A combined use of corneal ablation procedures and backscattering AO multiphoton microscopy using NIR fs pulses has been demonstrated. Intrastromal patterns can be monitored and tested in ex vivo corneas without the need of fixation or staining. Corneal ablation causes structural changes within the cornea in a controlled manner. Both epithelial cells and the corneal stroma morphology are visualized.
The high photon flux of these ultrafast pulsed light sources, together with a “reduced” mean power, allowed exploring the nonlinear interaction between light and matter. Ultrashort laser disruption of cells or tissues has become an effective technique with very innovative experimental applications [9
]. In particular, these laser systems make multiphoton imaging and micro-processing mechanisms effective when applied to corneal tissues [16
]. The NIR light provided by these sources is convenient due to the transparency of the human eye at this spectral range, increasing the penetration depth for both imaging and ablation.
A number of works have reported different photodisruption patterns produced within the corneal stroma. Their evaluation has often been carried out by means of histological sections [10
] and confocal microscopy [36
], but multiphoton imaging has not been used very often.
Han and colleagues reported individual cavitation bubbles and corneal cuttings induced in porcine corneas (Nd:glass laser, λ = 1.06 μm). These corneas were successfully imaged through nonlinear microscopy in transmission mode. They used a wavelength of 880 nm and an oil immersion objective (NA = 1.4) for signal collection [27
]. Wang and associates [28
] also visualized intrastromal cuts (Ti:sapphire laser, λ = 800 μm) in ex vivo
rabbit corneas. Although their microscope worked in backscattered mode, a high numerical aperture (1.3, oil) objective was used. In rabbit corneas, Morishige and co-authors reported some preliminary results on both backward and forward SHG images of corneal collagen following photodisruption produced by a commercial surgical laser [36
Unlike previous works using water or oil immersion objectives with larger NAs, our multiphoton microscope uses a non-immersion objective in a backscattered mode. Others have reported that this configuration provides weak nonlinear signals from corneas tissues [37
], however our setup provides us with high-quality images of the different corneal layers (see also [38
] for further information). The effectiveness of our microscope was mainly based on the optimization/correction of aberrations, combined with a backscattering configuration. This experimental configuration has been proven to provide high quality nonlinear images (both TPEF and SHG) [29
The ablation process is highly nonlinear and intensity dependent. Then, the affected area is reduced to a tiny region around the focal volume, what allows generating arbitrary geometries with improved accuracy and smoothness. When the analysis of the tissue effects is done through histological slices, artifacts in the size and the shape of the cavities of the ablation might appear as a result of the excessive manipulation of the samples [26
]. Alternatively, nonlinear microscopy offers an efficient tool to analyze the morphology of the corneal stroma in a minimally invasive manner [27
When visualizing the ablated corneal stroma, a decrease in the nonlinear signal of the areas where the ablation took place was observed. This loss of signal is a result of the disruption of the non-centrosymmetric structure of the collagen fibers within the stroma which is the structure associated with SHG signals. This was also shown in the work by Han and colleagues although they did no pay much attention to this “absence of signal” [16
]. Alternatively, other authors observed a strong increase of the nonlinear autofluorescence intensity (TPEF) of the ablated area in corneas [17
] and in other type I collagen tissues [40
]. This was related to the formation of new autofluorescent substances during the surgery process (such as tyrosine dimers). However this effect was not explored in depth either.
The clinical use of fs lasers in refractive surgery is basically restricted to corneal flap cutting [13
]. Ablation procedures are still performed by excimer laser systems [6
]. The flap creation is performed by fs lasers with low repetition rate (in the range from Hz to kHz) by inducing photodisruption and destructive optical breakdown in the more external corneal tissue. The use of fs lasers in conductive keratoplasty has also a great potential in clinical environments [21
]. The main advantage originates from the feasibility for precise cutting of any arbitrary geometry (zig-zag, top hat patterns,…) on both the donor and the recipient eye, increasing resistance and accelerating the healing process.
However recent techniques such as fs lentotomy [23
] and intratissue refractive index shaping (IRIS) [24
] have introduced new insights into research fields such as visual optics and ocular surgery. The former proposes a treatment for presbyopia (loss of flexibility in the crystalline lens with age) through the formation of small cuts inside the lens capsule by fs pulse photodisruption to recover flexibility. The latter creates periodic gratings in the corneal stroma or the cortex of the lens to change the refractive index. This technique may represent an alternative method to classical refractive surgery.
Based on the results reported here, the replacement of excimer laser systems by NIR fs lasers would be of high interest. This might be based on the fact that NIR light propagates with negligible absorption through the tissue. NIR fs ablation procedure (at a higher repetition frequency) provides precise intratissue processing without collateral damage such as mechanical or optical destruction of the epithelial layer.
Although actual experiments used ex vivo
corneas, the results found are indispensable to establish background knowledge, prior to an in vivo
examination approach. To our knowledge only preliminary studies have reported NIR ablation [35
] and multiphoton microscopy in living eyes of animal models [35
]. Future in vivo
applications would be helpful in exploring the collagen reorganization, regeneration, healing processes and disease progression.
To conclude, NIR fs laser technology has been used to perform intrastromal surgery in ex vivo chicken corneas and to image the ablated tissue through multiphoton microscopy. Corneal NIR ablation has been proven to be precise, with minimized thermal effects and surrounded regions hardly affected. The surgical performance has been visualized and precisely evaluated in a non-invasive manner without the use of staining procedures. Although in this work surgical and imaging investigations were conducted sequentially, a further combination of both surgical and monitoring functions into a single fs laser system has the potential to be an attractive tool. Potential in vivo implementations based on the combination of both techniques (ablation plus imaging) would be of special interest in vision research and future clinical environments, mainly for high-resolution imaging of ocular tissues, diagnosis and NIR corneal surgery monitoring.