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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 10, Iss. 3 — Feb. 11, 2002
  • pp: 171–176
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Intratissue surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared

Karsten König, Oliver Krauss, and Iris Riemann  »View Author Affiliations


Optics Express, Vol. 10, Issue 3, pp. 171-176 (2002)
http://dx.doi.org/10.1364/OE.10.000171


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Abstract

The use of 1 nanojoule near infrared 80 MHz femtosecond laser pulses for highly precise intratissue processing, in particular for intraocular refractive surgery, was evaluated. Destructive optical breakdown at TW/cm2 light intensities in a subfemtoliter intrastromal volume was obtained by diffraction-limited focussing with an 40x objective (N.A. 1.3) and beam scanning 50 to 140 μm below the epithelial surface. Using the same system at GW/cm2 intensities two-photon excited autofluorescence imaging was used to determine the target of interest and to visualize intraocular laser effects. Histological examination of laser-exposed porcine eyes reveal a minimum cut size below 1 μm without destructive effects to surrounding tissues.

© Optical Society of America

1. Introduction

By contrast, intense ultrashort pulses at the visible and near infrared (NIR) spectral range with no significant one-photon absorption in the cornea can be employed to realise non-invasive femtosecond optical breakdown and photodisruption within the tissue. These nonlinear effects have been used to ablate and to modify corneal tissue [3

3. 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] [PubMed]

,4

4. R.M. Kurtz, C. Horvath, H.H. Liu, R.R. Krueger, and T. Juhasz, “Lamellar refractive surgery with scanned intrastromal picosecond and femtosecond laser pulses in animal eyes,” J. Refract. Surg. 14, 541–548 (1998). [PubMed]

].

Plasma-mediated ablation of the cornea by photodisruption has been demonstrated with nanosecond, picosecond and femtosecond pulses. Photodisruption is based on the rapid expansion of the laser-induced plasma with Gigapascal pressures and the development and further collapse of cavitation bubbles accompanied with the formation of destructive shock waves. In order to realize a desired highly localized destructive effect without significant collateral damage, small bubble diameters and low optomechanical effects in the surroundings are required. It was shown that the threshold for optical breakdown in water is decreased by a factor of more than 100 when comparing 100 fs with 3 ns pulses accompanied with less transformation into destructive mechanical energy (factor 6) [5

5. A. Vogel, K. Nahen, D. Theisen, R. Birngruber, R.J. Thomas, and B.A. Rockwell, “Energy balance of optical breakdown in water at nanosecond to femtosecond time scales,“ Appl. Phys. B 68, 271–280 (1999). [CrossRef]

]. Kurtz et al. reported that femtosecond pulses require about one tenth the energy of picosecond and nanosecond pulses to produce corneal disruption [6

6. 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).

].

Clinical studies have been performed with nanosecond and picosecond laser pulses. Using the pulsed Nd:YLF laser at 1053 nm with a pulse width in the 30 to 60 ps range, better results where obtained as in the case of ns laser pulses. However a fully contiguous intrastromal photodisruption could not be obtained [7

7. M. Ito, A.J. Quantock, S. Malhan, D.J. Schanzlin, and R.R. Krueger, “Picosecond laser in situ keratomileusis with a 1053-nm Nd:YLF laser,” J. Refract. Surg. 12, 721–728 (1996). [PubMed]

,8

8. H. Gimbel, S. Coupland, and M. Ferensowisc,“Review of intrastromal photorefractive keratectomy with the neodymium-yttrium lithium fluoride laser,” Int. Ophthalmol. Clin. 37, 95–102 (1997). [CrossRef] [PubMed]

].

Using femtosecond laser pulses, precise intrastromal cuts in animal cadaver eyes could be performed, however, destructive effects by the bubble formation has been reported [3

3. 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] [PubMed]

,4

4. R.M. Kurtz, C. Horvath, H.H. Liu, R.R. Krueger, and T. Juhasz, “Lamellar refractive surgery with scanned intrastromal picosecond and femtosecond laser pulses in animal eyes,” J. Refract. Surg. 14, 541–548 (1998). [PubMed]

,9–12

9. H. Lubatschowski, G. Maatz, A. Heisterkamp, U. Hetzel, W. Drommer, H. Welling, and W. Ertmer, “Application of ultrashort laser pulses for intrastromal refractive surgery,“ Graefe’s Arch. Clin. Exp. Ophthalmol. 238, 33–39 (2000). [CrossRef]

]. Bubble diameters of more than 12 μm has been predicted [11

11. T. Juhasz, G.A. Kastis, C. Suarez, Z. Bor, and W.E. Brown, “Time-resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water,” Lasers Surg. Med. 19, 23–31(1996). [CrossRef] [PubMed]

]. The use of μJ pulses caused collateral damage by photodisruption.

To date, all femtosecond laser procedures on eyes have been based on the application of μJ pulses illumination, low repetition rate in the Hz and kHz range and spot diameters of more than 5 μm. These treatments have required extensive laser systems including laser resonator, pulse stretching/compression unit and amplifier. Another disadvantage is the danger of uncontrolled intratissue damage by self-focussing effects when using microjoule laser pulses and focussing optics of low numerical aperture (NA).

Recently we have shown that nanojoule NIR femtosecond laser pulses at high repetition rate may induce optical breakdown in DNA molecules which can be applied to perform chromosome ablation with a sub-100 nm cut size [13

13. K. König, I. Riemann, and W. Fritzsche, “Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,“ Opt. Lett. 26, 819–821 (2001). [CrossRef]

]. Here we report for the first time on highly precise tissue processing with 1 nJ near infrared laser pulses at MHz repetition frequency. To overcome the intensity threshold for optical breakdown the beam was focussed to its diffraction limit by objectives of 1.3 NA. In particular, intraocular laser treatment was performed with femtosecond laser pulses at 800 nm of a 80 MHz titanium:sapphire laser and submicron illumination spot sizes. The same laser system was also employed as diagnostic 3D tool with subcellular spatial resolution for target finding by autofluorescence detection, monitoring of plasma luminescence as well as by immediate analysis of intratissue femtosecond laser effects.

2. Materials and Methods

We studied the effect of material ablation with 1 nJ pulses on porcine eyes which were placed within a special tissue chamber with two 170 μm glass windows (MiniCeM, JenLab GmbH, Jena, Germany) on the stage of an inverted multiphoton laser microscope. The optical system consists of a solid state, diode-pumped, mode-locked compact 80 MHz titanium sapphire laser at 800 nm (Vitesse, Coherent Inc., USA), a modified laser scanning microscope (LSM 410, Carl Zeiss Jena GmbH, Jena, Germany) and an interface consisting of a beam expander, motorized beam attenuator, fast shutter, power control and synchronisation unit (JenLab GmbH, Jena, Germany). Luminescence (plasma radiation, two-photon excited autofluorescence) was detected with baseport photomultipliers whereas transillumination of light from a halogen lamp through the whole ex vivo porcine eye was on-line monitored with a baseport CCD camera.

The 800 nm laser beam was focussed to its submicron diffraction-limited spot size by a 40x objective of 1.3 numerical aperture (NA). The pulse width at the sample was determined to be 170 fs pulses by autocorrelation techniques. For two-photon autofluorescence imaging a mean power of 5 – 10 mW at the target surface was used. For material processing, a beam with a mean power of 80 mW has been employed which corresponds to 1 nJ pulse energy.

3. Results

Before laser processing, optical sectioning of fluorescent corneal structures with subcellular spatial resolution (512 × 512 pixels scans of 8 sec duration covering 320 × 320 μm2, 1 μm z steps) have been performed. As shown in Fig. 1A, 3D autofluorescence imaging based on non-resonant two-photon excitation of NAD(P)H, flavins and collagen12 enabled the discrimination of the various corneal tissue layers (epithelium, Bowman’s layer, stroma) and of individual cells and collagen fibers. After imaging, the laser beam was “parked” in an intratissue target region of interest. In order to perform a precise intrastromal cut, the mean laser power was increased to 80 mW and the beam was scanned along one line at a typical beam dwell time on a pixel of 4 μs which corresponds to about 320 pulses per pixel. In addition, knocking out of small intratissue structures have been performed by single-point-illumination. During laser exposure the baseport detector recorded an intense signal likely due the formation of plasma luminescence.

Fig. 1. Optical maging of intraocular tissue structures at different depths by two-photon excited autofluorescence. A: 3D fluorescence imaging enables discrimination between epithelial layer, Bowman’s layer and corneal stroma. Individual cells with fluorescent cytoplasm (c) and non-fluorescent nuclei (n) are visible as shown in the small image in the corner which reflects a 4x magnified region of the autofluorescence image of the epithelium. B: After laser treatment, an intratissue highly fluorescent structure along the cut with submicron lateral size is formed.
Fig. 2. Histological examination of a HE-stained cryosection after laser exposure by 488 nm laser scanning microscopy reveal precise <1 μm line cuts. No visible signs of collateral damage were found. The lower left image demonstrates an intratissue cut through a single nuclei at 90 μm tissue depth.

During and immediately following laser treatment, formation, localization and lifetime of intratissue bubbles have been monitored at video rate as well. Depending on intratissue structures, between 3 to 7 bubbles occurred at fluorescent sites along a 320 μm line. Using microsecond beam dwell times per pixel, a maximum mean diameter of 5 μm of intrastromal bubbles with mean lifetimes of 1.8±0.3 s have been recorded. The relatively long lifetime corresponds to observations by others who detected long-lived gas-filled bubbles (oxygen, hydrogen, methan) compared to short-lived cavitation bubbles.

Interestingly, the effect of the intratissue laser processing could be clearly visualized by 3D luminescence imaging at low light intensity. Obviously, the tissue borders along the cut became highly fluorescent during laser procedure. Fig. 1B demonstrates depth-resolved autofluorescence images after performing an intrastromal cut in 92 μm below the epithelial surface. The thickness of the fluorescent structure was found to be about 0.8±0.4 μm. Analysis of the autofluorescence of collateral structures revealed no significant modifications of fluorescence pattern and tissue morphology.

In order to study the laser effects more carefully, laser-exposed eyes underwent histological examination. Analysis of the hematoxilin/eosin (HE) stained cryosections showed clearly the presence of highly precise intraocular cuts without significant collateral damage. In some cases, a clear cut within the nuclei of individual cells became visible. Typical minimum cut sizes between 0.5 μm and 1 μm have been determined by 488 nm laser scanning microscopy (Fig. 2).

4. Summary and Conclusion

In contrast to PRK and LASIK techniques with UV laser pulses which have the drawback of mechanical or optical destruction of epithelial structures, and in contrast to amplified NIR femtosecond laser surgery with μJ pulse energies with the danger of significant damage to surrounding structures, the treatment with highly focused NIR beams of nanojoule femtosecond pulses at high repetition frequency have the potential of highly precise intratissue processing without collateral damage by intratissue self-focusing effects, large cavitation bubbles and destructive shock waves. Potential medical applications include highly precise intraocular and neuronal surgery. Further studies with a programmable 3D scanner are currently being pursued in our laboratory to remove laser-cutted micrometer-sized tissue blocks and to examine the quality of the cuts by electron and force microscopy.

Acknowledgments

This work was supported by the German Science Foundation (DFG, KO1361/10–3), the Ministry of Science, Research and Art of the State of Thuringia (TMWFK) and the Ministry of Science, Research and Technology of Germany (BMBF).

References and links

1.

I.G. Pallikaris and D.S. Saiganos, “Excimer laser in situ keratomileusis and photorefractive keratectomy for correction of high myopia, “ J. Refract. Surg. 10, 498–510 (1994).

2.

M. Mrochen, M. Kaemmerer, and T. Seiler, “Wavefront-guided laser in situ keratomileusis: early results in three eyes,“ J. Refrac. Surg. 16, 116–121 (2000).

3.

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] [PubMed]

4.

R.M. Kurtz, C. Horvath, H.H. Liu, R.R. Krueger, and T. Juhasz, “Lamellar refractive surgery with scanned intrastromal picosecond and femtosecond laser pulses in animal eyes,” J. Refract. Surg. 14, 541–548 (1998). [PubMed]

5.

A. Vogel, K. Nahen, D. Theisen, R. Birngruber, R.J. Thomas, and B.A. Rockwell, “Energy balance of optical breakdown in water at nanosecond to femtosecond time scales,“ Appl. Phys. B 68, 271–280 (1999). [CrossRef]

6.

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).

7.

M. Ito, A.J. Quantock, S. Malhan, D.J. Schanzlin, and R.R. Krueger, “Picosecond laser in situ keratomileusis with a 1053-nm Nd:YLF laser,” J. Refract. Surg. 12, 721–728 (1996). [PubMed]

8.

H. Gimbel, S. Coupland, and M. Ferensowisc,“Review of intrastromal photorefractive keratectomy with the neodymium-yttrium lithium fluoride laser,” Int. Ophthalmol. Clin. 37, 95–102 (1997). [CrossRef] [PubMed]

9.

H. Lubatschowski, G. Maatz, A. Heisterkamp, U. Hetzel, W. Drommer, H. Welling, and W. Ertmer, “Application of ultrashort laser pulses for intrastromal refractive surgery,“ Graefe’s Arch. Clin. Exp. Ophthalmol. 238, 33–39 (2000). [CrossRef]

10.

J. Noack and A. Vogel, “Laser-induced plasma formation in water at nanosecond to femtosecond time scales: calculation of thresholds, absorption coefficients, and energy densities,”IEEE J. Quantum Electron. 35, 1156–1167 (1999). [CrossRef]

11.

T. Juhasz, G.A. Kastis, C. Suarez, Z. Bor, and W.E. Brown, “Time-resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water,” Lasers Surg. Med. 19, 23–31(1996). [CrossRef] [PubMed]

12.

T. Juhasz, F.H. Loesel, R.M. Kurtz, C. Horvath, J.F. Bille, and G. Mourou, “Corneal refractive surgery with femtosecond lasers,” IEEE J. Quantum Electron. 5, 902–909 (1999). [CrossRef]

13.

K. König, I. Riemann, and W. Fritzsche, “Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,“ Opt. Lett. 26, 819–821 (2001). [CrossRef]

14.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000). [CrossRef] [PubMed]

OCIS Codes
(140.7090) Lasers and laser optics : Ultrafast lasers
(170.1020) Medical optics and biotechnology : Ablation of tissue

ToC Category:
Research Papers

History
Original Manuscript: January 4, 2002
Revised Manuscript: January 31, 2002
Published: February 11, 2002

Citation
Karsten Koenig, Oliver Krauss, and Iris Riemann, "Intratissue surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared," Opt. Express 10, 171-176 (2002)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-10-3-171


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References

  1. I. G. Pallikaris and D. S. Saiganos, ?Excimer laser in situ keratomileusis and photorefractive keratectomy for correction of high myopia," J. Refract. Surg. 10, 498-510 (1994).
  2. M. Mrochen, M. Kaemmerer and T. Seiler, ?Wavefront-guided laser in situ keratomileusis: early results in three eyes,? J. Refrac. Surg. 16, 116-121 (2000).
  3. D. Stern, C. A. Puliafito, E. T. Dobei andW. T. Reidy, ?Corneal ablation by nanosecond, picosecond and femtosecond laser pulses at 532 nm and 625 nm,? Arch. Ophthalmol. 107, 587-592 (1989). [CrossRef] [PubMed]
  4. R. M. Kurtz, C. Horvath, H. H. Liu, R. R. Krueger and T. Juhasz, ?Lamellar refractive surgery with scanned intrastromal picosecond and femtosecond laser pulses in animal eyes,? J. Refract. Surg. 14, 541-548 (1998). [PubMed]
  5. A. Vogel, K. Nahen, D. Theisen, R. Birngruber, R. J. Thomas and B. A. Rockwell, ?Energy balance of optical breakdown in water at nanosecond to femtosecond time scales,? Appl. Phys. B 68, 271-280 (1999). [CrossRef]
  6. 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).
  7. M. Ito, A. J. Quantock, S. Malhan, D. J. Schanzlin and R. R. Krueger, ?Picosecond laser in situ keratomileusis with a 1053-nm Nd:YLF laser,? J. Refract. Surg. 12, 721-728 (1996). [PubMed]
  8. H. Gimbel, S. Coupland and M. Ferensowisc, ?Review of intrastromal photorefractive keratectomy with the neodymium-yttrium lithium fluoride laser,? Int. Ophthalmol. Clin. 37, 95-102 (1997). [CrossRef] [PubMed]
  9. H. Lubatschowski, G. Maatz, A. Heisterkamp, U. Hetzel, W. Drommer, H. Welling and W. Ertmer, "Application of ultrashort laser pulses for intrastromal refractive surgery,? Graefe?s Arch. Clin. Exp. Ophthalmol. 238, 33-39 (2000). [CrossRef]
  10. J. Noack and A. Vogel, ?Laser-induced plasma formation in water at nanosecond to femtosecond time scales: calculation of thresholds, absorption coefficients, and energy densities,? IEEE J. Quantum Electron. 35, 1156-1167 (1999). [CrossRef]
  11. T. Juhasz, G. A. Kastis, C. Suarez, Z. Bor and W.E. Brown, ?Time-resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water,? Lasers Surg. Med. 19, 23-31(1996). [CrossRef] [PubMed]
  12. T. Juhasz, F. H. Loesel, R. M. Kurtz, C. Horvath, J. F. Bille and G. Mourou, ?Corneal refractive surgery with femtosecond lasers,? IEEE J. Quantum Electron. 5, 902-909 (1999). [CrossRef]
  13. K. K?nig, I. Riemann and W. Fritzsche, "Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,? Opt. Lett. 26, 819-821 (2001). [CrossRef]
  14. K. K?nig, "Multiphoton microscopy in life sciences," J. Microsc. 200, 83-104 (2000). [CrossRef] [PubMed]

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