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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 12, Iss. 18 — Sep. 6, 2004
  • pp: 4275–4281
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Mini-invasive corneal surgery and imaging with femtosecond lasers

Meng Han, Günter Giese, Leander Zickler, Hui Sun, and Josef F. Bille  »View Author Affiliations


Optics Express, Vol. 12, Issue 18, pp. 4275-4281 (2004)
http://dx.doi.org/10.1364/OPEX.12.004275


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Abstract

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

1. Introduction

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

1. T. Juhasz, F.H. Loesel, C. Horvath, R.M. Kurtz, and G. Mourou, “Corneal Refractive Surgery with Femtosecond Lasers,” IEEE J. Sel. Top. Quantum Electron. 5, 902–910 (1999) [CrossRef]

, 2

2. “New Frontiers in Vision and Aberration-Free Refractive Surgery,” edited by J.F. Bille, C.F.H Harner, and F. Loesel, Springer Press, Heidelberg, Germany (2002)

]. 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 [3

3. F.H. Loesel, M.H. Niemz, J.F. Bille, and T. Juhasz, “Laser-Induced Optical Breakdown on Hard and Soft Tissues and Its Dependence on the Pulse Duration: Experiment and Model,“ IEEE J. Quantum Electron. 32, 1717–1722 (1996) [CrossRef]

]. 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

4. A.T. Yeh, N. Nassif, A. Zoumi, and B.J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited f luorescence,” Opt. Lett. 27, 2082–2084 (2002) [CrossRef]

, 5

5. G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-Dimensional Imaging of Collagen Using Second Harmonic Generation,” J. Structure Biology 141, 53–62 (2003) [CrossRef]

, 6

6. R. Gauderon, P.B. Lukins, and C.J.R. Sheppard, “Optimization of second harmonic generation microscopy,” Micron. 32, 691–700 (2002) [CrossRef]

, 7

7. P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis, and L.M. Loew, “Second-harmonic Imaging Microscopy of Living Cells,” J. Biomed. Opt. 6, 277–286 (2001) [CrossRef] [PubMed]

, 8

8. M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter, and J. Bille, “Second Harmonic Corneal Imaging after femtosecond laser surgery,” J. Biomed. Opt. 9, 760–766 (2004) [CrossRef] [PubMed]

] 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 [8

8. M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter, and J. Bille, “Second Harmonic Corneal Imaging after femtosecond laser surgery,” J. Biomed. Opt. 9, 760–766 (2004) [CrossRef] [PubMed]

]. 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

Fig. 1. Schematic drawing of the diode pumped Nd:glass fs surgical laser system. The seed pulse from the Nd:glass fs pulse oscillator is amplified by a Chirped-Pulse-Amplification unit. The pulse is stretched and compressed by a single holographic transmission grating. FR: Faraday Rotator, PBS: Polarized Beam Splitter, λ/4: Quarter waveplate.

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 [10

10. S. Roth and I. Freund, ”Coherent Optical Harmonic Generation in Rat-tail,” Opt. Commun. 33, 292–296 (1980) [CrossRef]

]. 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 [8

8. M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter, and J. Bille, “Second Harmonic Corneal Imaging after femtosecond laser surgery,” J. Biomed. Opt. 9, 760–766 (2004) [CrossRef] [PubMed]

].

Fig. 2. (a) Two-photon excited fluorescence imaging of keratocyte cell nuclei stained with DAPI. (b) SHG imaging of the collagen fiber network surrounding the cell nuclei. The perimeters of the keratocyte nuclei are outlined. The image fields for both imaging modes are identical, located at the depth of 200 μm below the epithelium. Bars: 10 μm

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.

Fig. 3. (a) Corneal flap revealed by SHG imaging. The laser resection plane (posterior surface) is indicated by arrows. (b) Three dimensional SHG visualization of the intrastromal cavities and the collateral effects (microstreaks) through YZ optical sectioning. The microstreaks are pointed out by open arrows. Bars: 20 μm

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.

Acknowledgments

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.

References and links

1.

T. Juhasz, F.H. Loesel, C. Horvath, R.M. Kurtz, and G. Mourou, “Corneal Refractive Surgery with Femtosecond Lasers,” IEEE J. Sel. Top. Quantum Electron. 5, 902–910 (1999) [CrossRef]

2.

“New Frontiers in Vision and Aberration-Free Refractive Surgery,” edited by J.F. Bille, C.F.H Harner, and F. Loesel, Springer Press, Heidelberg, Germany (2002)

3.

F.H. Loesel, M.H. Niemz, J.F. Bille, and T. Juhasz, “Laser-Induced Optical Breakdown on Hard and Soft Tissues and Its Dependence on the Pulse Duration: Experiment and Model,“ IEEE J. Quantum Electron. 32, 1717–1722 (1996) [CrossRef]

4.

A.T. Yeh, N. Nassif, A. Zoumi, and B.J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited f luorescence,” Opt. Lett. 27, 2082–2084 (2002) [CrossRef]

5.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-Dimensional Imaging of Collagen Using Second Harmonic Generation,” J. Structure Biology 141, 53–62 (2003) [CrossRef]

6.

R. Gauderon, P.B. Lukins, and C.J.R. Sheppard, “Optimization of second harmonic generation microscopy,” Micron. 32, 691–700 (2002) [CrossRef]

7.

P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis, and L.M. Loew, “Second-harmonic Imaging Microscopy of Living Cells,” J. Biomed. Opt. 6, 277–286 (2001) [CrossRef] [PubMed]

8.

M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter, and J. Bille, “Second Harmonic Corneal Imaging after femtosecond laser surgery,” J. Biomed. Opt. 9, 760–766 (2004) [CrossRef] [PubMed]

9.

J.H. Marburger, “Self-focusing: theory,” Prog. Quantum Electron. 4, 35–110 (1975) [CrossRef]

10.

S. Roth and I. Freund, ”Coherent Optical Harmonic Generation in Rat-tail,” Opt. Commun. 33, 292–296 (1980) [CrossRef]

11.

C. Horvath, A. Braun, H. Liu, T. Juhasz, and G. Mourou, Compact Directly Diode-pumped Femtosecond Nd:glass Chirped-pulse-amplication Laser System, Opt. Lett. 22, 1790–1792 (1997) [CrossRef]

12.

G. Maatz, A. Heisterkamp, H. Lubatschowski, S. Barcikowski, C. Fallnich, H. Welling, and W Ertmer, “Chemical and Physical Side Effects at Application of Ultrashort Laser Pulses for Intrastromal Refracitve Surgery,” J. Opt. A 2, 59–64 (2000) [CrossRef]

OCIS Codes
(140.7090) Lasers and laser optics : Ultrafast lasers
(170.4470) Medical optics and biotechnology : Ophthalmology
(180.5810) Microscopy : Scanning microscopy

ToC Category:
Research Papers

History
Original Manuscript: June 18, 2004
Revised Manuscript: August 25, 2004
Published: September 6, 2004

Citation
Meng Han, Günter Giese, Leander Zickler, Hui Sun, and Josef Bille, "Mini-invasive corneal surgery and imaging with femtosecond lasers," Opt. Express 12, 4275-4281 (2004)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-18-4275


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References

  1. T. Juhasz, F. H. Loesel, C. Horvath, R. M. Kurtz, G. Mourou, �??Corneal Refractive Surgery with Femtosecond Lasers," IEEE J. Sel. Top. Quantum Electron. 5, 902-910(1999) [CrossRef]
  2. "New Frontiers in Vision and Aberration-Free Refractive Surgery,�?? edited by J.F. Bille, C.F.H Harner, F. Loesel, Springer Press, Heidelberg, Germany (2002)
  3. F. H. Loesel, M.H. Niemz, J.F. Bille and T. Juhasz, �??Laser-Induced Optical Breakdown on Hard and Soft Tissues and Its Dependence on the Pulse Duration: Experiment and Model," IEEE J. Quantum Electron. 32, 1717- 1722(1996) [CrossRef]
  4. A.T. Yeh, N. Nassif, A. Zoumi and B.J. Tromberg, �??Selective corneal imaging using combined second-harmonic generation and two-photon excited f luorescence," Opt. Lett. 27, 2082-2084 (2002) [CrossRef]
  5. G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi and M. D. Gorrell, �??3-Dimensional Imaging of Collagen Using Second Harmonic Generation,�?? J. Structure Biology 141, 53-62 (2003) [CrossRef]
  6. R. Gauderon, P.B. Lukins, C. J. R. Sheppard, �??Optimization of second harmonic generation microscopy,�?? Micron. 32, 691-700( 2002) [CrossRef]
  7. P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis and L.M. Loew, �??Second-harmonic Imaging Microscopy of Living Cells," J. Biomed. Opt. 6, 277-286 (2001) [CrossRef] [PubMed]
  8. M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter and J. Bille, �??Second Harmonic Corneal Imaging after femtosecond laser surgery,�?? J. Biomed. Opt. 9, 760-766(2004) [CrossRef] [PubMed]
  9. J. H. Marburger, "Self-focusing: theory," Prog. Quantum Electron. 4, 35-110 (1975) [CrossRef]
  10. S. Roth and I. Freund, �??Coherent Optical Harmonic Generation in Rat-tail,�?? Opt. Commun. 33, 292-296 (1980) [CrossRef]
  11. C. Horvath, A. Braun, H. Liu, T. Juhasz, and G. Mourou, "Compact Directly Diode-pumped Femtosecond Nd:glass Chirped-pulse-amplication Laser System", Opt. Lett. 22, 1790-1792 (1997) [CrossRef]
  12. G. Maatz, A. Heisterkamp, H. Lubatschowski, S. Barcikowski, C. Fallnich, H. Welling, W Ertmer, "Chemical and Physical Side Effects at Application of Ultrashort Laser Pulses for Intrastromal Refracitve Surgery,�?? J. Opt. A 2, 59-64 (2000) [CrossRef]

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