In this work, we investigate the non-ablative, non-thermal photo-modification of collagen fibers by femtosecond Ti:Sa laser. The effect was induced and simultaneously registered during the repetitive laser scanning of type I collagen (rat tail and bovine Achilles’ tendon), and bovine cornea. An irreversible increase in two-photon autofluorescence and a decrease in second harmonic generation intensities were associated with the collagen femtosecond laser photo-modification. Confocal spectral imaging revealed the formation of new fluorescent species. Controllable nonlinear photo-modification of collagen fibers and bovine cornea with ~2 µm spatial resolution was demonstrated.
©2008 Optical Society of America
Due to their unique optical properties, intense ultrafast near-infrared (NIR) laser pulses can induce nonlinear photophysical processes in living systems. Such phenomena can be used to conduct high precision, micro-surgical procedures deep inside tissue specimens. Specifically, the non-thermal disruption of tissues by focused high-power laser radiation is commonly applied in biomedical applications. In cases of high intensity laser illumination, the physical mechanism of photo-modification is laser-induced micro-plasma, which forms cavitation bubbles and disruptive shock waves. The dynamics of laser-induced optical breakdown (LIOB) in water and LIOB initiated photo-modification of biological macromolecules, neuro-fibers and other biological tissues have been studied using lasers with pulse duration in the 10-8-10-13 second range and pulse energy from 10-3 to 10-9 J [1–13]. Previously, minimally invasive and highly precise cornea tissue processing was achieved using a femtosecond (fs) laser system coupled with a high numerical 40×/NA 1.3 microscope objective. Through this process, the energy threshold of LIOB was estimated to be 1 nJ [7, 13].
Commercial medical laser systems based on the femtosecond (fs) technology have recently become available. The capabilities of these light sources stimulated biomedical research in improving the existing surgical protocols as there is considerable interest in expanding fs laser technology for additional medical applications. For example, in ophthalmology, the ability of stroma collagen in generating second harmonic generation (SHG) signals from the illuminating NIR, ultrafast laser sources offers a tremendous advantage for developing integrated fs laser systems for both precise eye surgery and monitoring of corneal conditions.
Since collagen is a major tissue component accessible to fs laser microsurgery, it is important to understand and characterize the photo-modification mechanism of collagen under fs laser illumination. In this report, non-ablative, non-thermal collagen fiber photo-modification was demonstrated using relatively weaker fs pulses (0.12–0.6 nJ) focused by an air immersion, long working distance objective. The effect was simultaneously induced and monitored using the repetitive laser scanning of pure collagen fibers and bovine cornea. Controlled photo-modification of collagen fibers and characterization using this method were demonstrated.
2. Materials and methods
Both the fs laser assisted microsurgery and multiphoton imaging (two-photon autofluorescence-TPAF and second harmonic generation-SHG) were achieved using a laser scanning microscope system (LSM 510 META, Zeiss, Jena, Germany) coupled to a fs, titanium:sapphire (Ti:Sa) laser operating at 780 nm and 80 MHz (Tsunami, Spectra-Physics, Mountain View, CA). The average power of the fs laser beam on the sample surface was varied between 10 to 50 mW and the nominal value of the laser pulse width is approximately 120 fs. The detection bandwidths of the SHG and TPAF signals were 380–400 nm and 435–700 nm, respectively. The effect of collagen photo-modification was observed in dry, type I collagen from rat tail (Fluka Biochemika, Chemie GmbH CH-9471 Buchs, 27666, UK), collagen from bovine Achilles’ tendon (Fluka, 27662, Switzerland) and partially dehydrated bovine cornea. An air immersion objective (Zeiss, Plan-Neofluar 20×/NA 0.5, WD=1.3 mm) was used in this study. Although multiphoton processes are most efficiently induced with high NA objectives, the use of low NA lenses is significant in photo-modification where in-depth and higher specimen processing speed are desired. For confocal examination of the photo-modified specimens, an argon laser (458 nm) was used in both reflectance and fluorescence modes. All of the illumination and imaging experiments were conducted at the ambient temperature.
For investigating fs induced photo-modification (Figs. 1–7, 11), simultaneous fs photo-modification and multiphoton imaging were performed using the fs Ti-Sa source. When appropriate, a new region of the sample was selected for each experiment in order to monitor the dynamics of fs photo-modification from the onset of the fs laser illumination. For fs lithographic demonstration in collagen (Figs. 8, 10, 12), selected regions within the specimens were scanned for localized photo-modification.
As shown in Figs. 1–4, key signatures of the fs photo-modification include an eventual and irreversible decrease in SHG and increase in TPAF intensities. Before performing quantitative measurements and analysis, we varied both the excitation laser intensity and wavelength to verify that the signal detected in the 380–400 nm bandwidth channel represented the SHG signal from collagen and the signal intensities in the both SHG and TPAF channels depended quadratically on the incident laser intensity.
Analysis of kinetic curves revealed the existence of different stages in nonlinear specimen responses from NIR, fs illumination. The characteristic temporal dependence of broadband TPAF (435–700 nm) showed marked increase following extended fs laser illumination. Concurrently, SHG intensity first increased slightly, and then sharply decreased. The SHG intensity degradation provided evidence for laser induced modification of the collagen fiber structure similar to that found in collagen thermal denaturation at around 60° C [14–19]. The process of the structural photo-modification began within the illuminated sites and can extend along the two opposite directions along single collagen fibers (Fig. 1 and supplemental movie) or radially in all directions in the case of cornea or other specimens with densely packed collagen fibers (Figs. 2, 3 and supplemental movie). When the modification extended radially (Fig. 2–4), the initial increase in TPAF intensity depended quadratically on the scan number n or dosage (total energy in Joules per scanning area). In this case, we defined the photo-modification efficiency parameter R to be the square root of the coefficient of the second-order TPAF profile in n (Fig. 4). In this manner, R can be used to estimate the initial rate of the photo-modification effect on scan number.
We also investigated the effects of photo-modification as functions of scan number (Fig. 4), pixel residence time (Fig. 5), scan frequencies (scan number per second) and illumination dosage (Fig. 6). Our results showed that the photo-modification efficiency, according to our definition, was proportional to fs laser pixel residence time when laser power and scan area were fixed (Fig. 5). Furthermore, it was found that the extent of photo-modification depended positively on the scanning frequency (Fig. 6).
Figure 6 shows that the temporal dependence curves (data series (1) and (3)) changed drastically when scanning frequency is changed from 5 Hz to 0.5 Hz (A) or from 0.5 Hz to 5 Hz (B) (the time points of the scanning frequency changes are denoted by black triangles), whereas such changes were not found in the dosage dependence curves (data series (2) and (4)). This observation suggests that the extent of photo-modification is cumulative and depends only on the dosage of the incident source.
Time sequence imaging (Fig. 7) showed that during a pause of 15 seconds (between the irradiation 22-nd and 37-th seconds) there was no essential change in the corresponding images at the two time points before and after the pause. In a similar fashion, we found (data not shown) that the fiber modification process stopped when the laser mode–locked regime was turned off for any period of time and was continued from the same state once the mode–locked regime restarted.
In order to elucidate the spectral changes in photo-modified collagen fiber network, confocal fluorescence imaging of bovine cornea sample treated with NIR, fs laser illumination was performed using 458 nm laser excitation (Figs. 8 and 9). It was found that the intensity and spectral composition of autofluorescence from the illuminated regions was different than that from a non-irradiated area. Similar to the TPAF results, autofluorescence emission from the illuminated regions was more intense and a new emission peak around 570 nm appeared (Fig. 9). Our observation supports the fact that new fluorescent species were formed during the illumination and photo-modification of collagen fibers.
The nonlinear photo-modification process demonstrated in this study provides a methodology for the photo-modification of collagen fibers with high spatial precision (around 2 µm). As demonstrations of the controlled photo-modification of collagen fibers, we show examples of photo-processing in collagen containing tissues in Figs. 10–12. Specifically, fs NIR illumination induced collagen cutting and bending were respectively demonstrated in rat tail (Fig. 10) and bovine Achilles’ tendon (Fig. 11). Furthermore, sculpturing of the letter “V” and “I” in bovine cornea is shown in Fig. 12.
In this work, we determined the optical characteristics of collagen fibers under femtosecond, NIR photo-modification from the illumination of 80 MHz Ti:Sa laser at 780 nm and with the pulse energy range of 10 to 50 mJ. Qualitatively, we did not detect signals in 380–400 nm (SHG channel) and 430–700 nm (TPAF channel) spectral bands when the Ti:Sa laser was operating in the continuous wave (CW) mode. No changes in TPAF signal, SHG intensity (λexc=780 nm) and corresponding images were registered, when the Ti:Sa laser was temporarily switched from mode-locked mode to CW mode for 0.1–2 minutes. These observations indicated that the detected signals and structural modifications of collagen fibers under NIR, fs illumination corresponded to nonlinear processes. The signal in the SHG channel disappeared when the Ti:Sa laser wavelength was tuned above 810 nm or below 750 nm. This observation and the quadratic dependence between the excitation laser power and the signal intensities in the two detection channels suggest that the registered signals represented SHG and TPAF of collagen in the case of collagen specimens.
Common LIOB features such as laser produced microstreaks, cavitations bubbles, or traces of carbonization [1–5] were not found in the illuminated area during the course of our experiments. In addition, for pulse energy up to 1 nJ, we did not observe bright scintillation associated with plasma formation. These factors along with quadratic dependence of registered signals in 380–700 nm range demonstrated that the laser pulse intensity was below the LIOB intensity threshold during the course of our experiments and that the collagen fiber modification was not due to microplasma-induced photodisruption.
Photobleaching of the TPAF signal observed in the initial stage of illumination provided evidence of an efficient, fs laser induced photochemical process in collagen fibers. The decrease in the autofluorescence intensity indicates the disruption of collagen architecture which could initiate subsequent photo-modification in the fiber. The level of structural modification became substantial with increased level of illumination (Fig. 2).
With increasing illumination, we observed a substantial increase in TPAF. The most probable mechanism of TPAF intensity increase in the illumination is the formation of new long-wavelength fluorophore of tyrosine dimers. It is likely the fs laser-induced photo-modification of collagen fiber structures resulted in the formation of bityrosine structures. A similar effect (increase in autofluorescence intensity with relatively the enhancement of long-wavelength fluorescence species) has been observed during UV laser irradiation of gelatin (partially hydrolyzed form of collagen) . These authors attributed these finding with dimerization of tyrosine residues and proved that fluorescence spectroscopy was sensitive test of the structural/conformation changes induced by laser irradiation in macromolecules.
The localized pattern of photo-modification along with the observation of dosage dependence on modification suggests that the disintegration of collagen fibers was non-thermal. A non-thermal disruption mechanism of collagen has previously been observed during the Er:glass laser (λ=1.56 µm) irradiation of the annulus fibrosus samples. It was demonstrated that quantitative differences existed between the effects of laser and hydrothermal treatments and suggested that the photomechanical processes played a dominant role in the disruption of the collagen tissue network .
The initial increase in SHG intensity immediately before collagen fiber degradation was observed in all specimens. This observation suggests that changes in the molecular packing of collagen fibrils had occurred and this result can be used for the investigation of collagen fiber micro-structures. One possibility of the increase in SHG intensity is the reduction of the collagen fiber volume. With an increase in the density of the collagen packing, the effective nonlinear coefficient grows proportionally. Generally, such growth in the nonlinearity would lead to a quadratic enhancement of the SHG intensity at the local site of the collagen fiber. In our case, the reduction of the lateral dimension of collagen fiber during fs laser illumination was observed (Fig. 11(B)). Concurrently, a monotonic increase in the SHG intensity was measured up to the point when the fiber underwent structural modification (Fig. 11). Our results correlate well to previous studies using x-ray diffractometry, where a reduction in the equatorial sizes of collagen fibrils from rat tail tendon and other tissues was detected during dehydration [23, 24]. It has been shown that upon air-drying, the intermolecular spacing in collagen fibrils decreased from 1.5 to 1.1 nm, which led to a decrease in the fibril volume. Dehydration also caused a minor shortening (~2%) in the fibril length .
Local maxima in SHG intensity have also been observed during thermal treatment of collagen specimens. It is well established that upon heating, collagen fibers gradually lose the ability to generate second harmonic signals in the temperature range between 50–75°C [14–20]. The decrease of SHG signals at higher temperatures is due to the unwinding of the collagen triple helical structures (helix–coil transition), with the result that the organized collagen structure transforms into an isotropic medium. In such cases, second harmonic signals cannot be effectively generated. However, distinct peaks of the SHG intensity have been observed [18, 19]. In particular, a local minimum at 45°C and an absolute maximum at 65°C were observed in the SHG intensity of porcine corneal stroma, when the specimens had been hydrothermally treated for 5 minutes . In addition, light and electron microscopic examinations revealed a shortening of collagen fibers along the length (shrinking), and up to a three-fold increase in the fibril diameter during thermal processing of human sclera samples . However, different rates of sclera shrinking and swelling induced several maxima and minima of the temperature-dependent specimen volume . In particular, a maximum density of collagen molecules corresponding was observed at 65°C, which coincides with the temperature of maximum SHG signal in cornea . Furthermore, a maximum of sclera specimen volume was observed at 55°C, close to the temperature when minimal SHG signal was detected. The correlation between relative SHG intensity and collagen density allowed us to explain the increase in SHG intensity during laser and hydrothermal processing of collagen samples. An interesting observation was that fs laser illumination reduced the lateral size of the collagen fiber, while a thermal treatment caused an increase. This also indicated that thermal processes played minor role in fs laser induced collagen fiber degradation. With sufficient illumination, SHG intensity eventually decreases (Figs. 1–4). This observation can be explained by the laser induced disruption of the non-centrosymmetric architecture of collagen responsible for generating the second harmonic signals.
Our results suggest that NIR, fs laser illumination initially dehydrates collagen fibers and destroys or weakens the cross-links which stabilize collagen fibers. At the same time, our data shows that during up to 50 mW of laser illumination, collagen microfibrils did not disintegrate in contrast to LIBR or thermal treatment. Our experimental results are in good agreement with the “low-density plasma” model of molecular bond breaking [10, 27]. According to this model, there is a wide illumination range when fs pulses with energies below the optical breakdown threshold are capable of generating free-electrons with sufficiently high density to induce photochemical reactions, but is insufficient to cause any thermal or thermo-mechanical effects.
Non-ablative fs laser modification technology may be applied in minimally invasive, high precision medical procedures such as intraocular, vascular and neuronal surgery, tissue engineering, cartilage reshaping, skin rejuvenation, and micromanipulation of biological systems. Moreover, surgical performance can be monitored in vivo in real-time as the SHG and TPAF imaging provide both the temporal and spatial images on the effects of photo-modification. In most cases this informative feedback can be used for the full automation and standardization of laser treatment as well as for tracing, evaluation and optimization the tissue healing process. The full potential of NIR, fs laser photo-modification in biology and medicine remains to be explored.
This work was supported by the National Research Program for Genomic Medicine (NRPGM) of the National Science Council (NSC) in Taiwan and was completed in the Optical Molecular Imaging Microscopy Core Facility (A5) of NRPGM.
References and links
1. D. Stern, C. A. Puliafito, E. T. Dobei, and W. T. Reidy, “Corneal ablation by nanosecond, picosecond and femtosecond laser pulses at 532 nm and 625 nm,” Arch. Ophthalmol. 107, 587–592 (1989). [CrossRef]
2. W. Kautek, S. Mitterer, J. Kruger, W. Husinsky, and G. Grabner, “Femtosecond-pulse laser ablation of human corneas,” Appl. Phys. A 58, 513–518 (1994). [CrossRef]
3. R. M. Kurtz, X. Liu, V. M. Elner, J. A. Squier, D. Du, and G. A. Mourou, “Photodisruption in the human cornea as a function of laser pulse width,” J. Refract. Surg. 13, 653–658 (1997).
4. T. Juhasz, F. H. Loesel, R. M. Kurtz, C. Horvath, J. F. Bille, and G. Mouro, “Corneal refractive surgery with femtosecond lasers,” IEEE J. Quantum Electron. 5, 902–909 (1999). [CrossRef]
5. K. König, I. Riemann, and W. Fritzsche, “Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,” Opt. Lett. 26, 819–821 (2001). [CrossRef]
6. C. Schaffer, N. Nishimura, E. Glezer, A. Kim, and E. Mazur, “Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds,” Opt. Express 10, 196–203 (2002). [PubMed]
7. K. König, O. Krauss, and I. Riemann, “Intratissue surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared,” Opt. Express. 10, 171–176 (2002).
8. M. F. Yanik, H. Cinar, N. Cinar, A. Chisholm, Y. Jin, and A. B. Yakar, “Neurosurgery: Functional regeneration after laser axotomy,” Nature 432, 882 (2004). [CrossRef]
10. A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]
11. B. G. Wang, I. Riemann, H. Schubert, K. J. Halbhuber, and K. Koenig, “In-vivo intratissue ablation by nanojoule near-infrared femtosecond laser pulses,” Cell Tissue Res. 328, 515–520 (2007). [CrossRef] [PubMed]
12. M. Sakakura, S. Kajiyama, M. Tsutsumi, J. Si, E. Fukusaki, Y. Tamaru, S. Akiyama, K. Miura, K. Hirao, and M. Ueda, “Femtosecond pulsed laser as a microscalpel for microdissection and isolation of specific sections from biological samples,” Jpn. J. Appl. Phys. 46, 5859–5864 (2007). [CrossRef]
13. A. Ehlers, I. Riemann, S. Martin, R. Le Harzic, A. Bartels, C. Janke, and K. König, “High (1 GHz) repetition rate compact femtosecond laser: A powerful multiphoton tool for nanomedicine and nanobiotechnology,” J. Appl. Phys. 102, 014701–6 (2007). [CrossRef]
14. V. Hovanessian and A. Lalayan, “Second harmonic generation in biofiber containing tissues,” in Proceeding of Int. Conf. Lasers ’96, (Society for Optical and Quantum Electronics, McLean, VA, 1996) pp. 107–110.
15. B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, “Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity,” Lasers Surg. Med. 27, 329–335 (2000). [CrossRef] [PubMed]
16. T. Theodossiou, G. S. Rapti, V. Hovhannisyan, E. Georgiou, K. Politopoulos, and D. Yova, “Thermally induced irreversible conformational changes in collagen probed by optical second harmonic generation and laser-induced fluorescence,” Lasers Med. Sci. 17, 34–41 (2002). [CrossRef] [PubMed]
17. S. J. Lin, C. Y. Hsiao, Y. Sun, W. Lo, W. C. Lin, G. J. Jan, S. H. Jee, and C. Y. Dong, “Monitoring the thermally induced structural transitions of collagen using second harmonic generation microscopy,” Opt. Lett. 30, 622–624 (2005). [CrossRef] [PubMed]
18. H. Y. Tan, S. W. Teng, W. Lo, W. C. Lin, S. J. Lin, S. H. Jee, and C. Y. Dong, “Characterizing the thermally induced structural changes to intact porcine eye, part 1: second harmonic generation imaging of cornea stroma,” J. Biomed. Opt. 10, 054019–5 (2005). [CrossRef] [PubMed]
19. Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. So, and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging,” Biophys J. 91, 2620–2625 (2006). [CrossRef] [PubMed]
20. T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91, 4665–4677 (2006). [CrossRef] [PubMed]
21. M. Oujja, E. Rebollar, C. Abrusci, A. Del Amo, F. Catalina, and M. Castillejo, “UV, visible and IR laser interaction with gelatine,” J. Phys.: Conf. Ser. 59, 571–574 (2007). [CrossRef]
22. N. Yu. Ignatieva, O. L. Zakharkina, I. V. Andreeva, E. N. Sobol, V. A. Kamensky, A. V. Myakov, S. V. Averkiev, and V. V. Lunin, “IR Laser and heat-induced changes in annulus fibrosus collagen structure,” Photochem. Photobiol. 83, 675–685 (2007). [CrossRef] [PubMed]
23. R. I. Price, S. Lees, and D. A. Kirschner, “X-ray diffraction analysis of tendon collagen at ambient and cryogenic temperatures: role of hydration,” Int. J. Biol. Macromol. 20, 23–33 (1997). [CrossRef] [PubMed]
24. E. D. Eanes and E. J. Miller, “Effect of covalent cross-linking on the X-ray diffraction properties of chick bone and rat tail tendon collagens,” Arch. Biochem. Biophys. 129, 769–771 (1969). [CrossRef] [PubMed]
26. A. I. Rem, J. A. Oosterhuis, H. G. Journée-de Korver, T. J van den Berg, and J. E. Keunen, “Temperature dependence of thermal damage to the sclera: exploring the heat tolerance of the sclera for transscleral thermotherapy,” Exp. Eye Res. 72, 153–62 (2001). [CrossRef] [PubMed]
27. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Femtosecond Plasma-Mediated Nanosurgery of Cells and Tissues” in Laser Ablation and its Applications (Springer Series in Optical Sciences, v. 129, Springer Berlin/Heidelberg, 2007).