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In Optogenetics, cells, e.g. neurons or cardiac cells, are genetically altered to produce for example the lightsensitive protein Channelrhodopsin-2. Illuminating these cells induces action potentials or contractions and therefore allows to control electrical activity. Thus, light-induced cell stimulation can be used to gain insight to various biological processes. Many optogenetics studies, however, use only full field illumination and thus gain no local information about their specimen. But using modern spatial light modulators (SLM) in conjunction with computer-generated holograms (CGH), cells may be stimulated locally, thus enabling the research of the foundations of cell networks and cell communications. In our contribution, we present a digital holographic system for the patterned, spatially resolved stimulation of cell networks. We employ a fast ferroelectric liquid crystal on silicon SLM to display CGH at up to 1.7 kHz. With an effective working distance of 33 mm, we achieve a focus of 10 µm at a positioning accuracy of the individual foci of about 8 µm. We utilized our setup for the optogenetic stimulation of clusters of cardiac cells derived from induced pluripotent stem cells and were able to observe contractions correlated to both temporal frequency and spatial power distribution of the light incident on the cell clusters.

In this work we present non-resonant four-wave mixing microscopy as an additional contrast mechanism innonlinear microscopy. The setup for this technique was based on a commerciallyavailable multiphoton microscopesetup equipped with a titanium:sapphire-laser and an optical parametric oscillator as light sources. Fundamentalsystem characteristics with respect to the spatio-temporal pulse overlap and the inuence of aberrations on theprocess are presented. Experiments regarding the directionality of the four-wave mixing signal performed onfresh porcine meat showed an average ratio of the backward to forward signal mean intensity of 0.16 0.01.Nevertheless, structural information is comparable for both detection modalities. This highlights the potential offour-wave mixing microscopy for in vivo applications. Furthermore, results on porcine meat show the additionalcontrast generated by four-wave mixing. In summary, the results show a great potential of non-resonant four-wavemixing microscopy as label-free imaging modality in the biomedical sciences.Keywords: Nonlinear microscopy, Four-wave mixing, Nonlinear Optics

The use of ultrashort-pulsed lasers for molecule delivery and transfection has proved to be a non-invasive and highly efficient technique for a wide range of mammalian cells. This present study investigates the effectiveness of femtosecond photoporation in plant cells, a hard-to-manipulate yet agriculturally relevant cell type, specifically suspension tobacco BY-2 cells. Both spatial and temporal shaping of the light field is employed to optimise the delivery of membrane impermeable molecules into plant cells using a reconfigurable optical system designed to be able to switch easily between different spatial modes and pulse durations. The use of a propagation invariant Bessel beam was found to increase the number of cells that could be viably optoinjected, when compared to the use of a Gaussian beam. Photoporation with a laser producing sub-12 fs pulses, coupled with a dispersion compensation system to retain the pulse duration at focus, reduced the power required for efficient optical injection by 1.5-1.8 times when compared to a photoporation with a 140 fs laser output.

Femtosecond photoporation is an optical, non-invasive method of injecting membrane impermeable substances contained within the surrounding medium into cells. The technique typically addresses individual cells in a static monolayer. While this gives excellent selectivity, it can be time consuming or impractical to treat larger samples. We build on previous work using a microfluidic platform, which allows for a suspension of cells to be dosed with femtosecond light as they flow through a microfluidic channel. A reusuable quartz chip is designed with an 's'-bend with facilitates the delivery of a 'non-diffracting' femtosecond Bessel beam along the centre of the channel. By implementing off-chip hydrodynamic focusing, cells are confined to the central region of the channel and pass along the Bessel beam core where they are photoporated. This new parallel approach allows for higher flow rates to be used compared to the previous, orthogonal, design whilst maintaining the necessary dwell time in the Bessel beam core. Optical injection of the cell membrane impermeable stain propidium iodide has been successful with two cell lines. These have yielded viable injection efficiencies of 31.0±9.5% Chinese hamster ovary cells (CHO-K1) and 20.4±4.2% human promyelocytic cells (HL60) with a cell throughput of up to 10 cells/second. This marks an order of magnitude increase compared to the previous microfluidic design.

New optical techniques have the potential to fill the gap between radiological and microscopic approaches to assess the lung's internal structure. Since its quantitative assessment requires unbiased sampling and measurement principles, imaging of the whole lung with sufficient resolution for visualizing details is important. To address this request, we applied scanning laser optical tomography (SLOT) for the three dimensional imaging of mouse lung ex vivo. SLOT is a highly efficient flourescence and transmission microscopy technique allowing for 3D imaging of specimen of sizes up to several millimeters. Previously fixed lung lobes and whole lungs were optically cleared and subsequently imaged with SLOT while making use of intrinsic contrast mechanisms like absorption and autofluorescence. Imaging of airways, blood vessels and parenchyma is demonstrated. Volumetric SLOT datasets of the lung's internal structure can be analyzed in any preferred planar orientation. Moreover, the sample preparation preserves microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, SLOT is a useful technique to visualize and survey the internal structure of mouse lung at different scales and with various contrast mechanisms. Potential applications of SLOT in lung research are e.g. quantitative phenotype analysis of mouse models of human lung disease in combination with stereological methods.

High precision femtosecond laser surgery is achieved by focusing femtosecond (fs) laser pulses in transparent tissues to create an optical breakdown leading to tissue dissection through photodisruption. For moving applications in ophthalmology from corneal or lental applications in the anterior eye to vitreal or retinal surgery in the posterior eye the applied pulse energy needs to be minimized in order to avoid harm to the retina. However, the aberrations of the anterior eye elements cause a distortion of the wave front and consequently an increase in size of the irradiated area and a decrease in photon density in the focal volume. Therefore, higher pulse energy is required to still surpass the threshold irradiance. In this work, aberrations in an eye model consisting of a plano-convex lens for focusing and 2-hydroxyethylmethacrylate (HEMA) in a water cuvette as eye tissue were corrected with a deformable mirror in combination with a Hartmann-Shack-sensor. The influence of an adaptive optics aberration correction on the pulse energy required for photodisruption was investigated. A reduction of the threshold energy was shown in the aberration-corrected case and the spatial confinement raised the irradiance at constant pulse energy. As less energy is required for photodisruption when correcting for wave front aberrations the potential risk of peripheral damage is reduced, especially for the retina during laser surgery in the posterior eye segment. This offers new possibilities for high precision fs-laser surgery in the treatment of several vitreal and retinal pathologies.

A today well-known laser based treatment in ophthalmology is the LASIK procedure which nowadays includes cutting of the corneal tissue with ultra-short laser pulses. Instead of disposing a microkeratome for cutting a corneal flap, a focused ultra-short laser pulse is scanned below the surface of biological tissue causing the effect of an optical breakdown and hence obtaining a dissection. Inside the tissue, the energy of the laser pulses is absorbed by non-linear processes; as a result a cavitation bubble expands and ruptures the tissue. Hence, positioning of several optical breakdowns side by side generates an incision. Due to a reduction of the amount of laser energy, with a moderate duration of treatment at the same time, the current development of ultra-short pulse laser systems points to higher repetition rates in the range of even Megahertz instead of tens or hundreds of Kilohertz. In turn, this results in a pulse overlap and therefor a probable occurrence of interaction between different optical breakdowns and respectively cavitation bubbles of adjacent optical breakdowns. While the interaction of one single laser pulse with biological tissue is analyzed reasonably well experimentally and theoretically, the interaction of several spatial and temporal following pulses is scarcely determined yet. Thus, the aim of this study is to analyse the dynamic and interaction of two cavitation bubbles by using high speed photography. The applied laser pulse energy, the energy ratio and the spot distance between different cavitation bubbles were varied. Depending on a change of these parameters different kinds of interactions such as a flattening and deformation of bubble shape or jet formation are observed. The effects will be discussed regarding the medical ophthalmic application of fs-lasers. Based on these results a further research seems to be inevitable to comprehend and optimize the cutting effect of ultra-short pulse laser systems with high (> 500 kHz) repetition rates.

The gold nanoparticle (AuNP) mediated ultrashort laser cell membrane perforation has been proven as an efficient delivery method to bring membrane impermeable molecules into the cytoplasm. Nevertheless, the underlying mechanisms have not been fully determined yet. Different effects may occur when irradiating a AuNP with ultrashort laser pulses and finally enable the molecule to transfer. Depending on the parameters (pulse length, laser fluence and wavelength, particle size and shape, etc.) light absorption or an enhanced near field scattering can lead to perforation of the cell membrane when the particle is in close vicinity. Here we present our experimental results to clarify the perforation initiating mechanisms. The generation of cavitation and gas bubbles due to the laser induced effects were observed via time resolved imaging. Additionally, pump-probe experiments for bubble detection was performed. Furthermore, in our patch clamp studies a depolarization of the membrane potential and the current through the membrane of AuNP loaded cell during laser treatment was detected. This indicates an exchange of extra- and intra cellular ions trough the perforated cell membrane for some milliseconds. Additionally investigations by ESEM imaging were applied to study the interaction of cells and AuNP after co incubation. The images show an attachment of AuNP at the cell membrane after several hours of incubation. Moreover, images of irradiated and AuNP loaded cells were taken to visualize the laser induced effects.

Manipulation of cells requires the delivery of membrane-impermeable substances like genetic materials or proteins into the cytoplasm. Thus delivery of molecules over the cell membrane barrier is one of the key technologies in molecular biology. Many techniques concerning especially the delivery foreign DNA have been developed. Notwithstanding there still is a range of applications where these standard techniques fail to raise the desired results due to low efficiencies, high toxicity or other safety issues. Especially the transfection of sensitive cell types like primary and stem cells can be problematic. Here we present an alternative, laser based technique to perforate the cell membrane and thus allowing efficient delivery of extra cellular molecules: Gold nanoparticles (GNP) are brought into close contact with the cell, were the laser-GNP interaction leads to membrane perforation. This allows the utilisation of a weakly focused laser beam leading to fast scanning of the sample and thus to a high throughput. To investigate the GNP-laser interaction in more detail we have compared membrane perforation obtained by different laser pulse lengths. From our results we assume strong light absorption for ps laser pulses and relatively small particles as the initiating perforation mechanism, whereas an enhanced near field scattering occurs at 200 nm GNP when using fs laser pulses. SEM and ESEM imaging were applied to give a deeper insight in the GNP-cell interaction and the effects of laser radiation on the GNP. Additionally dextran- FITC derivatives of varying sizes were used to investigate the impact of molecule size on delivery efficiency.

Although femtosecond laser cell surgery is widely used for fundamental research in cell biology, the mechanisms in the so-called low-density plasma regime are largely unknown. To date, it is still unclear on which time scales free electron and free radical-induced chemical effects take place leading to intracellular ablation. In this paper, we present our experimental study on the influence of laser parameters and staining on the ablation threshold. We found that the ablation effect resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. In addition, fluorescence staining of subcellular structures 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.

The delivery of extra cellular molecules into cells is essential for cell manipulation. For this purpose genetic materials (DNA/RNA) or proteins have to overcome the impermeable cell membrane. To increase the delivery efficiency and cell viability of common methods different nano- and micro material based approaches were applied. To manipulate the cells, the membrane is in contact with the biocompatible material. Due to a field enhancement of the laser light at the material and the resulting effect the cell membrane gets perforated and extracellular molecules can diffuse into the cytoplasm. Membrane impermeable dyes, fluorescent labelled siRNA, as well as plasmid vectors encoded for GFP expression were used as an indicator for successful perforation or transfection, respectively. Dependent on the used material, perforation efficiencies over 90 % with a cell viability of about 80 % can be achieved. Additionally, we observed similar efficiencies for siRNA transfection. Due to the larger molecule size and the essential transport of the DNA into the nucleus cells are more difficult to transfect with GFP plasmid vectors. Proof of principle experiments show promising and adequate efficiencies by applying micro materials for plasmid vector transfection. For all methods a weakly focused fs laser beam is used to enable a high manipulation throughput for adherent and suspension cells. Furthermore, with these alternative optical manipulation methods it is possible to perforate the membrane of sensitive cell types such as primary and stem cells with a high viability.

Cross-linking of stromal collagen with Riboflavin and UVA radiation is an alternative treatment of keratoconus. After the cross-linking a wound healing process starts with the regeneration of the abraded epithelial layer and the stromal keratocyte-network. To clarify possible side effects by visualization we established an imaging platform for the multimodal three-dimensional imaging of the cornea and looked for differences between normal and cross-linked rabbit corneae. The microscopy system utilizes femtosecond laser light for two photon excitation of autofluorescent metabolic compounds, second harmonic imaging in forward and backward direction for the study of stromal collagen-I structure and confocal detection of the backscattered femtosecond laser light for cell detection. Preliminary results show signatures of treatment 5 weeks after the intervention in all imaging modalities.

Light microscopy is one of the major tools in modern biology. The steady development of new microscopic techniques leads to an correspondent improvement of biological methods. To expand the catalog of biological experiments, we investigate the possibilities of optical projection tomography (OPT). This technique is based on the already established X-Ray computed tomography. In contrast to most other three-dimensional microscopy techniques it is able to create three dimensional data sets of the specimens natural absorption, staining and fluorescence. Unfortunately, these advantages are opposed by a low resolution, reconstruction artifacts, and a relatively big loss of fluorescence light. We reduced the disadvantage in resolution by applying physical filters in the Fourier plane of the image path, which is not possible in X-Ray imaging yet.

The resulting effects of the interaction between nanoparticles and laser irradiation are a current matter in research. Depending on the laser parameters as well as the particles properties several effects may occur e.g. bubble formation, melting, fragmentation or an optical breakdown at the surface of the nanoparticle. Besides the investigations of these effects, we employed them to perforate the membrane of different cell lines and investigated nanoparticle mediated laser cell perforation as an alternative optical transfection method. Therefore, the gold nanoparticles (GNP) of different shapes were applied. Furthermore, we varied the methods for attaching GNP to the membrane, i.e. co-incubation of pure gold nanoparticles and bioconjugation of the surface of GNP. The optimal incubation time and the location of the GNP at the cell membrane were evaluated by multiphoton microscopy. If these GNP loaded cells are irradiated with a fs laser beam, small areas of the membrane can be perforated. Following, extra cellular molecules such as membrane impermeable dyes or foreign DNA (GFP vectors) are able to diffuse through the perforated area into the treated cells. We studied the dependence of the laser fluence, GNP concentration, GNP size and shape for successful nanoparticle mediated laser cell perforation. Due to a weak focusing of the laser beam a gentle cell treatment with high cell viabilities and high perforation efficiencies can be achieved. A further advantage of this perforation technique is the high number of cells that can be treated simultaneously. Additionally, we show applications of this method to primary and stem cells.

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer over the last decade. However, this method still results in very low efficiencies originating from biological and technical aspects. The highly-invasive mechanical enucleation belongs to the technical aspects and requires considerable micromanipulation skill. In this paper, we present a novel non-invasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically determined the metaphase plate position and shape. Subsequent irradiation of this volume with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation. We show that functional fs laser-based enucleation of porcine oocytes completely inhibited further embryonic development while maintaining intact oocyte morphology. In contrast, non-irradiated oocytes were able to develop to the blastocyst stage without significant differences to control oocytes. Our results indicate that fs laser systems offer great potential for oocyte imaging and enucleation as a fast, easy to use and reliable tool which may improve the efficiency of somatic cell clone production.

Biocompatibility studies of percutanous implants in animal models usually involve numerous lethal biopsies for subsequent morphometric analysis of the implant-tissue interface. A common drawback of the study protocol is the restriction of the analysis to one final time point. In this study optical coherence tomography (OCT) was used to visualize and enable quantification of the local skin anatomy in the vicinity of a percutaneous implant in an animal model using hairless mice. Non invasive in vivo optical biopsies were taken on predetermined time points after implantation and ex vivo in situ at the day of noticeable inflammation. The custom made Fourier-domain OCT system was programmed for imaging with different scanning schemes. A spoke-pattern of 72 cross-sectional scans which was centred at the midpoint of the circular shaped implants was acquired and worked best for the in-vivo situation. Motion-artefact-free three-dimensional tomograms were obtained from the implant site before excision and preparation for histology. Morphometric parameters such as epithelial downgrowth, distance to normal growth and tissue thickness were extracted from the images with a simple segmentation algorithm. Qualitatively, the OCT B-Scans are in good agreement with histological sections. Therefore, OCT can provide additional valuable information about the implant-tissue interface at freely selectable time points before the lethal biopsy. Locally confined quantitative assessments of tissue-implant interaction for in vivo postoperative monitoring can be carried out.

Lately, several groups successfully used ultrashort laser pulses to selectively permeabilize the membrane of living cells to achieve transport of foreign molecules, like DNA, into the cells. For this, the high field intensities of tightly focused laser pulses are used to induce multiphoton absorption and the creation of a small scale optical breakdown at the membrane of the target cell. Afterwards, DNA or other foreign molecules are able to diffuse into the cell and achieve, for example, transfection of living cells. However, the cell throughput of this method is low, as, due to tight focusing. We present a technique to achieve fs-laser transfection in living cells at higher throughput by implementing optical traps into microfluidic chips. For this, a trapping laser beam, is coupled into a microscope setup and combined with a Ti:Sa fslaser beam to achieve simultaneous trapping and optical perforation.

The aim was to evaluate a method for visualizing fs laser pulse induced microincisions inside crystalline lens tissue. Porcine lenses were modified ex vivo by fs laser pulses to create defined planes at which lens fibers separate. Lens fiber orientation and fs laser-induced micro-incisions were examined using a confocal laser scanning microscope. Micro-incision visualization revealed different cutting effects depending on fs laser pulse energy, ranging from altered tissue scattering properties with all fibers intact to definite fiber separation with a wide gap. CLSM permits visualization and analysis and thereby control of fs laser pulse induced microincisions inside crystalline lens tissue.

To be able to understand and improve various optical systems including fs-laser lightsources, we developed a numerical simulation based on the coherent transfer function (CTF). Our work created a simulation tool, which is able to simulate all three dimensions and time while accounting for coherent light, short pulses, aberrations, chirp, pulse delay, numerical aperture, the intensity distribution and the polarization of the incident light. We explain the theoretical approach and aspects necessary for the simulations. Then we show the capabilities of our tool in simulation aberrations, 4Pi point spread functions, two objective fs-pulse interaction and multi objective confocal fluorescence microscopy.

Investigation on the interaction of small particles, e.g. gold nanoparticles with light is a current field of high interest. As light can be absorbed, enhanced or scattered by the nanoparticles a wide variety of possible applications become possible. If the electrons of such a nanoparticles oscillate with the incident light, plasmon resonances occur. Provided that these particles are brought very close to a cell, the cell membrane gets perforated due to the laser induced effect. We investigate nanoparticle mediated laser perforation as an alternative technique for cell transfection. By using weakly focussed femtosecond laser pulses, 150 nm gold particles were stimulated to perforate the cell membrane. Through the perforated area of the membrane macromolecules e.g. DNA are able to enter the cell. By this technique GFSHR-17 rat cells were successfully transfected with GFP vector and the dependence on laser parameters and concentration were studied. Even after 48 hours after manipulation the transfected cells show no indications of apoptosis or necrosis. This technique allows the transfection of cells by opto-perforation without the need of tight focusing conditions and single cell targeting- opening the way for a wide field of applications.

Ultrashort laser pulses recently found extensive application in micro- and nanostructuring, in refractive surgery of the eye, and in biophotonics. Due to the high laser intensity required to induce optical breakdown, nonlinear plasma formation is generally accompanied by a number of undesired nonlinear side-effects such as self-focusing, filamentation and plasma-defocusing, seriously limiting achievable precision and reproducibility. To reduce pulse energy, enhance precision, and limit nonlinear side effects, applications of ultrashort pulses have recently evolved towards tight focusing using high numerical aperture microscope objectives. However, from the theoretical and numerical point of view generation of optical breakdown at high numerical aperture focusing was barely studied. To simulate the interaction of ultrashort laser pulses with transparent materials, a comprehensive numerical model taking into account nonlinear propagation, plasma generation as well as the pulse's interaction with the generated plasma is introduced. By omitting the widely used scalar and paraxial approximations a novel nonlinear propagation equation is derived, especially suited to meet the conditions of high numerical aperture focusing. The multiple rate equation (MRE) model is used to simultaneously calculate the generation of free electrons. Nonparaxial and vectorial diffraction theory provides initial conditions. The theoretical model derived is applied to numerically study the generation of optical breakdown plasmas, concentrating on parameters usually found in experimental applications of cell surgery. Water is used as a model substance for biological soft tissue and cellular constituents. For focusing conditions of numerical aperture NA < 0.9 generation of optical breakdown is shown to be strongly influenced by plasma defocusing, resulting in spatially distorted breakdown plasmas of expanded size. For focusing conditions of numerical aperture NA ≥ 0.9 on the other hand generation of optical breakdown is found to be almost unaffected by distortive side-effects, perfectly suited for material manipulation of highest precision.

Fluorescence imaging of cells and cell organelles requires labeling by fluorophores. The labeling of living cells is often done by transfection of fluorescent proteins. Viral vectors are transferring the DNA into the cell. To avoid the use of viruses, it is possible to perforate the cell membrane for example by electro-shocks, the so called electroporation, so that the fluorescent proteins can diffuse into the cell. This method causes cell death in up to 50% of the treated cells because the damage of the outer membrane is too large. A less lethal perforation of the cell membrane with high efficiency can be realized by femtosecond (fs) laser pulses. Transient pores are created by focusing the laser beam for some milliseconds on the membrane. Through this pore, the proteins can enter into the cell. This was demonstrated in a proof of principle experiment for a few cells, but it is essential to develop an opto-perforation system for large numbers of cells in order to obtain statistically significant samples for biological experiments. The relationship between pulse energy, irradiation time, repetition rate and efficacy of the transfer of a chromophor into the cells as well as the viability of the cells was analysed. The cell viability was observed up to 90 minutes after manipulation.

Multiphoton microscopy is a very promising method for 3D imaging of living cells. The fluorochromes are solely excited at the laser focus by multiphoton absorption using near-infrared femtosecond laser pulses. The arising fluorescence serves for a pixel-to-pixel imaging with a resolution in the submicron range. At higher laser powers, the multiphoton absorption creates a micro plasma which induces an outwardly propagating shock wave. The rapidly expanding cavitation bubble causes disruption of the material, with hardly any interaction with the surrounding tissue as the optical breakdown proceeds faster than the thermal conduction. This combination offers the possibility of simultaneous manipulation and analysis of living cells or cell organelles. Manipulation is achieved using laser pulses with an energy of a few nanojoules while imaging is done at less than 1 nJ . The obtained resolution allows the precise cutting of single cell organelles without compromising the cells` viability. Thus, the implementation is excellently suited for cell surgery. We conducted ablation of different subcellular structures, like mitochondria, at different pulse energies within living cells while studying cell viability.

We selectively disrupted the cytoskeletal network of fixed and live bovine capillary endothelial cell using ultrashort laser pulses. We image the microtubules in the cytoskeleton of the cultured cells using green fluorescent protein. The cells are placed on a custom-built inverted fluorescence microscope setup, using a 1.4 NA oil-immersion objective to both image the cell and focus the laser radiation into the cell samples. The laser delivers 100-fs laser pulses centered at 800 nm at a repetition rate of 1 kHz; the typical energy delivered at the sample is 1-5nJ. The fluorescent image of the cell is captured with a CCD-camera at one frame per second. To determine the spatial discrimination of the laser cutting we ablated microtubules and actin fibers in fixed cells. At pulse energies below 2 nJ we obtain an ablation size of 200 nm. This low pulse energy and high spatial discrimination enable the application of this technique to live cells. We severed a single microtubule inside the live cells without affecting the cell's viability. The targeted microtubule snaps and depolymerizes after the cutting. This nanosurgery technique will further the understanding and modeling of stress and compression in the cytoskeletal network of live cells.

The most probable reason for presbyopia is an age-related loss of the elasticity of the lens. It develops through the whole life, but is first noticeable typically at the age of about 45. From that on it leads within 15 years to a total loss of the accommodation ability. However, both, the ciliary muscle and the lens capsule stay active and elastic, respectively. With respect to this, a possible treatment conception is to increase or regain the elasticity. The possibility to increase elasticity with ps-laser induced cuts inside the lens was already shown by Krueger. We made an improvement in cutting quality while using a fs laser with 5~kHz repetition rate emitting in the near infrared. Different fs-laser-induced μm smooth cuts inside fresh enucleated ex-vivo pig lenses will be presented.

When ultrashort laser pulses are focused inside transparent materials, extremely high field intensities can easily be achieved in the focal volume leading to nonlinear interaction with the material. In corneal tissue this nonlinear interaction results in an optical breakdown that may serve as a cutting mechanism in ophthalmology. As a side effect of optical breakdown in corneal tissue, streak-like structures have been observed as discoloration in histological sections under a light microscope. To investigate the streak formation, a numerical model including nonlinear pulse propagation due to self-focusing, group velocity dispersion, and plasma defocusing due to generated free electrons is presented. The model consists of a (3+1)-dimensional nonlinear Schroedinger equation, describing the pulse propagation coupled to an evolution equation covering the generation of free electrons. The rate equation contains multi photon ionization as well as avalanche ionization. The model is applicable to any transparent Kerr-medium.

The most probable reason for presbyopia, the age-related loss of the accomodation-ability of the eye, is an age-related loss of the elasticity of the lens. To increase the elasticity, resp. regain elasticity we performed different fs-laser-induced cuts with an near-infrared 5 kHz femtosecond laser inside ex-vivo and in-vivo rabbit lenses. Sagittal and annulus cutting patterns in the lens were produced by focusing the laser beam through the cornea inside the lens and creating a laser induced optical breakdown. The cutting results were recorded with light microscopy and finally, after euthanization, changes in the optical fibers of the lens tissue were described.

Due to the low energy threshold of photodisruption with fs laser pulses, thermal and mechanical side effects are limited to the sub μm range. The neglection of side effects enables the use of ultrashort laser pulses in a broad field of medical applications. Moreover, the interaction process based on nonlinear absorption offers the opportunity to process transparent tissue three dimensionally inside the bulk. We demonstrate the feasibility of surgical procedures in different fields of medical interest: In ophthalmology intrastromal cutting and preparing of corneal flaps for refractive surgery in living animals is presented. Besides, the very low mechanical side effects enables the use of fs-laser in otoralyngology to treat ocecular bones. Moreover, the precise cutting quality can be used in fields of cardiovascular surgery for the treatment of arteriosclerosis as well as in dentistry to remove caries from dental hard tissue.

Ultrafast lasers have become a promising tool for micromachining and extremely precise ablation of all kinds of materials. Due to the low energy threshold, thermal and mechanical side effects are limited to the bu micrometers range. The neglection of side effects enables the use of ultrashort laser pulses in a broad field of medical applications. Moreover, the interaction process based on nonlinear absorption offers the opportunity to process transparent tissue three dimensionally inside the bulk. We demonstrate the feasibility of surgical procedures in different fields of medical interest: in ophthalmology intrastromal cutting and preparing of cornael flaps for refractive surgery in living animals is presented. Besides, the very low mechanical side effects enables the use of fs- laser in otoralyngology to treat ocecular bones. Moreover, the precise cutting quality can be used in fields of cardiovascular surgery for the treatment of arteriosklerosis as well as in dentistry to remove caries from dental hard tissue.

In order to perform refractive surgery, fs-laser pulses were focussed into animal cornea. During histo-pathological analysis by light- and transmission-electron-microscopy new side effects of fs-photodisruption were found. Due to the high intensities at the focal region, self focussing followed by further nonlinear effects is observed, leading to streak formation inside the cornea and several micro-breakdowns on the sub-micron scale. The nature and origin of these streaks are further investigated.

In this work the dependence of the threshold energy and pulse duration for photodisruption was studied between 100 and 1500 femtoseconds using an erbium fiber laser system with subsequent chirped pulse amplification in a titanium-sapphire system. Cutting effects were analyzed in animal specimens like rabbit cornea and porcine eye globes. To perform intrastromal cuts, the laser beam was guided through a computer controlled delivery system. The treated tissue was analyzed by histological sections and by means of light and electron microscopy. Pressure transients were measured at the endothelium during the cutting procedure. Bubble formation and dynamics were studied using flash photography. The resulting gas bubbles were analyzed to their content, and photodissociation could be noticed. As a result of these studies a set of optimized parameters for pulse duration, scanning algorithm, pulse energy and the focusing and beam delivery system could be found. The thermal damage zone in the irradiated tissue was found to be smaller than 1 micrometer and maximum pressure transients at the endothelium could be determined to be in the range of some hundred millibars. Moreover, performing of intrastromal cuts and creation of a lenticule for achieving a refractive effect in the eye was demonstrated. The investigations show, that fs-pulses are a very precise tool for refractive surgery.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

International audience; Ultrashort pulses lasers are tools of choice for functionalizing the bulk of transparent materials. In particular, direct photoinscription of simple photonic functions have been demonstrated. Those elementary functions rely on the local refractive index change induced when focusing an ultrashort pulse in the volume of a transparent material. The range of possibilities offered by direct photoinscription is still under investigation. To help understanding, optimizing and assessing the full potential of this method, we developed a time-resolved phase contrast microscopy setup. The imaginary part (absorption) and the real part of the laser-induced complex refractive index can be visualized in the irradiated region. The setup is based on a commercially available phase contrast microscope extended into a pump-probe scheme. The originality of our approach is that the illumination is performed by using a pulsed laser source (i.e. a probe beam). Speckle-related issues are solved by employing adequate sets of diffusers. This laser-microscopy technique has a spatial resolution of 650 nm, and the impulse response is about 300 fs. The laser-induced refractive index changes can be tracked up to milliseconds after the energy deposition. The excitation beam (the pump) is focused with a microscope objective (numerical aperture of 0.45) into the bulk of an a-SiO2 sample. The pump beam can be temporally shaped with a SLM-based pulse shaping unit. This additional degree of flexibility allows for observing different interaction regimes. For instance, bulk material processing with femtosecond and picosecond duration pulses will be studied.