern. (B) Top view of cutting results (2 mm length) in PMMA, imaged by integrated surgical microscope. (C) Microscope image (10×) of an radial cut with spherical cavitation bubbles in lens tissue; 4.1 μJ pulse energy; 50 μm spot separation. (D) Zoomed-in side view details of gliding plane in PMMA, with 50 μm axial step of focal volume in Z direction.

3.3. Quantification of surgical softening effect using OCE

A total of 500 M-B scan acquisitions were performed over a 2.5 mm region on the crystalline lens surface. The scanning area was positioned as close as possible to the apex of the anterior capsule. The resulting structure and wave propagation process are shown in Fig. 7(A), where the dynamic process is briefly displayed at intervals of 0.2 ms. We can clearly observe the boundary of the anterior capsule, but due to the transparency of the lens cortex, the signals from deeper regions are less distinct. Using spatiotemporal displacement maps, the wave velocity was fitted within the lateral radius direction, as shown in Fig. 7(B) and (C). Further linear fitting of the wave velocity yielded the wave velocity and Young's modulus statistics, as illustrated in (D) and (E).

Fig. 7.

OCE measurement on intact porcine crystalline lens under different surgery pattern. (A) Motion snapshots of the vibration displacement of the elastic wave at different times. (B) & (C) The spatiotemporal displacement map of the surface elastic wave in the anterior lenses. (D) & (E) The group velocity of elastic wave and calculated Young’s modulus (as stiffness) results of the ex vivo intact porcine lens with different fs-laser inner cutting patterns.

The histograms presented in Fig. 7(D) and (E) are derived from the raw data points. With elastic wave group velocities expressed as “mean ± standard deviation”, it shows Control (3.07 ± 0.08 m/s), 4 cuts (2.92 ± 0.14 m/s), 8 cuts (2.89 ± 0.12 m/s), and 16 cuts (2.83 ± 0.09 m/s). After conversion, the corresponding Young's moduli were calculated as Control (36.88 ± 1.93 kPa), 4 cuts (32.84 ± 3.20 kPa), 8 cuts (32.03 ± 2.72 kPa), and 16 cuts (31.39 ± 2.11 kPa). These results demonstrate a progressive reduction in both wave velocity and Young's modulus with increasing numbers of femtosecond laser cuts, indicating a softening effect on the lens tissue. Here, one-way analysis of variance (ANOVA) and Tukey HSD tests were used in SPSS to analyze whether there were significant differences among each experimental pattern. It has a significant difference on elastic wave group velocity between control group and treatment groups (p ≤ 0.045 < 0.05). Additionally, fs-laser cutting may reduce the intensity of vibrational displacement, as shown in Fig. 7(B) and (C). Compared with other reports of OCE measurements on the porcine lens, the elastic wave velocities (∼3 m/s) are nearly same the excitation frequency (∼1.5 kHz) [45], while the elastic modulus values obtained are slightly higher due to the calculation model and ex vivo conditions [35,45].

4. Discussion

Presbyopia is receiving increasing attention, as an age-related eye health challenge. Its primary cause may stem from the hardening of the crystalline lens, and the corresponding fs-lento surgery, which emerged 20 years ago, remains promising, with intriguing clinical trial results recently reported. However, the precise quantitative assessment of the softening effect of this surgery is still lacking. To date, measurement techniques have only reflected relative changes in mechanical properties induced by the surgery rather than providing specific quantitative values. As a result, although the surgery is highly promising, its progress in clinical Phase II trials has not been as rapid as expected, with existing studies achieving only about a 40% restoration rate in the amplitude of accommodation (AoA from 0 to 2 D) [12]. This work majorly focuses on the development of instrument systems, enabling fs-laser cutting and optical coherence elastography research for presbyopia. We have further refined the quantitative assessment of the surgical effects of fs-lento surgery, achieving the characterization of subtle changes in stiffness of less than 5 kPa. It is worth acknowledging that the results of this study will have limitations due to the ex vivo measurement device and the mechanical boundary conditions that may not be the same as the real physiological conditions. And the following research will be orientated towards dynamic and in vivo.

Due to the limited research on the relationship between laser parameters and the effects of fs-lento surgery, we will subsequently conduct innovative studies on spot spacing, threshold effects, and pulse train, etc. The ultrafast laser self-focusing properties [46] and other optical side effects [47] in deep ocular tissues need to be considered, and methods to improve processing accuracy, such as simultaneous spatiotemporal focusing [48], deserve further investigation. Combined with adaptive optics, fs-laser eye microscopy and surgery also hold great promise [49,50]. Additionally, this reported work is limited by the shallow imaging depth of SD-OCT and the OCT beam has not yet been implemented in surgery system, which prevents experiments on intact porcine eyes. Therefore, large depth and 3D ophthalmic imaging methods such as Scheimpflug imaging and SS-OCT [51] could be integrated into next systems.

In the field of optical elastography, it is undeniable that non-contact excitation, such as ARF-OCE [43,52] is more suitable for studying the crystalline lens, which is highly sensitive to temperature and humidity [44], compared to the PZT-contact OCE used in this work. Besides, this study is based on the two limited assumptions, that the crystalline lens is homogeneous and the measured value of Young's modulus is equivalent to stiffness. It should be clarified that this is a simplified quantitative approach to mechanics and ignores multilayer structures [53].

Furthermore, we aim to achieve higher-accuracy characterization of the softening effects of fs-lento surgery, enabling real-time surgery and measurement in vivo and on heterogeneous lens samples, while considering the pre-stressed condition [45]. The prestress in the lens capsule and cortex is partially released after the cuts. Given the crucial influence of prestress on wave speed, it is valuable to quantify the prestress changes following fs-lento surgery and decouple it from the intrinsic mechanical properties. It is worth anticipating that with the continuous advancement of non-contact, high-sensitivity, multi-frequency OCE technologies [54,55], as well as in-depth research on optical and mechanical models [56] of the lens [57], new opportunities will emerge for understanding the mechanisms, diagnosis, and treatment of presbyopia.

In future studies, combined with finite element analysis (FEA) as well as ray tracing methods, the surgically improved accommodation and visual quality should be quantitatively simulated based on real fine geometries and mechanical property characterization.

5. Conclusion

In this work, we established a fs-laser ocular surgery research system and developed an OCE system capable of assessing tissue stiffness. Utilizing these two systems, we employed OCE to evaluate changes in the biomechanical properties of porcine crystalline lenses before and after fs-lento surgery. Compared to lenses that did not undergo surgery, those treated with fs-lento exhibited a reduction in the speed of elastic wave propagation, corresponding to a softening in stiffness. Furthermore, the best of our knowledge, this study provides the very first report for quantified modulus measurements of fs-lento surgery. Moving forward, by leveraging more refined non-contact OCE measurement methods to investigate high-efficient and low-damage parameters for femtosecond laser-induced lens softening outcomes, there is potential to pioneer innovative technological approaches for the treatment of presbyopia.

Funding

National Natural Science Foundation of China 10.13039/501100001809 ( 82227805, 62275094); Ministry of Science and Technology of the People's Republic of China 10.13039/501100002855 ( 2022YFC2404500).

Disclosures

The authors have no conflicts to disclose.

Data availability

Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.