Open Access
Add to BibSonomyAbstract
The use of nanojoule femtosecond pulses (NFP) for highly precise proceeding in anti-glaucoma surgery was evaluated. According to the observation of scanning electron microscopy (SEM), four types of incision patterns, including subsurface, slit-like, spot and cuboid ablations, were accomplished on in vitro sclera by NFP with little collateral damage. In comparison to microjoule femtosecond pulses (MFP), NFP can make extremely precise incisions with smoother inner surface with less peak power density. The present study first illustrates the potential use of NFP in minimally invasive laser sclerectomy for glaucoma therapy.
© 2015 Optical Society of America
1. Introduction
Glaucoma is the leading cause of irreversible blindness in the world [1]. Filtration surgery is the main treatment when medicine cannot control the disease. It decreases the intraocular pressure by constructing a transscleral aqueous outflow passway between anterior chamber and subconjunctival space. However, the surgery risks to fail due to fibrosis of incisions resulting from wound healing process in response to surgically-induced tissue trauma [2]. Hence, the fewer traumas the sclera suffers during the surgery, the less fibrosis will be [3].
Femtosecond laser provides us a favorable way for minimally invasive surgery. It has been demonstrated to be a promising tool to create precise incisions with little collateral damage in various ophthalmic tissues such as cornea, lens and sclera [4–6 ]. To date, several groups have managed to do some research operating on sclera with femtosecond laser. Sacks et al. have reported subsurface sclera photodisruption by either using a 1700 nm wavelength femtosecond laser or dehydrating the sclera in vitro [7, 8 ]. Bahar et al. have achieved non-penetrating deep sclerectomy on in vitro eye ball [9]. Chai et al. have demonstrated that in vivo subsurface ablation of sclera can reduce intraocular pressure [10]. Our group have proven the ability to perform precise and minimally invasive sclerectomy with femtoseond laser in rabbits [11, 12 ]. However, the pulse energies applied in the above-mentioned experiments have been at least at the level of microjoule.
It has been illustrated that the low energy minimizes collateral damage [13]. Nanojoule femtosecond pulses (NFP), compared to the microjoule femtosecond pulses (MFP), attenuated cell death, wound repairing reaction and inflammation adjacent to the photodisruptive site [14]. Given to the fact that inflammatory process plays an important role in the pathogenesis of fibrosis [15], we infer that the use of nanojoule pulses may minimize fibrosis and provide better outcome with filtration surgery. In addition, as pulse energy threshold decreases with the increase of repetition rate [16], we assume that NFP with an enough high repetition rate of 50 MHz may also help minimize collateral damage to the target tissue. To date, however, few reports have described successful sclerectomy with such a low pulse energy and high repetition rate.
The present study aims to explore the feasibility of photodisruption of sclera with NFP of 50 MHz and observe the morphological and biological changes of sclera. Furthermore, we compare the sclera incision created with NFP and MFP to seek for the differences.
2. Methods and materials
2.1 Experimental setup of micro-fabrication system
The setup of the system was illustrated in Fig. 1 . The laser source was an Yb-doped large-mode-area (LMA) photonic crystal fiber (PCF) femtosecond laser amplifier system. The system consisted of a LMA PCF oscillator and an amplifier. The amplifier provided an output of 85 fs pulses at a wavelength of 1040 nm with a repetition rate of 50 MHz. The maximum average output power was 10 W.
A combination of one half-waveplate (λ/2) and a polarization-beam-splitter (PBS) were inserted after femtosecond laser source to adjust power of laser on the sclera samples, which were placed on a computer controlled three dimensional (3D) micro-displacement stage. The spot sizes on the samples were 3-4 μm after passing through an objective lens (OL) with 0.4 NA and 20 mm working distance (20 × Mitutoyo Plan Apo NIR Infinity). A charge coupled device (CCD, Chameleon CMLN-13S2M) provided real time view of ablation processes. Dichroic mirrors (DM) were used to lead the laser to the OL and help combine a CCD to the system. A beam splitter (BS) between DM and CCD separated the laser light before it arrived at CCD. And the Light Emitting Diode (LED) under the BS functioned as a lighting device. The computer controlled the movement of 3D micro-displacement stage, received and recorded signs from the CCD. Spot energy distribution obeyed Gaussian distribution.
Sclera samples were mounted between two 1-mm-thick microscope slides with inner surface facing outward. The slides were fixed on the computer controlled 3D micro-displacement stage. The stage could move as we designed in computer in order to enable different patterns of the laser focus and scan on sclera pieces.
2.2 Preparation of sclera
New Zealand white rabbits used in this study were provided by Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Rabbits were sacrificed with injection of excessive 3% pentobarbital sodium solution. The eyes were enucleated immediately. After carefully distracting of conjunctiva and choroid, the full thickness sclera was cut into several 3 × 10 mm sized rectangle pieces. The sample pieces were stored in physiological saline solution in 4°C before use. All experiment operations were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were treated in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care.
2.3 Four different patterns of scanning
2.3.1 Linear scanning
In linear scanning, sclera samples were irradiated in a 1.5 mm-long straight line pattern by NFP.
In order to test the threshold pulse energy for sclera photodisruption with NFP, pulse energies ranging from 5 to 35 nJ were applied on the surface of sclera with a scan speed of 100 μm/s and corresponding exposure time of 50 ms.
NFP with various pulse energies (10-40 nJ), exposure times (10-50 ms) and focal depths (20-60 μm subsurface) were applied to seek relationships between the laser parameters and the depth of incisions.
2.3.2 Zoom scanning
The samples were scanned in the pattern of Fig. 2(a) . The focal depth of first line was 10 μm subsurface. The focal depth of last line was 45 μm subsurface. The distance of two adjacent lines was 5 μm. The length of each line was 1 mm. The exposure time was 25 ms. Pulse energies ranged from 10 nJ to 30 nJ.
Fig. 2 Two types of scanning patterns (a) Zoom scanning; (b) Raster scanning. The arrows represent the direction of laser scan.
2.3.3 Raster scanning
The pattern of raster scanning was shown in Fig. 2(b). The focus depth was 10 μm subsurface. The pulse energy was 30 nJ. The exposure time was 50 ms. The length of each line was 200 μm. The number of scanning lines was 25. The distance of two adjacent lines varied from 3 μm to 10 μm.
2.3.4 Spot scanning
The laser beam was focused at one fixed point to achieve spot ablation. The pulse energy was 30 nJ, The exposure time was 100 ms, The focus depth was 10 μm subsurface.
2.4 Observations of samples
The sclera samples exposed to femtosecond laser were soaked in 2% glutaraldehyde solution for 24h. After a series of standard process, the samples were examined by scanning electron microscopy (SEM, VEGA 3 LMU, TESCAN) to observe morphological change.
2.5 Bubble formation induced by NFP
The formation and localization of NFP-induced bubbles following linear scanning treatment on the surface of the sclera were real-time monitored with CCD. The observation results were acquired from the focal site in the condition of 30 nJ pulses with 50 ms exposure time.
2.6 Comparison of ablations by NFP and MFP
The results of MFP photodisruption on sclera in our previous studies were used for the comparison. In linear scanning pattern, the threshold peak power density needed for creating detectable morphological change on sclera with the two laser sources was investigated. In addition, the peak power density used to achieve similar depth of incision with the two lasers was discussed. Furthermore, in raster scanning pattern, the inner surface roughness of incisions induced by NFP and MFP was compared.
2.7 Surface roughness measurements
An image analysis software Image J was used to obtain the contour line of the surface on the SEM cross section image of incision induced by raster and zoom scanning. Five contour lines of the surface were taken on each sample with an evaluation length of 100 μm. Arithmetical mean deviation of the profile (Ra) was calculated according to ISO 4288 standards.
2.8 Statistics
For roughness, the values of Ra were expressed as the mean and SD. The unpaired t-test with Welch's correction was used to assess the difference of the surface roughness between NFP and MFP induced incisions. P < 0.05 was considered statistically significant. For the relation between the incision depth and laser parameters, the data was analyzed statistically with a linear correlation model by applying the SPSS for Windows in the version of 12.0, with P < 0.05 indicating significance.
3. Results
3.1 Linear scanning
3.1.1 Threshold pulse energy for sclera photodisruption by NFP
As observed by CCD after scanning immediately [Figs. 3(a) and 3(b) ], when pulse energy dropped to 5 nJ from 15 nJ, there was no detectable lesion on the surface of sample. Two additional pulses with pulse energy of 10 nJ and 7 nJ were applied as followed. It was shown in the Figs. 3(c) and 3(d) that when pulse energy was 7 nJ with corresponding power density of 4.46 × 1011 W/cm2, the pulses just began to produce detectable incision on the sclera. Consequently, the pulse energy of 7 nJ with the corresponding power density of 4.46 × 1011 W/cm2 could be determined as the threshold level for linear scanning in this experiment.
Fig. 3 (a-c) CCD micrographs of sclera surface after linear scanning by 15 nJ, 5 nJ and 7 nJ pulses respectively; (d) SEM image of cross section of incisions created by NFP (the exposure time was 10 ms) with pulse energies ranging from 5 to 35 nJ.
3.1.2 Correlations between NFP parameters and depth of incisions
The incisions made by NFP were 6.7-22.25 μm in width and 4.77-31.27 μm in depth. Of note, the edge of the incision was sharp and smooth. The data regarding the depths of incisions with various pulse energies, focal depths and exposure times were exhibited in Fig. 4 . Quantitative analysis revealed a significant positive correlation between the depths of the incision and both pulse energies [Figs. 4(a) and 4(d), r = 0.9944, P = 0.0028] and laser exposure times [Figs. 4(c) and 4(f), r = 0.9056, P = 0.0083]. In addition, the lesion depths were negatively correlated with the focal depths [Figs. 4(b) and 4(e), r = −0.9783, P = 0.0019].
Fig. 4 Cross section SEM images of NFP-induced linear incisions (a-c) and the correlation between laser parameters and the incision depth (d-f). Arrow: incisions created by NFP. (a) Pulse energies were 10 nJ, 20 nJ, 30 nJ and 40 nJ from left to right (the focal depth was 0 μm subsurface, the exposure time was 50 ms); (b) Focal depths were 60 μm, 50 μm, 40 μm, 30 μm, 20 μm below the surface from left to right (45 nJ pulse energy, 10 ms exposure time); (c) The exposure time was 10 ms, 12.5 ms, 17 ms, 25 ms and 50 ms from left to right (the pulse energy was 45 nJ, the focal depth was 60 μm subsurface); (d) The positive correlation obtained between the depths of the incision and pulse energies (r = 0.9944, P = 0.0028); (e) The negative correlation found between incision depths and focal depths (r = −0.9783, P = 0.0019); (f) The positive correlation obtained between depths of the incision and exposure times (r = 0.9056, P = 0.0083).
3.2 Slit-like and subsurface incisions induced by zoom scanning of the laser focus
Three types of incisions came into being after zoom scanning. When the pulse energy was 10 nJ, subsurface photodisruption without destructive damage on the surface of sclera was produced [Fig. 5(a) ]. When the pulse energy turned to 20 nJ, a slit-like incision came into being [Fig. 5(b)], which possessed of following features. Firstly, it was a precise incision, of which the inner and external widths were nearly the same in a small value of 7.09 μm. Furthermore, the inner surface of incision was extremely smooth (Ra = 0.93 ± 0.14 μm). In addition, the ablation could reach the level as deep as the beam had focused on. When the pulse energy was increased to 30 nJ, strong thermal and mechanical collateral damage such as coagulation, shrinking and lose of normal layer appeared in the tissue adjacent to the incision [Fig. 5(c)].
Fig. 5 NFP created incisions in zoom scanning pattern. Laser parameters: the exposure time was 25 ms, the focal depth of first line was 10 μm subsurface, the focal depth of last line was 45 μm subsurface, distance of two adjacent lines was 5 μm; (a-c) The pulse energy was 10 nJ, 20 nJ and 30 nJ respectively.
3.3 Raster scanning
A cuboid ablation which size was 200 μm long and 150 μm wide was successfully created by 30 nJ laser pulses with exposure time of 10 ms and the focal depth of 10 μm subsurface. The SEM observation indicated that a regular rectangular solid tissue removal could be achieved with little evidence of collateral damage to the surrounding tissue (Fig. 6 ) by applying a 6-μm-wide interspace between the collateral lines while scanning. The inner surface of the photodisruption was measured to be extremely smooth (Ra = 1.26 ± 0.20 μm).
Fig. 6 Raster scanning (the pulse energy was 30 nJ, the exposure time was 50 ms, the focal depth was 10 μm subsurface). (a) Cross section of the incision; (b) Plane observation of the incision.
3.4 Spot ablation
As shown in Fig. 7 , a full thickness spot photodisruption formed a channel perpendicular to the sclera surface. The diameter of spot was 18.8 μm. The exposure time of the spot was 100 ms with 30 nJ pulses. The photodisruption of the spot was nearly ideal round in shape with smooth inner surface, causing minimal damage to neighbouring tissues.
Fig. 7 Transscleral spot ablation image. (the pulse energy was 30 nJ, the exposure time was 100 ms, the focal depth was 10 μm).
3.5 Bubble formation induced by NFP
A maximum mean diameter of 6.1 ± 1.32 µm of intratissue bubbles was recorded. Figure 8 shows the CCD image of the bubble in the focal region of the sclera by 30 nJ pulses with an exposure time of 50 ms.
Fig. 8 The CCD image of the intratissue bubble induced by NFP with 30 nJ pulse energy and 50 ms exposure time in the focal region.
3.6 Comparison of ablations by NFP and MFP
In the present study, the differences between the NFP and MFP used for creating photodisruption in sclera were concluded. Firstly, the threshold peak power density for sclera photodisruption was 4.06 × 1014 W/cm2 with MFP, which was three orders of magnitude larger than that with NFP (4.46 × 1011 W/cm2). Second, to achieve similar depth of ablation, peak power density needed with MFP was 3.84 × 102 – 5.64 × 102 times larger than that with NFP (Table 1 ). Third, the average of five Ra measurements for the inner surface roughness of incisions induced by the raster pattern of scanning was 1.26 ± 0.20 μm with NFP and 7.05 ± 1.09 μm with MFP. The difference of surface roughness is of significance (P = 0.0135)
Table 1. Differences of Parameters between NFP and MFP in Achieving Similar Incision Depth
4. Discussion
Femtosecond laser has been widely used in medical field [17]. It is a promising tool to create precise incisions with little collateral damage. Femtosecond-induced tissue dissection is achieved by photodisruption based on plasma-mediated ablation associated with mechanical effects (shock waves and cavitation bubbles) [5]. Unwanted femtosecond-induced collateral damage is mainly induced by photodisruptive effects of bubble dynamics [18], which is positively related to laser pulse energy applied [19].
So far, most of femtosecond laser operations have been based on utilization of laser devices with single-pulse energy around microjoule-scale, with which destructive effects by bubble formation and self-focusing effects may occur [16, 20 ]. It is reported that the maximum diameters of MFP-induced intratissue bubbles in cornea range from 23μm to more than 50 μm [19, 21, 22 ]. The formation of large intratissue bubble has been proved to be harmful for the highly precise tissue processing by inducing destructive shock waves and streak formation [20, 22 ].
To solve these problems, NFP are taken into consideration. NFP induced intratissue bubbles in cornea with a maximum diameter of 5 µm have been reported [20]. The formation of smaller bubbles with NFP makes it possible to create incisions with minimized intratissue damage and higher precision [22]. In addition, NFP-induced low-density plasmas offer the possibility to produce localized dissection of tissue because of the nonlinearity of the plasma formation process which could produce the plasma in a volume smaller than the diffraction limited focus [23]. The well localized precise dissection with little collateral damage contributes to the formation of fairly smooth inner surface of incisions by NFP. Furthermore, NFP tissue ablation supplies more predictable and reproductive outcomes while excluding severe thermal damage and mechanical effects [24, 25 ].
To date, NFP have been applied in precise operations in tissues such as corneas [20, 26, 27 ], lens [28], retinas [29], muscles [30], intestines [31] and skins [16], nanosurgery in cells [32, 33 ], and targeted transfection [34, 35 ]. In ophthalmology, NFP are mainly used in refractive surgery by focusing tissue through transparent medium. Yet NFP ablation on highly scattering sclera, which plays a vital role in anti-glaucoma surgery, has not been accomplished so far.
In this study, we explore the feasibility of NFP to create highly precise and minimally invasive incisions on in vitro sclera and investigate the advantage of NFP on sclerectomy over MFP.
4.1 Linear scanning
In the present study, only when pulse energy reached 7 nJ or above, photodisruption began to appear in hydrated sclera in vitro. Below this value, NFP focused on the target sample without tissue removal. Consequently, the pulse energy of 7 nJ with the corresponding power intensity of 4.46 × 1011 W/cm2 could be determined as the threshold level for linear scanning with NFP.
Furthermore, we can conclude that the depth of photodisruption increases with both pulse energy and exposure time, and falls with increasing focal depth of laser beam. The seeking for corresponding relationship of laser parameters and sclera morphological change will help set laser parameters for sclera surgery. In addition, improved accuracy of laser fabrication and more controllable ocular laser surgery by NFP may provide more predictable and reproductive surgical outcomes.
4.2 Subsurface and slit-like incisions induced by zoom scanning
As mentioned above, Sacks et al. have reported that subsurface ablation is possible at a wavelength of 1700 nm on hydrated sclera as absorption and scattering of sclera is minimized at that wavelength [8]. In addition, with femtosecond laser at 1040 nm, subsurface removal could also be accomplished on dehydrated sclera with improved translucency by the utilization of the dehydrating agent [7]. However, the water absorption of pulses with long wavelength reduces the ablation efficiency [8] and the dehydrating agent would alter the nature optical characteristics of the sclera. As to our experiment, samples we utilized are hydrated sclera with highly scattering property at a wavelength of 1040 nm. The current study demonstrates the feasibility of subsurface photodisruption on hydrated sclera, for the first time to our knowledge, with femtosecond laser at 1040 nm. Possible explanation of this phenomenon may be illustrated as following. During the proceeding of zoom scanning, the first linear scan on the surface of sclera with pulse energy of 10 nJ and exposure time of 25 ms might create a incision less than 5 μm in depth. When the beam focused on the next level, which was 5μm deeper than the previous one, there might not be enough power density for photodisruption on the sclera tissue. In this condition, dehydration of sample might occur due to low peak power on the target [36]. Given to the fact that dehydration increases sclera transparency by reducing scattering, we infer that the target layer of sclera might be focusable after several scans of NFP with low peak power. As a result, subsurface ablation with NFP at 1040 nm is realized. Such a phenomenon suggests the possibility of transscleral sclerectomy without damaging the outer layers, which may aid in attenuating post-operation fibrosis by avoiding conjunctival and episcleral damage.
Intriguingly, the same pattern of zoom scanning with increased pulse energies resulted in other two types of incisions. When the pulse energy turned to 20 nJ, a slit-like incision came into being. This type of incision is characterized by smooth surface, high precision and minimal collateral damage. These advantages of the incision may make NFP a suitable alternative to mechanical blade for sclera flap creation in filtration surgery. The other type of incision was the one with apparent collateral damage (structural damage of tissue around the incision) shown in Fig. 5(c), when the pulse energy was increased to 30 nJ. These observations may be a useful guide for selecting the most effective laser parameters for future clinical applications of NFP sclerectomy surgery.
4.3 Raster scanning
In raster scanning pattern, a cuboid ablation sclera removal with precise geometry and extremely smooth inner surface was produced. It indicated that the NFP may be a promising tool for precisely and efficiently skiving scleral tissue, which allows controlled escape of aqueous humor from the sclerectomy site into the subconjunctival space. Furthermore, the smooth surface which helps decrease the attachment of fibroblast and aids in less post-operation fibrosis could reinforce the treatment efficiency of filtration surgery.
4.4 Spot ablation
Full thickness sclera spot photodisruption created by NFP in this study has managed to form a fistula on the sclera, which has the potential to function as a micro aqueous drainage passway in glaucoma treatment. For its distinct advantage of precise ablation and little collateral damage, this spot ablation with NFP is expected to take the place of the outflowing instruments, which are currently widely used in anti-glaucoma surgery yet usually cause significant destructive damage to scleral and conjunctival regions and consequently scar formation.
4.5 Bubble formation induced by NFP
As is observed in this study, the maximum diameter of the intratissue bubble is measured 6.1 ± 1.32 µm, which is much smaller than previously reported diameters of intratissue bubbles generated by MFP [19, 21, 22 ]. The relatively small bubble size obtained in our study is within reasonable agreement with the measured value reported by König et al. [20]. The formation of small bubbles in scleral tissue with NFP may make it possible to realize a desired highly localized disruptive effect with little collateral damage.
4.6 The comparison between NFP and MFP
In the present study, by comparing photodisruption created by NFP and MFP, NFP exhibited distinct advantages in performing laser sclerectomy surgery [11]. First, NFP decrease the threshold power density for sclera ablation by almost two orders of magnitude in comparison to microjoule pulses. Second, the peak power density needed to achieve similar depth of ablation with NFP is two orders of magnitude lower than that with MFP. Remarkable decrease of peak power density and pulse energy needed for ablation means notable reduction of lesion of surrounding tissues [36]. Third, inner surface roughness of NFP-induced incision decreases significantly (P = 0.0135) in comparison to that induced by MFP, which may take the advantage of the absence of large intratissue bubbles during ablation. Conceivably, NFP are able to create more precise incisions with less collateral damage than MFP. Additionally, the smoother surface of incision induced by NFP may help prevent fibrosis by impeding fibroblast cell adhesion [37].
5. Conclusion
In the present study, we have demonstrated extremely precise and minimally invasive photodisruption on hydrated sclera in vitro with NFP using an Yb-doped LMA PCF femtosecond laser amplifier system at 1040 nm wavelength. In addition, four types of incision patterns, including subsurface photodisruption, slit-like incision, spot and cuboid ablation have been accomplished by NFP with potential use for anti-glaucoma surgeries. Finally, the comparison of NFP and MFP sclerectomy suggests that the potential anti-fibrosis effect of NFP is superior to MFP in the filtration surgery. Our goal is not only to provide parameter basis for in vivo study, but also to further detail the size, shape, placement and design of micro-fabrication patterns of sclerectomy with NFP for future clinical applications.
Acknowledgments
The work described in this paper has been supported from the National Natural Science Foundation of China, the Fundamental Research Funds for the Central Universities of China (No.2011JC044) by the Grant no. 81100701 and the Tianjin City S&T Project (Grant No.13RCGFGX01122). None of the authors have any actual or potential conflict of interests to disclose.
References and links
1. D. Y. Yu, W. H. Morgan, X. Sun, E. N. Su, S. J. Cringle, P. K. Yu, P. House, W. Guo, and X. Yu, “The critical role of the conjunctiva in glaucoma filtration surgery,” Prog. Retin. Eye Res. 28(5), 303–328 (2009). [CrossRef] [PubMed]
2. L. K. Seibold, M. B. Sherwood, and M. Y. Kahook, “Wound modulation after filtration surgery,” Surv. Ophthalmol. 57(6), 530–550 (2012). [CrossRef] [PubMed]
3. S. Georgoulas, A. Dahlmann-Noor, S. Brocchini, and P. T. Khaw, “Modulation of wound healing during and after glaucoma surgery,” Prog. Brain Res. 173, 237–254 (2008). [CrossRef] [PubMed]
4. H. K. Soong and J. B. Malta, “Femtosecond lasers in ophthalmology,” Am. J. Ophthalmol. 147(2), 189–197 (2009). [CrossRef] [PubMed]
5. A. Vogel and V. Venugopalan, “Mechanisms of pulsed laser ablation of biological tissues,” Chem. Rev. 103(2), 577–644 (2003). [CrossRef] [PubMed]
6. G. Kullman and R. Pineda 2nd, “Alternative applications of the femtosecond laser in ophthalmology,” Semin. Ophthalmol. 25(5-6), 256–264 (2010). [CrossRef] [PubMed]
7. Z. S. Sacks, R. M. Kurtz, T. Juhasz, and G. A. Mourau, “High precision subsurface photodisruption in human sclera,” J. Biomed. Opt. 7(3), 442–450 (2002). [CrossRef] [PubMed]
8. Z. S. Sacks, R. M. Kurtz, T. Juhasz, G. Spooner, and G. A. Mouroua, “Subsurface photodisruption in human sclera: wavelength dependence,” Ophthalmic Surg. Lasers Imaging 34(2), 104–113 (2003). [PubMed]
9. I. Bahar, I. Kaiserman, G. E. Trope, and D. Rootman, “Non-penetrating deep sclerectomy for glaucoma surgery using the femtosecond laser: a laboratory model,” Br. J. Ophthalmol. 91(12), 1713–1714 (2007). [CrossRef] [PubMed]
10. D. Chai, G. Chaudhary, E. Mikula, H. Sun, R. Kurtz, and T. Juhasz, “In vivo femtosecond laser subsurface scleral treatment in rabbit eyes,” Lasers Surg. Med. 42(7), 647–651 (2010). [CrossRef] [PubMed]
11. X. Yang, N. Dai, H. Long, P. Lu, W. Li, and F. Jiang, “Experimental femtosecond laser photodisruption of rabbit sclera for minimally invasive laser sclerostomy: An in vitro study,” Opt. Lasers Eng. 48(7-8), 806–810 (2010). [CrossRef]
12. Y. Shi, X. B. Yang, N. L. Dai, H. Long, P. X. Lu, L. Jin, and F. G. Jiang, “External sclerostomy with the femtosecond laser versus a surgical knife in rabbits,” Int. J. Ophthalmol. 5(3), 258–265 (2012). [PubMed]
13. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13(10), 3690–3696 (2005). [CrossRef] [PubMed]
14. A. K. Riau, Y. C. Liu, N. C. Lwin, H. P. Ang, N. Y. Tan, G. H. Yam, D. T. Tan, and J. S. Mehta, “Comparative study of nJ- and μJ-energy level femtosecond lasers: evaluation of flap adhesion strength, stromal bed quality, and tissue responses,” Invest. Ophthalmol. Vis. Sci. 55(5), 3186–3194 (2014). [CrossRef] [PubMed]
15. G. C. Gurtner, S. Werner, Y. Barrandon, and M. T. Longaker, “Wound repair and regeneration,” Nature 453(7193), 314–321 (2008). [CrossRef] [PubMed]
16. D. X. Hou, D. L. Butler, L. M. He, and H. Y. Zheng, “Experimental study on low pulse energy processing with femtosecond lasers for glaucoma treatment,” Lasers Med. Sci. 24(2), 151–154 (2009). [CrossRef] [PubMed]
17. S. H. Chung and E. Mazur, “Surgical applications of femtosecond lasers,” J. Biophotonics 2(10), 557–572 (2009). [CrossRef] [PubMed]
18. K. Konig, B. Wang, O. Krauss, I. Riemann, H. Schubert, S. Kirste, and P. Fischer, “First in vivo animal studies on intraocular nanosurgery and multiphoton tomography with low-energy 80-MHz near-infrared femtosecond laser pulses,” Proc. SPIE 5314, 262–269 (2004). [CrossRef]
19. H. Lubatschowski, G. Maatz, A. Heisterkamp, U. Hetzel, W. Drommer, H. Welling, and W. Ertmer, “Application of ultrashort laser pulses for intrastromal refractive surgery,” Graefes Arch. Clin. Exp. Ophthalmol. 238(1), 33–39 (2000). [CrossRef] [PubMed]
20. K. Koenig, O. Krauss, and I. Riemann, “Intratissue surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared,” Opt. Express 10(3), 171–176 (2002). [CrossRef] [PubMed]
21. T. Juhasz, G. A. Kastis, C. Suárez, Z. Bor, and W. E. Bron, “Time-resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water,” Lasers Surg. Med. 19(1), 23–31 (1996). [CrossRef] [PubMed]
22. B. G. Wang, C. P. Lohmann, I. Riemann, H. Schubert, K. J. Halbhuber, and K. König, “Multiphoton-mediated corneal flap generation using the 80 MHz nanojoule femtosecond near-infrared laser,” J. Refract. Surg. 24(8), 833–839 (2008). [PubMed]
23. A. Vogel, J. Noack, G. Hüttmann, and G. Paltauf, “Femtosecond-laser-produced low-density plasmas in transparent biological media: A tool for the creation of chemical, thermal and thermomechanical effects below the optical breakdown threshold,” Proc. SPIE 4633, 23–37 (2002). [CrossRef]
24. B. G. Wang and K. J. Halbhuber, “Corneal multiphoton microscopy and intratissue optical nanosurgery by nanojoule femtosecond near-infrared pulsed lasers,” Ann. Anat. 188(5), 395–409 (2006). [CrossRef] [PubMed]
25. M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, “Neurosurgery: functional regeneration after laser axotomy,” Nature 432(7019), 822 (2004). [CrossRef] [PubMed]
26. L. Xu, K. R. Huxlin, M. DeMagistris, N. Wang, L. Ding, and W. H. Knox, “Non-invasive Blue Intra-tissue Refractive Index Shaping (IRIS) In Living, Excised Cornea,” in Frontiers in Optics 2010/Laser Science XXVI, OSA Technical Digest (CD) (Optical Society of America, 2010), paper PDPA11.
27. L. Xu, W. H. Knox, and K. R. Huxlin, “Exogenous and endogenous two-photon absorption for Intra-tissue Refractive Index Shaping (IRIS) in live corneal tissue [Invited],” Opt. Mater. Express 1(7), 1159–1164 (2011). [CrossRef]
28. L. Ding, W. H. Knox, J. Bühren, L. J. Nagy, and K. R. Huxlin, “Intratissue refractive index shaping (IRIS) of the cornea and lens using a low-pulse-energy femtosecond laser oscillator,” Invest. Ophthalmol. Vis. Sci. 49(12), 5332–5339 (2008). [CrossRef] [PubMed]
29. M. Hild, M. Krause, I. Riemann, P. Mestres, S. Toropygin, U. Löw, K. Brückner, B. Seitz, C. Jonescu-Cuypers, and K. König, “Femtosecond laser-assisted retinal imaging and ablation: experimental pilot study,” Curr. Eye Res. 33(4), 351–363 (2008). [CrossRef] [PubMed]
30. S. Yavaş, M. Erdogan, K. Gürel, F. O. Ilday, Y. B. Eldeniz, and U. H. Tazebay, “Fiber laser-microscope system for femtosecond photodisruption of biological samples,” Biomed. Opt. Express 3(3), 605–611 (2012). [CrossRef] [PubMed]
31. M. Choi and S. H. Yun, “In vivo femtosecond endosurgery: an intestinal epithelial regeneration-after-injury model,” Opt. Express 21(25), 30842–30848 (2013). [CrossRef] [PubMed]
32. W. Watanabe, N. Arakawa, S. Matsunaga, T. Higashi, K. Fukui, K. Isobe, and K. Itoh, “Femtosecond laser disruption of subcellular organelles in a living cell,” Opt. Express 12(18), 4203–4213 (2004). [CrossRef] [PubMed]
33. H. He, S. Y. Li, S. Y. Wang, M. L. Hu, Y. J. Cao, and C. Y. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012). [CrossRef]
34. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002). [CrossRef] [PubMed]
35. H. He, S. K. Kong, R. K. Lee, Y. K. Suen, and K. T. Chan, “Targeted photoporation and transfection in human HepG2 cells by a fiber femtosecond laser at 1554 nm,” Opt. Lett. 33(24), 2961–2963 (2008). [CrossRef] [PubMed]
36. A. Hansen, R. Géneaux, A. Günther, A. Krüger, and T. Ripken, “Lowered threshold energy for femtosecond laser induced optical breakdown in a water based eye model by aberration correction with adaptive optics,” Biomed. Opt. Express 4(6), 852–867 (2013). [CrossRef] [PubMed]
37. G. Bolortuya, A. Ebihara, S. Ichinose, S. Watanabe, T. Anjo, C. Kokuzawa, H. Saegusa, N. Kawashima, and H. Suda, “Effects of dentin surface modifications treated with Er:YAG and Nd:YAG laser irradiation on fibroblast cell adhesion,” Photomed. Laser Surg. 30(2), 63–70 (2012). [CrossRef] [PubMed]
