Based on the transparency of corneal tissue and on laser plasma mediated non-thermal tissue ablation, near infrared femtosecond lasers are promising tools for minimally invasive intrastromal refractive surgery. Femtosecond lasers also enable novel nonlinear optical imaging methods like second harmonic corneal imaging. The microscopic effects of femtosecond laser intrastromal surgery were successfully visualized by using second harmonic corneal imaging with diffraction limited resolution, strong imaging contrast and large sensing depth, without requiring tissue fixation or sectioning. The performance of femtosecond laser intrastromal surgery proved to be precise, repeatable and predictable. It might be possible to integrate both surgical and probing functions into a single femtosecond laser system.
©2004 Optical Society of America
In the past decade, the rapid development of femtosecond (fs) pulsed lasers has highlighted the laser science. Fs lasers are powerful tools not only for fundamental research in nonlinear optics, chemical dynamics and laser spectrometry, but also for novel biomedical applications in mini-invasive surgery and tissue imaging. Due to the transparency of the human eye to visible and near infrared light, the ocular tissues are ideal objects for laser-based diagnostic and therapeutic applications. Ultraviolet nanosecond (ns) excimer lasers have been used with great success for Photorefractive Keratectomy (PRK) and Laser In-situ Keratomileusis (LASIK) in refractive surgery. Recently, there has been increasing interest in exploring novel applications of fs lasers for refractive surgery [1, 2]. Initiated by multiphoton absorption and laser induced optical breakdown, the high pressure laser plasma non-thermally dissociates the dense corneal tissue thereby enabling mini-invasive intrastromal cornea surgery. The laser-affected region is highly localized, leading to precise ablation with minimized side effects . Theoretically fs lasers offer numerous advantages over excimer lasers, but providing direct experimental evidence for the feasibility of fs laser intrastromal surgery is crucial before starting clinical applications. Since the intrastromal structures induced by fs laser are sensitive to external stress, the cornea tissue must be probed under the condition closest to its natural physiological state. Unfortunately, typical high resolution histological analysis methods like scanning/transmission electron microscopy (SEM/TEM) may introduce artificial effects during fixation and slicing.
It is interesting that fs lasers also play the most important role in nonlinear laser scanning microscopy. Among various implementations, Second Harmonic Generation (SHG) microscopy [4, 5, 6, 7, 8] turned out to be particularly well suited to explore the microscopic performance of novel intrastromal surgical approaches. The full thickness of the cornea tissue can be probed with high resolution and strong contrast, without requiring sectioning, staining or labelling . In this article, first the layout of the Neodymium:glass (Nd:glass) fs surgical laser system is briefly introduced. The pros and cons of Nd:glass lasers and Titanium:Sapphire (Ti:S) lasers are discussed. The microscopic performance of fs laser intrastromal surgery was evaluated by high resolution, non-invasive corneal SHG imaging of fs laser treated, enucleated porcine eye.
2. All-solid-state Nd:glass femtosecond surgical laser system
To meet the requirements of real world applications, the fs surgical lasers have to be compact and simple. As the dominant fs laser type, Ti:S lasers require expensive pump laser sources, and typically the Kerr-Lens Mode-locking (KLM) in Ti:S laser is not self-starting: end mirror translating or prism acoustic modulation is required to initiate KLM. Compared to Ti:S lasers, laser diode pumped Nd:glass lasers are more suitable for ophthalmologic applications . The main arguments in favor of Nd:glass lasers can be summarized as follows: System compactness and price. The absorption band of Nd:glass is around 800 nm, fitting perfectly into today’s high power AlGaAs laser diode technique. Direct laser diode pumping allows compact laser setup and low costs. Self Starting mode-locking. A Semiconductor Saturable Absorber Mirror (SESAM) is employed in Nd:glass lasers as a second passive mode-locking device, thus the strict resonator cavity alignment requirement for pure KLM is considerably relaxed. The fs pulse generation in Nd:glass lasers is self starting and maintenance free. Capability for high power applications. Nd:glass is an ideal gain medium for regenerative amplification due to its excellent energy storage capability (the upper level lifetime of Nd:glass exceeds 300 μs). Compared to Ti:S, the main drawbacks of Nd:glass are narrow emission bandwidth and poor thermal conduction. The emission bandwidth of Nd:glass is less than 30 nm. Taking into account gain narrowing and large dispersion in the regenerative amplifier, the eventual pulse width easily exceeds 500 fs. Furthermore, poor thermal conduction of glass may produce severe heat load or thermal lensing effects. In spite of the disadvantages mentioned above, Nd:glass fs lasers are competitive for ophthalmologic applications. At the center wavelength of Nd:glass laser (1.06 μm), both protein and water show high transparency, and ocular tissues ranging from cornea to retina can be readily accessed without significant attenuation. Previous study reveal that the corneal ablation threshold fluence scales with the square root of pulse width τ , therefore shorter fs pulse can achieve higher ablation precision. However, ultrashort pulse may also induce undesired nonlinear effects like self focusing or self phase modulation. Since the threshold power for self focusing is on the order of a few MW  and the typical pulse energy for corneal surgery is on the order of a few μJ, the Nd:glass lasers (τ=500 fs ~ 1 ps) appear more favorable than Ti:S lasers (τ = 10 fs ~ 150 fs) to avoid self focusing. It turns out that Nd:glass lasers will never be general purpose ultrafast lasers like Ti:S lasers, but they seems to be better suited for specific applications in ophthalmology.
The basic layout of the Nd:glass fs surgical laser system is schematically illustrated in Fig. 1. The fs seed pulse is generated by a commercial Nd:glass oscillator pumped by two 1.2 W laser diodes (High Q, Vienna, Austria). The self-starting, stable mode-locking is accomplished by SESAM as one of the end mirrors. The pulse width of the mode-locked pulses is 180 fs (measured by an APE Autocorrelator, APE, Berlin, Germany). The laser repetition rate is 90 MHz, carrying an average power of 90 mW. The seed pulses are amplified by Chirp-Pulse-Amplification (CPA). For a compact setup, a single transmission holographic grating (Littrow angle incidence) is utilized for both stretcher and compressor. After the Faraday isolator, the stretched pulses are coupled to the regenerative amplifier through a 4 KHz Lithium Niobate (LiNbO3) Pockels Cell (LaserMetrics, Saddle Brook, U.S.A.), which is synchronized to the oscillator’s mode-locking frequency. The Nd:glass regenerative amplifier is based on a V-cavity configuration. The Brewster-cut Nd:glass crystal is end-pumped by a 2 W laser diode and water cooled to reduce the thermal load. As soon as the pulse energy approaches the maximum value of about 25 μJ after approximate 100 round trips in the regenerative amplifier, the amplified pulse is rejected from the cavity. After the compressor, the final amplified laser pulse (500 fs FWHM) is guided to the target tissue (fresh enucleated porcine eye) through a lens pair with variable focal length (Z scan) and two galvanometer mirrors enabling rapid XY scan. The diameter of the focused laser spot is around 5 μm (single TEM00 mode).
3. Nonlinear second harmonic cornea imaging
Collagen, as the major component of corneal tissue, displays the unique properties of second harmonic generation . Since SHG is an intrinsic process, fixation or staining procedures are not necessary. As a two-photon excited process, most advantages of multi-photon laser scanning microscopy are shared by SHG imaging. The experimental implementation of SHG imaging usually is rather straightforward: in this study, only a detector filter change in the multi-photon laser scanning microscope is required. SHG imaging was performed on a Zeiss LSM 510 NLO laser scanning multi-photon microscope (Zeiss, Jena, Germany). The excitation laser source was a mode-locked Ti:S laser (Coherent Mira, Coherent Inc, Santa Clara, USA), tunable from 720 to 980 nm, pumped by a solid state laser (Verdi, 8 W, Coherent Inc.). The Ti:S laser emission wavelength was set to 820 nm, which generated the strongest SHG signals in cornea samples. Laser intensity attenuation was implemented using an Acoustic Optic Modulator (AOM, Zeiss). A 40×/0.8 numerical aperture (N.A.) water immersion objective was used for high resolution imaging of the cornea sample. Due to the coherent generation of the second harmonic signal from bulk collagen, the signal is emitted predominantly in transmission direction. A Zeiss 1.4 N.A. oil immersion condenser was employed to collect the transmission SHG signal. Two IR beam block filters in sequence (Zeiss KP685) and a narrow bandpass filter (410/10 nm) in front of the transmission light path photomultiplier tube ensured that illumination light was rejected and only second harmonic signals from the corneal tissue were recorded. The acquisition of a single 512 × 512 pixel image was generally achieved within a few seconds (fast laser scan with galvanometer scanners) and a typical image stack of porcine cornea could be acquired within 20 minutes (Z Stack size ≈ 1.5 mm, Z step size: 5 μm). More detailed descriptions of SHG corneal imaging can be found in our recent publication .
Figure 2 shows an image pair of fluorescence (a) and SHG (b) imaging of a corneal specimen stained with 4′,6-Diamidino-2-phenylindole (DAPI). In Fig. 2(a), two nuclei of the keratocyte cells (the most important cells for generating new collagen fibers) were visualized with strong contrast against the surrounding stromal tissue. However, due to lack of fluorescence signal, the corneal stroma remained invisible. Various staining protocols have been tried but proved unsuccessful. In contrast, SHG imaging nicely revealed the collagen fiber structure with diffraction limited resolution and satisfactory image contrast, up to a depth of 1500 μm. As shown in Fig. 2(b), at the depth of 200 μm, most collagen fibers shared the orientation with their neighbors, except that in a few places the adjacent collagen fibers were arranged in right angles, agreeing well with the histological findings. SHG imaging appears to be an efficient, simple and reliable method to non-invasively analyze the fine structures of corneal stroma. Therefore, in the following sections, all laser treated corneal samples were probed by SHG imaging without applying any staining.
4. Microscopic performance of femtosecond laser intrastromal surgery
One preliminary application of Nd:glass fs laser in refractive surgery was flap cutting for LASIK, which was carried out by spirally scanning the Nd:glass laser at a fixed focusing depth. The laser produced flap was manually separated from the corneal substrate and kept in Phosphate Buffered Saline (PBS, pH 7.4) solution for microscopic investigations. An optical stack was recorded in the central flap region. The YZ section of the corneal flap is presented in Fig. 3(a). The posterior surface of the corneal flap corresponds to the laser resection plane (indicated by arrows). The fs laser-produced corneal flap demonstrated a homogenous thickness of 260 μm, agreeing well with the expected value. The average roughness was below 5 μm, which is comparable to the performance of mechanical microkeratome. However, fs laser flap cutting is more deterministic, safe and independent on surgical skills.
Besides their applications for flap cutting in LASIK, fs lasers also offer the novel concept of intrastromal vision correction . By applying specific intrastromal ablation patterns, the corneal curvature and refractive power can be modified by removal of corneal tissue or deformation of the collagen fibers through laser plasma or shock wave compression. One of the corneal intrastromal cavities generated by laser ablations is shown in Fig. 3(b). The laser ablation plane was located in the middle of the image. Due to missing of the collagen tissue, high image contrast was produced at the boundaries of fs laser induced intrastromal cavities. The anterior and posterior surfaces of the intrastromal cavities are well separated, explaining the easy opening of the corneal flap. Immediately after laser treatment, the cornea appeared to be opaque due to the cavitation bubbles induced by laser plasma. These gas bubbles subsequently vanished, presumably by either diffusion or by pumping by the endothelium cell layer. After collapse of the intrastromal cavities, cornea regained its transparency within hours. Concerning side effects, laser produced microstreaks were found both in front and behind the laser focus, with a mean diameter of 2 μm and an average length exceeding 60 μm(Fig. 3(b)). Previous TEM studies after fs Ti:S laser treatment revealed similar streak formation . Most likely the streaks resulted from the low numerical aperture (N.A.) focusing optics. In order to achieve a large scanning field up to 10 mm, a low N.A. (0.2) lens was utilized to guide the surgical beam. The laser power along the Z axis outside the focus center probably was still high enough to induce these photodamage effects, either by destroy of collagen fibers or by impairing their SHG ability in the microstreaks. The tissue healing process and the influences of these microstreaks on the visual acuity and on the long term outcome of refractive surgery are still under investigation.
5. Conclusion and discussions
The main goals of biomedical laser applications in ophthalmology are mini-invasive surgery and non-invasive diagnostics. Fs lasers may play important roles in both fields. Nd:glass fs lasers give rise to several mini-invasive intrastromal surgical strategies like flap cutting, penetrating keratoplasty and intrastromal vision correction. Compared with the Ti:S laser, the Nd:glass fs laser appears to be more suitable for clinical applications with the advantages of self-starting mode locking, direct diode pumping and low cost. With NIR wavelength, Nd:glass lasers enable intrastromal corneal or retinal surgery. With fs pulsewidth, multiphoton absorption-initiated ablation leads to more repeatable and predictable surgical outcomes and the laser plasma mediated ablation excludes the involvement of severe thermal damage or heating effects. As an all-optical-surgical process, intrastromal vision correction can be particularly valuable for accurate corrections of high order aberrations or post LASIK or -PRK enhancement. Complications as incomplete flap, tissue scar and cutting-induced aberrations associated with LASIK may be avoided. To prove the concept of fs laser intrastromal surgery, the corneal sample was non-invasively probed by SHG imaging. Corneal intrastromal structures induced by fs laser ablations were successfully revealed with diffraction limited resolution, high penetration depth and strong image contrast. Neither fixation nor any slicing or labelling procedure were required. The high precision of intrastromal fs laser surgery and minimal tissue damages were nicely confirmed by SHG imaging.
In this study, surgical and imaging investigations were conducted separately. Thus the corneal structure could not be recorded immediately after laser ablation. Also, an exact comparison between pre- and post-laser treatment in the same region of interest was impossible since the sample had to be dislocated. It might be interesting to integrate both surgical and probing functions into a single fs laser system. Since the fs Nd:glass surgical laser system consists of an oscillator and a regenerable amplifier, the seed pulse from the oscillator (τ ≈ 180 fs, E ≈ 1 nJ, f = 90 MHz) may be utilized as the probing beam, and the amplified pulses (τ ≈ 500 fs, E ≈ 1~5 μJ, f = 1 ~ 4 kHz) could serve as a surgical beam. Thus the surgical performance can be visualized in vivo by temporally subdividing the laser treatment procedure into a surgical and an imaging phase. However, SHG from cornea is predominantly in the transmission direction, the backscattered second harmonic signals from the corneal stroma, intraocular lens or retinal layer might be too weak. Immediate clinical applications of SHG imaging for refractive surgery appears not feasible at the moment. Compared with living human eye, the excised cornea sample in appropriate tissue culture media seems more preferable for in vivo study. The intrastromal cavity dynamics, the collagen fiber distortion due to laser ablation and the tissue healing process could be monitored as the functions of time by transmission SHG imaging. The combination of fs laser surgery and SHG imaging might therefore be useful to understand ultrafast laser-tissue interactions, to provide simultaneous guide to the development of next generation fs surgical lasers and to explore novel fs laser intrastromal surgical strategies.
The authors thank M. Walter for stimulating discussions, F. Loesel, M. Weinacht and R. Kessler from 20/10 PerfectVision for laser treatment, and W. Denk from Max-Planck Institute for Medical Research for giving generous access to the microscopic facility. This work was partially supported by the BMBF Femtosecond Technology (FST) project.
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