Femtosecond laser induced step-like structures inside transparent hydrogel due to laser induced threshold reduction.

In the area of laser material processing, versatile applications for cutting glasses and transparent polymers exist. However, parasitic effects such as the creation of step-like structures appear when laser cutting inside a transparent material. To date, these structures were only described empirically. This work establishes the physical and chemical mechanisms behind the observed effects and describes the influence of process and material parameters onto the creation of step-like structures in hydrogel, Dihydroxyethylmethacrylat (HEMA). By focusing laser pulses in HEMA, reduced pulse separation distance below 50 nm and rise in pulse energy enhances the creation of unintended step-like structures. Spatial resolved Raman-spectroscopy was used to measure the laser induced chemical modification, which results into a reduced breakdown threshold. The reduction in threshold influences the position of optical breakdown for the succeeding laser pulses and consequently leads to the step-like structures. Additionally, the experimental findings were supplemented with numerical simulations of the influence of reduced damage threshold onto the position of optical breakdown. In summary, chemical material change was defined as cause of the step-like structures. Furthermore, the parameters to avoid these structures were identified.

Comparison of two prototypes of a magnetically adjustable glaucoma implant in rabbits.

Glaucoma drainage devices are used in surgical glaucoma therapy. Success of controlling the intraocular pressure is limited due to fibrous implant encapsulation and fibrin coating on the implant which lead to drainage obstructions. An innovative implant with a magnetically adjustable valve was developed. The valve opening of the implant should eliminate inflammatory products from the outflow area and affect fibrous tissue formation to achieve a sufficient long-term aqueous humour outflow. Lifting of this valve should disturb cell adhesion by exerting mechanical forces. Before testing this hypothesis, the flow characteristics of glaucoma drainage devices, especially the outflow resistance by regular IOP, should be considered in a pilot study, as they are important in preventing too low postoperative intraocular pressure known as ocular hypotony. Therefore, two prototypes of the innovative implant differing in their valve area design were examined regarding their flow characteristics in a limited animal experiment lasting two weeks. Ten healthy New Zealand White rabbits were divided into two groups (A & B) with different implanted prototypes. Daily, tonometry and direct ophthalmoscopy were performed to assess the intraocular pressure and the inflammatory reaction of the eye. After two weeks, the rabbits were euthanised to evaluate the initially histological inflammatory reaction to the implant. In group A, one case of hypotony emerged. When considering the entire observation period, a highly statistically significant difference between the intraocular pressure in the operated eye and that in the control eye was detected in group A (p < 0.0001) in contrast to group B (p = 0.0063). The postoperative inflammatory signs decreased within two weeks. Histologically, a typical but low level foreign body reaction with macrophages and lymphocytes as well as mild to moderate fibrosis was seen after the short experimental period. Based on our tonometric results, prototype B seems to be the system of choice for further research assessing its long-term function and biocompatibility.

Measurement of Ex Vivo Porcine Lens Shape During Simulated Accommodation, Before and After fs-Laser Treatment.

PURPOSE: According to Helmholtz, accommodation is based on the flexibility of the crystalline lens, which decreases with age, causing presbyopia. With femtosecond (fs)-lentotomy treatment, it is possible to restore the flexibility of presbyopic lenses. The efficiency of the treatment can be systematically evaluated using the finite element method based on experimental data. The purpose of this study was to quantify the shape change of ex vivo lenses in different accommodation states according to the fs-lentotomy treatment. METHODS: Five lenses with ciliary body excised from ex vivo porcine eyes (age: approximately 6 months, exact age unknown) were stretched in an accommodation device before and after laser treatment. Depending on the accommodation state, the lens shape, reconstructed from lens thickness, diameter, and anterior and posterior curvature, was measured using optical coherence tomography (OCT). The complete lens shape was parameterized and each measured parameter was compared to the results of a control group (n = 5, age: approximately 6 months, exact age unknown) without treatment. RESULTS: The amplitudes of the parameters thickness (+140%), diameter (+54%), and anterior radius of curvature (+57%) significantly increased after treatment (P < 0.05), and showed no significant change for the control group. By contrast, the amplitude of the posterior radius of curvature showed no change after treatment (P > 0.05). CONCLUSIONS: Measurement of the lens shape in different accommodation states was successful and showed significant changes after the treatment. The resulting data will be utilized as input for a finite element model to systematically evaluate the effect of fs-lentotomy treatment in future work.

Effects of cavitation bubble interaction with temporally separated fs-laser pulses.

We present a time-resolved photographic analysis of the pulse-to-pulse interaction. In particular, we studied the influence of the cavitation bubble induced by a fs-pulse on the optical focusing of the consecutive pulse and its cavitation bubble dynamics in dependence on temporal pulse separation in water. As a first result, by decreasing the temporal separation of laser pulses, there is a diminishment of the laser-induced optical breakdown (LIOB) efficiency in terms of energy conversion, caused by disturbed focusing into persisting gas bubbles at the focal volume. A LIOB at the focal spot is finally suppressed by impinging the expanding or collapsing cavitation bubble of the preceding pulse. These results could be additionally confirmed in porcine gelatin solution with various concentrations. Hence, the interaction between the laser and transparent ophthalmic tissue may be accompanied by a raised central laser energy transmission, which could be observed in case of a temporal pulse overlap. In conclusion, our experimental results are of particular importance for the optimization of the prospective ophthalmic surgical process with future generation fs-lasers.

Femtosecond lentotomy: generating gliding planes inside the crystalline lens to regain accommodation ability.

Based on Helmholtz Theory for accommodation the increasing sclerosis of lens nucleus and cortex is the main cause for the developments of presbyopia. Existing therapies, however, do not reverse the stiffness of the crystalline lens and thus do not regain real accommodation ability. A new approach to restore the flexibility of the lens could be realized by photodisruption using ultrafast laser pulses. This process, known as fs-lentotomy, was used to create micro-incisions which act as gliding planes inside the crystalline lens without opening the eye globe.

Control of femtosecond thin-flap LASIK using OCT in human donor eyes.

PURPOSE: Thin-flap keratomileusis is a procedure that minimizes LASIK flap thickness to preserve both the corneal epithelium and the maximum residual stroma. This study investigated the usefulness of optical coherence tomography (OCT) as a tool in guiding the femtosecond laser in the creation of a thin flap in human eyes in a non-randomized case series. METHODS: In a private research laboratory, an in vitro investigation was performed on human autopsy eyes. Five human cadaver eyes, unsuitable for transplantation, underwent flap creation with a femtosecond laser. The laser procedure was controlled in real-time with an OCT system (Thorlabs HL AG) to ensure that the cut was placed just underneath Bowman's layer. The repetition rate of the femtosecond laser was 10 MHz with a single-pulse duration of <400 femtoseconds (pulse energy in the nJ range). As a control, all eyes underwent histological dissection and were examined using light microscopy. RESULTS: Video monitoring of the flap creation supported the feasibility of real-time OCT monitoring of the femtosecond laser flap creation process. A clear distinction of the corneal epithelium was possible in all eyes. Bowman's layer was not identified in all donor eyes at the given resolution of the OCT device used in this study. Light microscopy demonstrated flaps approximately 50-microm thick, confirming that the real-time monitoring assured a positioning of the cutting plane at minimum distance underneath Bowman's layer. CONCLUSIONS: This study of five human cadaver eyes shows that real-time OCT monitoring of the creation of thin-flaps in LASIK using a femtosecond laser is possible, thus ensuring that the flap is created at the desired depth.

Interaction dynamics of spatially separated cavitation bubbles in water.

We present a high-speed photographic analysis of the interaction of cavitation bubbles generated in two spatially separated regions by femtosecond laser-induced optical breakdown in water. Depending on the relative energies of the femtosecond laser pulses and their spatial separation, different kinds of interactions, such as a flattening and deformation of the bubbles, asymmetric water flows, and jet formation were observed. The results presented have a strong impact on understanding and optimizing the cutting effect of modern femtosecond lasers with high repetition rates (>1 MHz).

Visualization of femtosecond laser pulse-induced microincisions inside crystalline lens tissue.

PURPOSE: To evaluate a new method for visualizing femtosecond laser pulse-induced microincisions inside crystalline lens tissue. SETTING: Laser Zentrum Hannover e.V., Hannover, Germany. METHOD: Lenses removed from porcine eyes were modified ex vivo by femtosecond laser pulses (wavelength 1040 nm, pulse duration 306 femtoseconds, pulse energy 1.0 to 2.5 microJ, repetition rate 100 kHz) to create defined planes at which lens fibers separate. The femtosecond laser pulses were delivered by a 3-dimension (3-D) scanning unit and transmitted by focusing optics (numerical aperture 0.18) into the lens tissue. Lens fiber orientation and femtosecond laser-induced microincisions were examined using a confocal laser scanning microscope (CLSM) based on a Rostock Cornea Module attached to a Heidelberg Retina Tomograph II. Optical sections were analyzed in 3-D using Amira software (version 4.1.1). RESULTS: Normal lens fibers showed a parallel pattern with diameters between 3 microm and 9 microm, depending on scanning location. Microincision visualization showed different cutting effects depending on pulse energy of the femtosecond laser. The effects ranged from altered tissue-scattering properties with all fibers intact to definite fiber separation by a wide gap. Pulse energies that were too high or overlapped too tightly produced an incomplete cutting plane due to extensive microbubble generation. CONCLUSIONS: The 3-D CLSM method permitted visualization and analysis of femtosecond laser pulse-induced microincisions inside crystalline lens tissue. Thus, 3-D CLSM may help optimize femtosecond laser-based procedures in the treatment of presbyopia.

Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery.

Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.

Green laser light activates the inner ear.

The hearing performance with conventional hearing aids and cochlear implants is dramatically reduced in noisy environments and for sounds more complex than speech (e. g. music), partially due to the lack of localized sensorineural activation across different frequency regions with these devices. Laser light can be focused in a controlled manner and may provide more localized activation of the inner ear, the cochlea. We sought to assess whether visible light with parameters that could induce an optoacoustic effect (532 nm, 10-ns pulses) would activate the cochlea. Auditory brainstem responses (ABRs) were recorded preoperatively in anesthetized guinea pigs to confirm normal hearing. After opening the bulla, a 50-microm core-diameter optical fiber was positioned in the round window niche and directed toward the basilar membrane. Optically induced ABRs (OABRs), similar in shape to those of acoustic stimulation, were elicited with single pulses. The OABR peaks increased with energy level (0.6 to 23 microJ/pulse) and remained consistent even after 30 minutes of continuous stimulation at 13 microJ, indicating minimal or no stimulation-induced damage within the cochlea. Our findings demonstrate that visible light can effectively and reliably activate the cochlea without any apparent damage. Further studies are in progress to investigate the frequency-specific nature and mechanism of green light cochlear activation.

Femtosecond laser induced flexibility change of human donor lenses.

BACKGROUND: According to the Helmholtz theory of accommodation the loss of accommodation amplitude is caused by the growing sclerosis of the crystalline lens, whereas the ciliary muscle and the lens capsule are mainly uneffected by age. A permanent treatment method for presbyopia which offers a dynamic accommodation ability is a recent field of study. The concept followed in this paper uses femtosecond laser pulses to potentially overcome the loss of deformation ability of the crystalline lens by creating gliding planes inside the lens tissue to improve its flexibility. METHODS: The aim of the study is to show that the flexibility of human donor lenses can be increased by applying tightly focused near infrared femtosecond laser pulses into the lens tissue. Thereby the tissue is separated by the photodisruption effect. A certain pattern of gliding planes is cut inside the tissue of 41 human donor lenses and the deformation ability of the lenses are compared using the Fisher spinning test before and after laser treatment. RESULTS: The laser treatment results in an increased deformation ability of the crystalline lens. The lens a-p thickness increases on average by 97 microm+/-14 microm after the treatment. The Fisher spinning test shows an increase of 16% in deformation ability of the lens at a rotational speed of 1620 rpm. CONCLUSION: The creation of gliding planes with a fs laser inside the crystalline lens tissue can change the deformation ability of the lens. This might be an indication for a possible method to treat presbyopia in future.

Purinergic signalling in rat GFSHR-17 granulosa cells: an in vitro model of granulosa cells in maturing follicles.

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca(2+)-imaging and perforated patch-clamp. We observed a resting intracellular Ca(2+)-concentration ([Ca(2+)](i)) of 100 nM and a membrane potential of -40 mV. This was consistent with high K(+)- and Cl(-) permeability and a high intracellular Cl(-) concentration of 40 mM. Application of ATP for 5-15 s every 3 min induced repeated [Ca(2+)](i) increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP(3)-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y(2) and P2Y(4) receptors and that the [Ca(2+)](i) increase was a prerequisite for hyperpolarization. Inhibitors of Ca(2+)-activated channels or K(+) channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl(-) channels hyperpolarized cells to -70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y(2) and P2Y(4) receptors in granulosa cells modulate Cl(-) permeability by regulating Ca(2+)-release.

In vivo application and imaging of intralenticular femtosecond laser pulses for the restoration of accommodation.

PURPOSE: According to the Helmholtz theory of accommodation, one major cause of the development of presbyopia is the increasing sclerosis of the crystalline lens. One concept for regaining the elasticity of the sclerosing lens is intralenticular treatment by femtosecond laser pulses. METHODS: The feasibility of applying and imaging in vivo microincisions by femtosecond laser pulses was evaluated in five rabbit lenses with a new high repetition rate (100 kHz) femtosecond laser unit. The treated eyes were monitored using optical coherence tomography (OCT) and Scheimpflug imaging for localizing and studying the tissue effects of the incisions. The rabbits were investigated preoperatively, immediately postoperatively, and 14 days after treatment. RESULTS: The procedure, termed femtosecond-lentotomy, was successfully applied to the left lens of each rabbit. The laser microincisions within the crystalline lens were detectable with OCT and Scheimpflug imaging, which emphasizes the integral role these technologies play in targeting and characterizing postoperative tissue effects. The imaging within the lens showed a progressive fading of the incisional opacities generated by the femtosecond laser after 14 days with no detectable cataract formation. CONCLUSIONS: It is possible to create microincisions inside the crystalline lens within an acceptably short treatment time (<30 seconds). The 14-day follow-up did not show undesirable side effects, such as cataract formation, after intralenticular laser treatment.

Clinical value of optical coherence tomography in laryngology.

BACKGROUND: Optical coherence tomography (OCT) is a new, noninvasive imaging technology for the evaluation of superficial lesions. The objective of this study is to evaluate microlaryngoscopy in combination with OCT compared with microlaryngoscopy alone (ie, without OCT) in supplying a specific diagnosis, predicting invasive tumor growth and epithelial dysplasia in the larynx. METHODS: This was a prospective study including 217 laryngeal lesions in a total of 193 patients undergoing surgery. Intraoperative suspicion diagnosis gained by microlaryngoscopy with and without OCT was compared with conventional histopathology after excisional biopsy. RESULTS: Microlaryngoscopy with OCT supplied a specific diagnosis in 89% of cases, but in only 80% of cases with microlaryngoscopy alone. In particular, our results in malignant and benign pathologies were correct in 93% each, and the exact grade of dysplasia could be predicted in 71% of precancerous lesions. Microlaryngoscopy with OCT presented a higher sensitivity than microlaryngoscopy alone in predicting invasive tumor growth (93% vs 87%) and epithelial dysplasia (78% vs 66%), but the specificity and accuracy were comparable in both methods. CONCLUSIONS: OCT is a simple, rapid, and reliable aid in the diagnostic investigation and intraoperative monitoring of laryngeal disease.

Real-time optical coherence tomography-guided femtosecond laser sub-Bowman keratomileusis on human donor eyes.

PURPOSE: To determine the usefulness of optical coherence tomography (OCT) as a tool in guiding the femtosecond (fs) laser in the creation of a sub-Bowman keratomileusis (SBK) flap in human eyes. DESIGN: A nonrandomized case series. METHODS: In a private research laboratory setting, we performed an in vitro investigation on human autopsy eyes. Five human cadaver eyes, unsuitable for transplantation, underwent flap creation with a fs laser. The laser procedure was controlled in real-time with an OCT system (Thorlabs HL AG, Luebeck, Germany) to ensure that the cut was placed just underneath the Bowman layer. The fs laser worked at a repetition rate of 10 MHz with a single-pulse duration of < 400 fs (pulse energy in the nanoJoule range). As a control, all eyes underwent histologic dissection (toluidine blue) and were examined using light microscopy (LM). RESULTS: Video monitoring of the flap creation supported the feasibility of real-time OCT monitoring of the fs laser flap creation process. A clear distinction of the corneal epithelium was possible in all eyes. The Bowman membrane was not identified in all donor eyes at the given resolution of the OCT used in this study. Still, LM examination confirmed that the real-time monitoring assured a positioning of the cutting plane at minimum distance underneath the Bowman layer. CONCLUSION: This small laboratory test offers evidence that real-time OCT monitoring of creation of a SBK flap using a fs laser is possible, thus ensuring that the flap is created at the proper depth.

Overview of commercially available femtosecond lasers in refractive surgery.

PURPOSE: To report on four different commercially available femtosecond laser systems used in refractive surgery. METHODS: Four systems were analyzed: Ziemer DaVinci system, Zeiss VisuMax, IntraLase FS laser, and 20/10 Perfect Vision FEMTEC. RESULTS: The DaVinci system attracts attention with its concept of low laser pulse energy and flexible mirror arm as a beam delivery. The low pulse energy makes it compact and robust, because only an oscillator as a laser source is required. Another important feature of the low energy pulses is the reduced bubble formation during the cutting process. The smaller the bubbles, the more precise the cut can be positioned. However, the compact setup limits the system in its flexibility in cutting geometry. The VisuMax also uses the new generation of femtosecond technology, which should make it more immune to environmental and temperature changes. However, it is a bulky system, and the patient has to be moved to the excimer laser system after the flap is created. The IntraLase is the laser with the most clinical experience by far, which should be an important issue in terms of safety and technical support. On the other hand, it uses older femtosecond laser technology. This technology is sensitive to changes in temperature or humidity, which can be an important factor if the surgical environment is not air conditioned. The 20/10 Perfect Vision FEMTEC system is comparable with the IntraLase with respect to its technical parameters. However, no data are available about the amount of clinical experience. CONCLUSIONS: Although the systems are based on the same principle of photodisruption in corneal tissue, differences exist regarding the concept of these devices.

fs-Laser induced elasticity changes to improve presbyopic lens accommodation.

BACKGROUND: According to the Helmholtz theory of accommodation, one of the major reasons for the development of presbyopia is the progressive sclerosis of the crystalline lens. However, both the ciliary muscle and the lens capsule stay active and elastic. Thus, the concept for regaining the deformation-ability of the crystalline lens is to create microincisions inside lens tissue to achieve gliding planes. METHODS: For the preparation of the microincisions, near-infrared femtosecond laser pulses are used, generating laser-induced optical breakdowns. Different cutting patterns were performed, and the elasticity regain of the lenses were measured with Fisher's spinning test for thickness determination. RESULTS: The creation of gliding planes inside lens tissue shows very good results in terms of increasing the deformation-ability. The optimization of laser parameters leads to a minimally invasive surgery with no remarkable side effects like residual gas bubbles. Furthermore, ex vivo elasticity measurements of untreated and treated pig lenses show an improvement in the flexibility of the lens. The deformation-ability increases up to 26% with a very low standard deviation (1.6%) and a high significance (p < 0.05). CONCLUSION: Generating particular cutting patterns inside lens tissue can increase the deformation-ability of the crystalline lens. Thus, it might be one possible way to treat presbyopia.

Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics.

The use of ultrashort laser pulses for microscopy has steadily increased over the past years. In this so-called multiphoton microscopy, laser pulses with pulse duration around 100 femtoseconds (fs) are used to excite fluorescence within the samples. Due to the high peak powers of fs lasers, the absorption mechanism of the laser light is based on nonlinear absorption. Therefore, the fluorescence signal is highly localized within the bulk of biological materials, similar to a confocal microscope. However, this nonlinear absorption mechanism can not only be used for imaging but for selective alteration of the material at the laser focus: The absorption can on one hand lead to the excitation of fluorescent molecules of fluorescently tagged cells by the simultaneous absorption of two or three photons or on the other hand, in case of higher order processes, to the creation of free-electron plasmas and, consequently, plasma-mediated ablation. Typical imaging powers are in the range of tens of milliwatts using 100-fs pulses at a repetition rate of 80-90 MHz, while pulse energies needed for ablation powers are as low as a few nanojoules when using high numerical aperture microscope objectives for focusing the laser radiation into the sample. Since the first demonstration of this technique, numerous applications of fs lasers have emerged within the field of cellular biology and microscopy. As the typical wavelengths of ultrashort laser systems lie in the near infrared between 800 and 1000 nm, high penetration depth can be achieved and can provide the possibility of imaging and manipulating the biological samples with one single laser system.