DNA Damage in Bone Marrow Cells Induced by Femtosecond and Nanosecond Ultraviolet Laser Pulses.

OBJECTIVE: The purpose of this study was to investigate the possible genotoxic impact of new generation 205 nm femtosecond solid-state laser irradiation on the DNA of murine bone marrow cells in vitro, and to compare the DNA damage caused by both femtosecond and nanosecond UV laser pulses. BACKGROUND DATA: Recent experiments of corneal stromal ablation in vitro and in vivo applying femtosecond UV pulses showed results comparable with or superior to those obtained using nanosecond UV lasers. However, the possible genotoxic effect of ultrashort laser pulses was not investigated. METHODS: Mouse bone marrow cells were exposed to different doses of 205 nm femtosecond, 213 and 266 nm nanosecond lasers, and 254 nm UV lamp irradiation. The comet assay was used for the evaluation of DNA damage. RESULTS: All types of irradiation demonstrated intensity-dependent genotoxic impact. The DNA damage induced depended mainly upon wavelength rather than on other parameters such as pulse duration, repetition rate, or beam delivery to a target. CONCLUSIONS: Both 205 nm femtosecond and clinically applied 213 nm nanosecond lasers' pulses induced a comparable amount of DNA breakage in cells exposed to the same irradiation dose. To further evaluate the suitability of femtosecond UV laser sources for microsurgery, a separate investigation of the genotoxic and mutagenic effects on corneal cells in vitro and, particularly, in vivo is needed.

High-speed photorefractive keratectomy with femtosecond ultraviolet pulses.

Femtosecond near-infrared lasers are widely used for a number of ophthalmic procedures, with flap cutting in the laser-assisted in situ keratomileusis (LASIK) surgery being the most frequent one. At the same time, lasers of this type, equipped with harmonic generators, have been shown to deliver enough ultraviolet (UV) power for the second stage of the LASIK procedure, the stromal ablation. However, the speed of the ablation reported so far was well below the currently accepted standards. Our purpose was to perform high-speed photorefractive keratectomy (PRK) with femtosecond UV pulses in rabbits and to evaluate its predictability, reproducibility and healing response. The laser source delivered femtosecond 206 nm pulses with a repetition rate of 50 kHz and an average power of 400 mW. Transepithelial PRK was performed using two different ablation protocols, to a total depth of 110 and 150 µm. The surface temperature was monitored during ablation; haze dynamics and histological samples were evaluated to assess outcomes of the PRK procedure. For comparison, analogous excimer ablation was performed. Increase of the ablation speed up to 1.6 s/diopter for a 6 mm optical zone using femtosecond UV pulses did not significantly impact the healing process.

Corneal stromal ablation with femtosecond ultraviolet pulses in rabbits.

PURPOSE: To determine the effectiveness of femtosecond ultraviolet (UV) pulses in ablating corneal stroma in a rabbit model and to compare the healing response between eyes treated with femtosecond UV pulses and eyes treated with standard excimer photorefractive keratectomy. SETTING: Laser Research Center, Vilnius University, Vilnius, Lithuania. DESIGN: Experimental study. METHODS: Myopic photoablation using a femtosecond UV solid-state laser system was applied to corneas of pigmented rabbits. Experiments in 16 eyes were performed for optimization of the laser system parameters (fluence, spot size, pulse repetition rate) and calibration of ablation rate. In 7 rabbits, deep femtosecond UV ablation (∼130 µm) in 1 eye and shallow ablation (∼30 µm) in the contralateral eye were performed. Nine rabbits received an approximately 30 µm ablation with femtosecond UV pulses in 1 eye and with a conventional excimer system in the contralateral eye. Two eyes were used as controls. The ablation process and surface-temperature dynamics were monitored and recorded. Surface quality and haze development were evaluated. Rabbits were humanely killed 0 to 6 months after surgery, and eyes were enucleated for histological examination. RESULTS: Rabbit corneas ablated with femtosecond UV pulses or excimer laser radiation were similar in terms of the corneal wound-healing process, surface quality, and histology. CONCLUSIONS: The experiments indicate the feasibility of clinical application of femtosecond UV lasers for stromal ablation. The ability to switch between laser harmonics allows fast changeover from infrared to the UV mode, implying that a wide range of ophthalmic procedures can be performed using a single solid-state laser device. FINANCIAL DISCLOSURE: Ms. Gabryte and Mr. Danielius are paid employees of Light Conversion Ltd. Mr. Danielius is a shareholder of Light Conversion Ltd. No other author has a financial or proprietary interest in any material or method mentioned.

DNA damage in bone marrow cells induced by ultraviolet femtosecond laser irradiation.

BACKGROUND DATA: Research on the damaging effect of ultraviolet (UV) laser irradiation on the DNA of live organisms is still scarce, although UV lasers are increasingly being used in therapeutics and surgical treatment. OBJECTIVE: In this study we investigated the effect of new-generation 205-nm femtosecond solid-state laser irradiation on the DNA of murine bone marrow cells in vitro. MATERIALS AND METHODS: Mouse bone marrow cells in distinct plates were exposed to different doses of 205-nm femtosecond laser irradiation. Single-cell gel electrophoresis, or comet, assay was used for DNA damage measurement. RESULTS: Our study revealed intensity-dependent genotoxic, genotoxic-cytotoxic, or cytotoxic impact of laser irradiation. The lowest doses we used (0.0175-0.105 J/cm(2)) induced DNA photodamage in irradiated cells directly, medial doses (0.175 and 0.35 J/cm(2)) caused both direct damage of genetic material and irreversible injury of cell's structure whereas the highest doses (1.05-4.2 J/cm(2)) caused the death of most irradiated cells. It is worrisome that even comparatively low doses of irradiation were genotoxic. Exposure to the lowest-intensity irradiation (0.0175 J/cm(2)) caused a highly significant (p < 0.0001) increase in DNA strand breaks of bone marrow cells: the mean ± SEM %DNA score in the comet tail was 9.96 ± 0.56 compared with 3.58 ± 0.80 for controls. Investigation of the effects of low and medial intensities of irradiation showed a dosage-effect relationship of R(2) = 0.84, P < 0.01. CONCLUSION: New-generation 205-nm femtosecond laser irradiation produced a genotoxic effect by inducing strand breaks in the DNA of murine bone marrow cells in vitro. Research on the possible genotoxic effects of this laser on corneal and skin epithelial cells in vivo is needed.

Corneal shaping and ablation of transparent media by femtosecond pulses in deep ultraviolet range.

PURPOSE: To assess the performance of a newly developed solid-state femtosecond ultraviolet (UV) laser system in corneal ablation. SETTING: Vilnius University, Laser Research Centre, Vilnius, Lithuania. METHODS: Femtosecond pulses in the deep UV range (205 nm) were obtained by the generation of the fifth-harmonic of an amplified Yb:KGW laser system (fundamental output at 1027 nm). Coupled with galvanometric beam-scanning mirrors, this system allowed ablation shaping of transparent media, including poly(methyl methacrylate) (PMMA), collagen, and ex vivo porcine corneas. The surfaces of ablated structures were characterized using a noncontact confocal optical profiler. RESULTS: Spherical structures were successfully formed in all 3 materials tested. A 10.0 diopter refraction change in the cornea was produced in 180 seconds. The resulting surface quality was significantly higher (roughness length >100 microm versus approximately 6 microm) in gelatin and ex vivo corneas than in PMMA. CONCLUSION: The solid-state femtosecond UV laser system seems an attractive alternative to the currently used ophthalmic argon-fluoride excimer laser system because of its small footprint, silent operation, and ability to generate femtosecond light pulses at both 1027 nm (suitable for flap creation) and 205 nm (corneal ablation).