[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.

Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery.

Femtosecond (fs) laser-based intracellular nanosurgery has become an important tool in cell biology, albeit the mechanisms in the so-called low-density plasma regime are largely unknown. Previous calculations of free-electron densities for intracellular surgery used water as a model substance for biological media and neglected the presence of dye and biomolecules. In addition, it is still unclear on which time scales free-electron and free-radical induced chemical effects take place in a cellular environment. Here, we present our experimental study on the influence of laser parameters and staining on the intracellular ablation threshold in the low-density plasma regime. We found that the ablation effect of fs laser pulse trains resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. At the threshold, the number of applied pulses was inversely proportional to a higher order of the irradiance, depending on the laser repetition rate and wavelength. Furthermore, fluorescence staining of subcellular structures before surgery significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade. Consequently, future calculations of free-electron densities for intracellular nanosurgery have to take them into account, especially in the calculations of multiphoton ionization rates.

[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.

[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.

[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.

[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.

[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.

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.

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.

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.

[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.

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.

[Optical coherence tomography in the diagnosis of vocal folds]. / Charakterisierung von Stimmlippen mittels optischer Kohärenztomographie.

Optical coherence tomography (OCT) is a new, non-invasive method for investigating biological tissue, with a beam penetration of up to 3 mm. In this study, OCT was used on porcine and human larynxes. The porcine vocal folds were assigned to defined areas and examined by OCT followed by traditional histomorphological analysis. We were able to validate this new method by demonstrating that both OCT and the histological assays showed a clear demarcation of the epithelium mucosae from the deeper layers. OCT application to human vocal folds delivered same results. The current standard procedure for ensuring an accurate diagnosis if tissue is malignant, is still an invasive one. Our results show that OCT allows the collection of crucial information on the quality of morphological changes on a vocal fold without the necessity of being invasive. Thus, we propose that OCT be introduced as a new method for the detection of morphological changes in the vocal fold complementary to other established methods.