[Update on fs laser technology in ophthalmology]. / Update Femtosekundenlaser-Technologien in der Augenheilkunde.

Femtosecond (fs) lasers for LASIK flap creation have increased their market share from 0 percent in 2001 to over 55 percent in 2010. Now, in cataract surgery a similar revolution may take place. What makes this technology so special and which new areas of surgery will furthermore be entered in the future? This review elucidates the most important technical features of the fs lasers and interaction processes with the tissue. This understanding is intended to provide a better overview of the current market situation on the one hand. In addition, it can be estimated what kind of short- and long-term development this technology could take in the future.

Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection.

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

[Optical coherence tomography : significance of a new method for assessing unclear laryngeal pathologies]. / Optische Kohärenztomographie : Stellenwert einer neuen Methode bei der Abklärung unklarer Kehlkopfveränderungen.

BACKGROUND: Optical coherence tomography (OCT) is a new imaging technology of as yet unknown significance in laryngology. The objective of this study was to evaluate the ability of this noninvasive method to reliably predict diagnosis and possible malignancy in laryngeal disease. PATIENTS AND METHODS: In a prospective study, a total of 225 laryngeal lesions in 201 consecutive patients were examined during elective microlaryngoscopy. Clinical assessment under the operating microscope with and without OCT was compared with conventional histopathology. RESULTS: For all cases, diagnosis gained by OCT was significantly superior to microlaryngoscopy alone. In particular, the exact grade of dysplasia could be better determined with the help of OCT, whereas statistical significance was just barely missed for the prediction of benign lesions. Additionally, OCT proved to be a very helpful method for identifying malignant tumors of the larynx. CONCLUSIONS: OCT is a simple, rapid, and reliable aid in the diagnostic investigation of unclear laryngeal pathologies, especially of laryngeal cancer and its precursor lesions.

[Morphologic classification of Reinke's edema through optical coherence tomography]. / Morphologische Klassifikation des Reinke-Odems mittels optischer Kohärenztomografie.

OBJECTIVE: Several classification systems for Reinke's edema have been proposed in the past, which are somewhat less than morphologically ideal. The objective of the present study is to evaluate the ability of optical coherence tomography (OCT) to attain a reproducible graduation of Reinke's edema. MATERIAL AND METHODS: In a prospective study, 30 consecutive patients underwent endolaryngeal, microsurgical resection of their Reinke's edema. Graduation was performed through OCT based on morphologic criteria, where each result was compared with that of other classification systems. RESULTS: In Reinke's edema grade I according to Glanz, a feathered pattern is found, while Reinke's edema grade II demonstrates a lacunar and Reinke's edema grade III a confluent pattern. Correlation between the different classification systems was uniformly weak and not significant. CONCLUSIONS: Due to the three different recognizable patterns, OCT qualifies as an objective and reproducible aid for the graduation of Reinke's edema and gives certain insights in its pathogenesis.

Optoacoustic induced vibrations within the inner ear.

An acoustic transient can be generated inside an absorbing tissue as a result of laser-tissue interaction after pulsed laser irradiation. Herein we report a novel application of this physical process, the optoacoustic wave generation in the inner ear and subsequently the induction of basilar membrane vibrations. These laser induced vibrations show a direct correlation to the laser energy and an indirect correlation to the distance from the irradiation focus. Through these characteristics they may be used, in a new generation of cochlear implants, to improve the frequency specific cochlear activation and consequently improve speech perception in hearing impaired patients with residual hearing.

[Real-time monitoring of a femtosecond laser in Sub-Bowman Keratomileusis on human donor eyes using OCT]. / Echtzeitsteuerung einer Femtosekundenlaser Sub-Bowman-Keratomileusis an humanen Spenderaugen mittels optischer Kohärenztomografie.

PURPOSE: In LASIK (laser in situ keratomileusis) the thickness of the corneal flap is important since it is the residual corneal bed that determines corneal stability. The introduction of real-time OCT visualisation of the corneal layers during the fs-laser cut should enable the surgeon to control and monitor the position of the plane of laser-tissue-interaction during operation. To prove that optical coherence tomography (OCT) can be useful to guide femtosecond (fs)-laser in Sub-Bowman-Keratomileusis (SBK) an in-vitro experimental study was performed on human autopsy eyes in a research laboratory set-up. METHODS: Five human autopsy eyes, unsuitable for transplantation, received fs-laser keratomileusis (flap) cuts. The laser procedure was controlled in real-time with an OCT system (Thorlabs HL AG, Lübeck, Germany) to ensure that the cut was placed just underneath Bowman's layer. As a control all eyes were dissected histologically (H & E staining) and examined under the light microscope (LM). RESULTS: Videomonitoring of the laser process supported the feasibility of the concept to online monitor the fs-laser cutting process via OCT. A clear distinction of the corneal epithelium was possible in all eyes. Bowman's membrane was not identified in all autopsy eyes at the given resolution of the OCT used in this study. Still, LM sections confirmed that the online monitoring assured a positioning of the cutting plane at minimum distance underneath Bowman's membrane. CONCLUSION: It was proven that real-time OCT monitoring of fs-laser SBK on human eyes is in principle possible.

[fs-Lentotomy: presbyopia reversal by generating gliding planes inside the crystalline lens]. / Lentotomie mittels fs-Laserpulsen: Behandlung der Presbyopie durch Erzeugen von Gleitebenen in der Linse.

Based on the Helmholtz theory for accommodation, increasing sclerosis of the lens nucleus and cortex is the main cause for the development 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 has been realised by utilising the non-linear interaction of ultrafast laser pulses with transparent tissue, the so-called photodisruption. This process has been used to create micro-incisions which act as gliding planes inside the crystalline lens without opening the eye globe. This treatment method, known as fs-lentotomy, enables regeneration of real dynamic accommodation. For the first time, 3D structures for gliding planes were successfully generated in experiments with human donor lenses of different ages. An average increase in anterior-posterior lens thickness of 100 mum accompanied by a decrease of equatorial lens diameter was observed as a direct consequence of fs-lentotomy. This is attributed to the increased flexibility, as the force of the capsule bag moulds the lens tissue more spherically. Moreover, in vivo experiments on rabbit eye lenses did not induce an increasing opacification (cataract) over a six-month follow-up period. However, the incisions were still detectable using Scheimpflug imaging and histopathological techniques, although the visibility of the incisions was declining. Furthermore, no side effects were observed during the wound healing process and during a six-months follow-up period. Based on these findings fs-lentotomy might have the potential to become a procedure for the reversal of presbyopia.

[Two-photon microscopy of the cornea using intrinsic contrast]. / Farbstofffreie Zweiphotonenmikroskopie der Augenhornhaut.

BACKGROUND: Three-dimensional imaging of the cornea under physiological conditions is best performed with intrinsic contrast mechanisms for the visualisation of cells and extracellular matrix. However, the unique transparency of the cornea goes along with a lack of contrast for the extracellular matrix (ECM) in reflective mode microscopy and optical coherence tomography. METHODS: Femtosecond laser-based non-linear microscopy provides novel contrast mechanisms for the visualisation of ECM. The confinement of the non-linear contrast to the focus volume provides an intrinsic sectioning property for 3D imaging. Further advantages of the infrared light are lower phototoxicity and higher penetration depth into the tissue. For the visualisation of the cornea and its layered substructures two non-linear contrast mechanisms are of main interest: Two-photon excited autofluorescence of NAD(P)H in the cytoplasma and second harmonic generation (SHG) in the collagen-I fibres of the stroma. Ex-vivo corneas of the rabbit were imaged to demonstrate the abilities of non-linear microscopy. RESULTS: Using the autofluorescence of NAD(P)H the corneal epithelium with squamous cells, wing cells and basal cells is visualised in three dimensions without additional exogenoeus staining. Stromal keratocytes are also imaged using the NAD(P)H autofluoresecence. The layered structure of lamella in the stroma is visible after virtual resclicing of the 3D volume data. The en-face SHG images detected through the transparent cornea in forward direction show areas of parallel streaks, which increase in size and periodically alter in orientation (90 degrees , 45 degrees) with increasing depth from anterior to posterior. These streaks are not visible in the backward SHG signal. First results on rabbit corneas, which were cross-linked with Rivoflavin and UV application showed a signature of treatment five weeks post treatment. There were zones in the stroma totally lacking NAD(P)H autofluorescence and the abundance of keratocytes was less homogeneous than in control corneas. CONCLUSION: These results and current reports on applications in the literature show that femtosecond laser-based non-linear microscopy is an emerging imaging modality which provides dye-free imaging of the corneal ECM and therefore complements scattering imaging modalities such as optical coherence tomography and confocal laser scanning microscopy in the reflective mode.

Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

[Mucosal lesions in the larynx: predictive value of new imaging modalities for a histological diagnosis]. / Schleimhautveränderungen im Kehlkopf: Prädiktionswert neuerer bildgebender Verfahren für eine histologische Diagnose.

BACKGROUND: Optical coherence tomography (OCT) and high-frequency ultrasound are promising new methods in the early diagnosis of laryngeal cancer. However, no reliable values are given in the literature for epithelial thickness in early laryngeal cancer and its precursor lesions of the vocal folds. PATIENTS AND METHODS: In the present study, epithelial thickness in different benign and malignant lesions of the vocal folds was determined histologically using a normal white light microscope. RESULTS: The vocal fold mucosa showed progressive thickening over the different grades of dysplasia up to microinvasive carcinoma, while additional inflammation did not have any significant influence on the total epithelial thickness. In moderate dysplasia, however, the mean thickness of the epithelium was found to be double that of normal mucosa, and in severe dysplasia and carcinoma in situ, as much as three times that of normal mucosa. In the presence of microinvasive carcinoma, the average thickness of the epithelium was found to be as much as six times that in healthy mucosa. On the other hand, in case of benign lesions such as Reinke's edema, polyps, chronic laryngitis, and papillomas there was only slight epithelial thickening. CONCLUSIONS: Determination of epithelial thickness by OCT or high-frequency ultrasound may allow conclusions on whether or not a laryngeal lesion is malignant.

Computational model for nonlinear plasma formation in high NA micromachining of transparent materials and biological cells.

Cell surgery based on ultrashort laser pulses is a fast evolving field in biophotonics. Noninvasive intra cellular dissection at sub-diffraction resolution can be performed within vital cells with very little hazardous effects to adjacent cell organelles. Microscope objectives of high numerical aperture (NA) are used to focus ultrashort pulses to a small spot. Due to the high order of nonlinearity, plasma formation and thus material manipulation is limited to the very focus. Nonetheless nonlinear plasma formation is generally accompanied by a number of additional nonlinear effects like self-focusing and filamentation. These parasitic effects limit the achievable precision and reproducibility of applications. Experimentally it is known that the intensity of these effects decreases with increasing NA of the focusing optics, but the process of nonlinear plasma formation at high NA has not been studied numerically in detail yet. To simulate the interaction of ultrashort laser pulses with transparent materials at high NA a novel nonlinear Schr odinger equation is derived; the multiple rate equation (MRE) model is used to simultaneously calculate the generation of free electrons. Nonparaxial and vectorial effects are taken into account to accurately include tight focusing conditions. Parasitic effects are shown to get stronger and increasingly distortive for NA < 0.9, using water as a model substance for biological soft tissue and cellular constituents.

[In vitro and in vivo investigations on the treatment of presbyopia using femtosecond lasers]. / In-vitro- und In-vivo-Untersuchungen zur Presbyopiebehandlung mit Femtosekundenlasern.

BACKGROUND: Ultrashort (femtosecond) laser pulses can generate precise cuts in biological tissue without damaging the surface. The application of femtosecond laser technology at the lens was evaluated with respect to a possible treatment of presbyopia. MATERIALS AND METHODS: Femtosecond laser lentotomy was performed on 150 pig lenses in vitro. Cutting geometry and laser settings were optimized to generate smooth cuts with a minimum of produced gas bubbles. Four rabbit lenses were treated afterwards in vivo and were controlled for 3 months post-treatment. The lenses were then extracted and evaluated. RESULTS: With suitable laser settings, light scattering due to residual gas bubbles could be almost completely avoided in pig lenses. A pulse energy of less than 1.2 microJ and a cutting geometry with spot separations of more than 5 microm are important. The rabbit lenses stayed macroscopically clear for 3 months in vivo. Only the cell structures directly adjacent to the laser focus were cut; structures 5-10 microm away appeared to be intact. No cataract formation occurred during this time. CONCLUSION: Femtosecond laser application allows precise and smooth cuts inside pig and rabbit lenses without damage to adjacent tissue.

[Erbium:YAG laser surgery on vocal fold tissue]. / Erbium:YAG-Laserchirurgie an Stimmlippengewebe.

The application of lasers in microsurgery of the vocal fold has very special requirements. Easy handling as well as a precise cutting quality with a small laser induced thermal necrosis zone are necessary. In this study, an Erbium:YAG laser was evaluated for phonosurgery. For this, vocal folds from the porcine larynx were irradiated with the Erbium laser wavelength of 3 mum. The ablation rate, as well as the mechanical and thermal damage were investigated as a function of pulse duration, repetition rate and laser fluence. The results show that Erbium lasers are well suited for application on the vocal fold. The laser induced thermal damage was restricted to a superficial layer of only about 20-30 microm, resulting in a loss of the multilayered epithelium. Thermal necrosis had a range of about 5 microm. Further investigations on the vocal folds of humans must be performed to determine whether this laser is qualified for clinical applications.

Development of a drug delivery device: using the femtosecond laser to modify cochlear implant electrodes.

Animal experiments suggest that pharmacological intervention could possibly enhance cochlear implant performance. One of the key aspects is therefore a drug delivery device for the human inner ear. The objective of this study was to investigate the possibility of using the femtosecond laser for modifying a cochlear implant electrode for the purpose of drug delivery to the cochlea. Using silicone sheets, the best parameters for creating defined channels at calculated diameters were investigated using a femtosecond laser. The results were transferred to a cochlear implant electrode array (Nucleus Contour). The capability of delivering substances through the drilled openings was tested in vitro. By variation of the output of the laser, spot distance, repetition rate, number of cycles and introducing several focus planes, it was possible to drill holes with nearly vertical walls in the silicone sheets. Transferring these data to the cochlear implant electrode resulted in prototypes for drug delivery with various openings along the array. The use of the femtosecond laser allows rapid modification and adaptation of designs to experimental prototypes of cochlear implant electrodes for the purpose of drug delivery to the inner ear.

Pulsed photothermal radiometry as a method for investigating blood vessel-like structures.

Pulsed photothermal radiometry (PPTR) is known to be suitable for in vivo investigations of tissue optical properties. As a noncontact, nondestructive method it is a very attractive candidate for on-line dosimetry of laser treatments that rely on thermal laser-tissue interaction. In this article, we extend the one-dimensional (1D) analytical formalism that has widely been used to describe PPTR signals to a two-dimensional treatment of a simplified model of a blood vessel. This approach leads to quantitative description of a PPTR signal that, unlike in an 1D treatment, not only shows changes in time, but also varies in space. Using this approach, we are able to gain instructive understanding on how target characteristics of a blood vessel-like structure influence such a spatiotemporal PPTR signal. Likewise, the ability of extracting target features from those measurements is evaluated. Subsequently, we present experimental realization of the idealized model of a blood vessel as used in our theory. Comparison of actual PPTR measurements with theoretical predictions allow vessel localization laterally and in depth. Using our setup, we furthermore demonstrate the influence of flow inside the vessel on the measured signal.

A scanning and rotating slit arF excimer laser delivery system for refractive surgery.

PURPOSE: This study was designed to investigate the quality of a scanning and rotating slit delivery system of an ArF excimer laser (Nidek EC-5000). METHODS: The ablation patterns on polymethylmethacrylate (PMMA) wafers were examined by scanning electron microscopy. The influence of inhomogeneities in the beam profile was simulated on a computer and compared with a conventional large-area ablation system. The impairment of the ablation rate by radiation absorption of the ablation plume was measured as a function of the repetition rate and the application of a fixation ring. RESULTS: The scanning and rotating slit delivery system is tolerant of small-beam non-homogeneities. The ablation rate is sensitive to the dynamics of the ablation plume. CONCLUSIONS: Although the operating procedure takes less time with a large-area ablation system, a scanning and rotating delivery system has the advantage of reliable and homogeneous removal of corneal tissue.

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy.

PURPOSE: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. SETTING: Medical Laser Center Lübeck, Germany. METHODS: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 microns) and a pulsed chromium-thulium-holmium: YAG laser (2.1 microns) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbium: YSGG laser (2.70 microns), a free-running erbium: YAG laser (2.94 microns), and an argon-fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson's, and Masson-Goldner's trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius-red staining. Sirius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. RESULTS: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius-red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius-red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence. CONCLUSION: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

[193 nm Excimer laser photoablation of the cornea. Spectrum and transmission behavior of secondary irradiation]. / 193 nm Excimer-laserphotoablation der Hornhaut. Spektrum und Transmissionsverhalten von Sekundärstrahlung.

In order to estimate the photomutagenic and phototoxic potential involved we analyzed the spectrum and intensity of secondary radiation induced with a 193 nm excimer laser in photoablation of corneal tissue. The spectrum of secondary radiation was analyzed by an optical multichannel analyzer. A pyroelectrical detector was used to evaluate the energy of the secondary radiation. We found the secondary radiation to be in the range of ultraviolet to infrared. The spectrum has a relatively high quantity (20%) of radiation between 240 and 280 nm. The intensity of the secondary radiation depends on the energy fluence of the primary laser pulse and can reach a quantum yield of up to 10(-3) (at 800 mJ/cm2). At a fluence of 230 mJ/cm2 (193 nm), the secondary radiation in the range of 240-280 nm is approximately 5 microJ/cm2. Radiation below 300 nm was not transmitted through the cornea. Since the high genetic activity of UV radiation in the range from 240-280 nm is well known, evaluation of the potential mutagenicity of 193 nm excimer laser radiation should take into consideration the presence of secondary radiation in this problematic spectral range. However, there is no incidence known for specific UV-induced corneal cancers, which supports the argument that the potential mutgenic risk of clinical excimer laser application can be considered to be low. Because of the full corneal absorption of radiation below 300 nm, there is no risk known for pigmented intraocular structures.

[Photoablation of the cornea with pulsed 2790 nm ErCr:YSGG laser irradiation. Basic studies]. / Zur Photoablation der Hornhaut mit gepulster 2790 nm ErCr:YSGG-Laserstrahlung. Grundlegende Untersuchungen.

The potential of 3 microns solid-state lasers as an alternative to excimer lasers for photoablative corneal surgery was investigated. A Q-switched ErCr:YSGG laser (2790 nm, 200 ns) was used for irradiation of porcine corneas and agar-agar samples. Mechanical tissue effects (stroma, endothelium) were documented by micromorphology. Laser-induced shock-waves were analyzed by piezo-electric transducers. No sharp ablation threshold, as in excimer laser photoablation, could be determined. Energy fluences < 2 J/cm2 led to dehydration of the irradiated samples. Higher fluences are necessary for the evaporation of tissue water to be so vigorous that the tissue matrix is expelled along with the organic matrix. At high fluences, the ablation rate exceeds the absorption depth of the laser radiation (up to 25 microns/pulse). At fluences between 2.5 and 28 J/cm2 the thermal necrosis zone adjacent to the crater was 7 +/- 3 microns. The intensity of the laser-induced acoustic shock waves can peak to some hundred bar. Small gas bubbles up to 1 mm were found in the surrounding area of the ablation crater. Apparently, they were pressed between the collagen lamellas by the explosive force of the ablative process. In deep excisions (> 75%) endothelial defects underneath the beam axis could be documented. Large-area tissue ablation, with a resolution in the range of 1 micron, as necessary in myopia correction, will not be possible with the present generation of ErCr:YSGG lasers. Its high ablation rate makes this laser suitable as a cutting (astigmatism, keratoplasty, vitreous surgery) and drilling (glaucoma) device.

[Principle and mechanism of action of an ArF excimer laser system with a scanning and rotating slit aperture]. / Prinzip und Wirkungsweise eines ArF-Excimerlasersystems mit scannender und rotierender Schlitzblende.

This study was designed to investigate the quality of a scanning and rotating slit delivery system of an ArF-Excimer laser (Nidek EC 5000). The ablation patterns on PMMA samples were examined by REM. The influence of inhomogeneities in the beam profile was simulated on a computer and compared with a conventional large-area ablation system. The impairment of the ablation rate by radiation absorption of the ablation plume was measured as a function of the repetition rate and the application of a fixation ring. The scanning and rotating slit delivery system creates extremely smooth ablation patterns. The delivery system is very tolerant of small-beam inhomogeneities. The ablation rate is very sensitive to the dynamics of the ablation plume. Although the operating procedure takes less time with a large-area ablation system, a scanning and rotating delivery system has the advantage of reliable and homogeneous removal of corneal tissue.