This PDF file contains the front matter associated with SPIE-OSA Proceedings Volume 6632, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Drilling and surface processing of bone and tooth tissue belongs to standard medical procedures (bores and embeddings for implants, trepanation etc.). Small circular bores can be generally quickly produced with mechanical drills. However problems arise at angled drilling, the need to execute drilling procedures without damaging of sensitive soft tissue structures underneath the bone or the attempt to mill small non-circular cavities in hard tissue with high precision. We present investigations on laser hard tissue "milling", which can be advantageous for solving these problems. The processing of bone is done with a CO₂ laser (10.6 &mgr;m) with pulse durations of 50 - 100 &mgr;s, combined with a PC-controlled fast galvanic laser beam scanner and a fine water-spray, which helps keeping the ablation process effective and without thermal side-effects. Laser "milling" of non-circular cavities with 1 - 4 mm width and about 10 mm depth can be especially interesting for dental implantology. In ex-vivo investigations we found conditions for fast laser processing of these cavities without thermal damage and with minimised tapering. It included the exploration of different filling patterns (concentric rings, crosshatch, parallel lines, etc.), definition of maximal pulse duration, repetition rate and laser power, and optimal water spray position. The optimised results give evidence for the applicability of pulsed CO₂ lasers for biologically tolerable effective processing of deep cavities in hard tissue.

During the last few years, ultra-short laser pulses have proven their potential for application in medical tissue treatment in many ways. In hard tissue ablation, their aptitude for material ablation with negligible collateral damage provides many advantages. Especially teeth representing an anatomically and physiologically very special region with less blood circulation and lower healing rates than other tissues require most careful treatment. Hence, overheating of the pulp and induction of microcracks are some of the most problematic issues in dental preparation. Up till now it was shown by many authors that the application of picosecond or femtosecond pulses allows to perform ablation with very low damaging potential also fitting to the physiological requirements indicated. Beside the short interaction time with the irradiated matter, scanning of the ultra-short pulse trains turned out to be crucial for ablating cavities of the required quality. One main reason for this can be seen in the fact that during scanning the time period between two subsequent pulses incident on the same spot is so much extended that no heat accumulation effects occur and each pulse can be treated as a first one with respect to its local impact. Extension of this advantageous technique to biocompatible materials, i.e. in this case dental restoration materials and titanium plasma-sprayed implants, is just a matter of consequence. Recently published results on composites fit well with earlier data on dental hard tissue. In case of plaque which has to be removed from implants, it turns out that removal of at least the calcified version is harder than tissue removal. Therefore, besides ultra-short lasers, also Diode and Neodymium lasers, in cw and pulsed modes, have been studied with respect to plaque removal and sterilization. The temperature increase during laser exposure has been experimentally evaluated in parallel.

A 1.94 μm Laser-Scalpel system (Starmedtec, Starnberg, Germany) emitting at a wavelength at 1.94 μm and a max. laser power 18 W (cw mode) was used for partial resection of porcine kidney. Partial resection was performed on 9 kidneys. Six kidneys were extracted directly after resection and pigs were sacrificed. The remaining three pigs were kept alive for 2 weeks post resection in order to investigate the healing process. No complications during the operation and during the 2 weeks survival period were found. Total resection time including haemostasis of the remaining tissue was 10.2±6.5 min, blood lost was 14.7±29 ml. Haemostatic property of the 1.94 μm Laser-Scalpel was sufficient. Histological evaluation with H&E stained tissue samples showed medium carbonization and a thermal damage zone of 1mm. In conclusion, the first experiments show that the 1.94 μm Laser-Scalpel is a very promising device for bloodless and fast kidney resection.

Introduction and objectives: The aim was to identify the capability and the laser parameter of under water tissue vaporisation by means of a diode laser (1470 nm). Afterwards the feasibility and postoperative clinical outcome of vaporization of the prostate was investigated. Method: After acquiring suitable laser parameters in in-vitro experiments using a perfused tissue model patients (n=10) suffering from bladder outlet obstruction due to benign prostatic hyperplasia (BPH) were treated by diode laser. Their clinical outcome, in terms of acceptance and post-operatively voiding were evaluated. The diode laser emitted light of the wavelength of 1470 nm at 50 W (Biolitec GmbH) and delivered to the tissue by means of a side-fire fibre introduced through a 24F continuous-flow cystoscope. Normal saline was used for irrigation with an additive of 1% ethanol. The prostatic lobes (volume range 35-80ml) were vaporized within the prostatic capsular using sweeping and push and pull technique. The mean time of laser application was 2400 sec (1220-4000 sec) resulting in applied energies of 121 kJ in the mean (range: 61-200kJ). Results: During laser treatment none of the 10 patients showed any significant blood loss or any fluid absorption (no ethanol uptake). Foley catheters were removed between 18 and 168 hours postoperatively (mean: 49.8h±46h). After removal of the catheter the mean peak urine flow rate increased from 8.9ml/s ± 2.9ml/s pre-operatively in comparison to 15.7ml/s ± 5 ml/s (p=0.049) post-operatively. 8/10 patients were satisfied with their voiding outcome. None of the patients showed appearance of urgency, dysuria, hematuria, or incontinence but two patients required re-catheterization. After a follow-up of 1month, 8/10 patients showed evidence of good results and are satisfied with the outcome. Two patients required consecutive TUR-P. After a follow-up of 6-month the 8 patients are still satisfied. Conclusions: This very early and limited experience using a 50W-Diode laser emitting at 1470 nm indicates a safe and effective treatment modality for quickly relieving bladder outlet obstruction due to BPH. Larger randomized clinical trials to compare this technique with standard transurethral resection of the prostate and increased follow-up data are needed to determine its long-term efficacy and durability.

Surgery of benign pathological alterations of the vocal folds results in permanent disphonia if the bounderies of the vocal fold layers are disregarded. Precise cutting with a femtosecond laser (fs-laser) combined with simultanous imaging of the layered structure enables accurate resections with respect to the layer boundaries. Earlier works demonstrated the capability of optical coherence tomography (OCT) for utilization on vocal folds. The layered structure can be imaged with a spatial resolution of 10-20&mgr;m up to a depth of 1.5mm. The performance of fs-laser cutting was analyzed on extracted porcine vocal folds with OCT monitoring. Histopathological sections of the same processed samples could be well correlated with the OCT images. With adequate laser parameters thermal effects induced only negligable damage to the processed tissue. The dimensions of the thermal necrosis were determined to be smaller than 1&mgr;m. OCT contolled fs-laser cutting of porcine vocal fold tissue in the &mgr;m range with minimal tissue damage is presented.

Laser cartilage reshaping is a temperature dependent process that results in stress relaxation with subsequent formation of a new and stable specimen's geometry. This temperature dependent process results in mechanical stress relaxation and is characteristic of a phase transformation. The objective of this study was to quantitavely measure changes in tensile elongation and elastic modulus of rabbit auricle cartilage reshaped via diode laser (980 nm) and irradiated in two different protocols. The results revealed that the laser irradiation parameter used in cartilage reshaping does not produce significant irreversible changes in mechanical properties of the cartilage tissue. So diode laser can be considered as one of efficient tools in cartilage reshaping.

In the present paper feasibility and potential advantages of using diode laser for surgical treatment of cerebral ischemia and intracranial aneurysms will be evaluated. At this purpose non linear mathematical model was developed and experimentally validated to investigate the effects of the changes in tissue physical properties, in terms of operating time, tensile strength and tissue damage during medical laser application. The numerical simulations have been carried on by a finite-elements based software package (FEMLAB). In vitro results of human saphenous veins of inferior limbs (n=55) after 799 nm diode laser soldering, combined with an indocyanine green-enhanced, will be presented. The simulations results and their comparison with experimental measurements will be reported.

Our working hypothesis is that a dual-wavelength Nd:YAG laser, emitting simultaneously at 1064 and 532 nm, may induce stronger heating of PWS blood vessels relative to the epidermis than the customary KTP laser, due to conversion of hemoglobin to met-hemoglobin in the target blood vessels and the associated increase in NIR absorption. We apply pulsed photothermal radiometry to determine temperature depth profiles induced in PWS lesions by a dual-wavelength laser at sub-therapeutic radiant exposures. The results indicate no effect at 1 ms pulse duration and low radiant exposures (1-2 J/cm²). Increased radiant exposure (3-4 J/cm²) and extended pulse duration (20-25 ms) result in increased energy deposition. In addition, two PWS lesions and one healthy skin site were irradiated at incrementally increasing radiant exposures, up to 9 J/cm². Analysis of the laser-induced temperature profiles clearly revealed irreversible changes of tissue properties. Formation of met-hemoglobin and consequent increase of IR absorption was however not reliably detected.

Optical techniques applied to biological tissue allow the development of new tools in medical praxis, either in tissue characterization or treatment. Examples of the latter are Photodynamic Therapy (PDT) or Low Intensity Laser Treatment (LILT), and also a promising technique called thermotherapy, that tries to control temperature increase in a pathological tissue in order to reduce or even eliminate pathological effects. The application of thermotherapy requires a previous analysis in order to avoid collateral damage to the patient, and also to choose the appropriate optical source parameters. Among different implementations of opto-thermal models, the one we use consists of a three dimensional Beer-Lambert law for the optical part, and a bio-heat equation, that models heat transference, conduction, convection, radiation, blood perfusion and vaporization, solved via a numerical spatial-temporal explicit finite difference approach, for the thermal part. The usual drawback of the numerical method of the thermal model is that convergence constraints make spatial and temporal steps very small, with the natural consequence of slow processing. In this work, a new algorithm implementation is used for the bio-heat equation solution, in such a way that the simulation time decreases considerably. Thermal damage based on the Arrhenius integral damage is also considered.

This work presents results on the modeling of the photon diffusion in a three-layered model, (skin, fat and muscle). The Finite Element method was performed in order to calculate the temporal response of the above-mentioned structure. The thickness of the fat layer was varied from 1 to 15 mm to investigate the effects of increasing fat thickness on the muscle layer absorption coefficient measurements for a source-detector spacing of 30 mm. The simulated time-resolved reflectance data, at different wavelengths, were fitted to the diffusion model to yield the scattering and absorption coefficients of muscle. The errors in estimating muscle absorption coefficients &mgr;_α depend on the thickness of the fat layer and its optical properties. In addition, it was shown that it is possible to recover with a good precision (~2.6 % of error) the absorption coefficient of muscle and this up to a thickness of the fat layer not exceeding 4mm. Beyond this limit a correction is proposed in order to make measurements coherent. The muscle-corrected absorption coefficient can be then used to calculate hemoglobin oxygenation.

We demonstrate significant differences in the propagation of polarized laser light through biological tissue phantom. The Stokes vectors along with degree of linearly and circularly polarized light were measured with stokes polarimetry techniques. The measurements were performed on dense and diluted tissue phantoms that consisted of soybean oil interloped. Liquid crystal variable retarder (LCVR) Stokes polarimeter is used for either rotating the major axis of elliptically polarized light or for converting an input linearly polarized beam into an arbitrary elliptically polarized beam. This system makes possible a direct measurement of a component of the Stokes vector with phase change detection of polarization modulation for polarimetric measurements of turbid media and biological tissue.

In this work we tried to create a mathematical model of thermal response of laser irradiated multilayer biological tissue. The tissue has 4 horizontal radial symmetric layers with its own optical-physical characteristics. We used the results of Monte Carlo modeling to describe the propagation of light (laser beams) in tissue and receive the function of heat source after we multiply the density of thermal emission by absorption coefficient. As we usually have radial symmetric laser beams we can use cylindrical coordinates. The solution of the 2D heat conduction equation is based on finite-element theory with using square finite elements. We simulated constant laser fluency and as result there are temperature fields. The analysis of the results represents, that heat does not localize on the surface, but collects inside of the tissue. By varying the boundary condition on the surface and type of laser irradiation we can reach high temperature inside the tissue without formation of necrosis at the same time.

Mesenchymal stromal cells (MSCs) are multipotent cells, which are present in adult bone marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, and muscle. Their rapid and selective differentiation should provide the potential of new therapeutic approaches for the restoration of damaged or diseased tissue. However, several fundamental questions must be answered before it will be feasible to usefully predict and control MSCs responses to exogenous cytokines or genes. In particular, a better understanding of how specific factor may alter the fate of differentiation of MSCs is needed. In recent reports, circadian clock protein controls osteogenesis in vitro and in vivo. Here we show that a stimulation of a blue-violet laser irradiation regulates the differentiation of mouse MSCs to osteoblasts by change of the localization of a circadian rhythm protein, mouse Cryptochrome 1 (mCRY1). We found that a blue laser irradiation accelerated osteogenesis of MSCs. After laser irradiation, mCRY1 protein was translocated from cytoplasm to nucleus and mCRY1 mRNA level was downregulated thereafter. These results indicate that mCRY1, a blue-violet-light receptor and a master regulator of circadian rhythm, plays important roles in the regulation of the differentiation of MSCs. Since the differentiation of MSCs was easily regulated only by a laser irradiation, the potential of new therapeutic approaches for the restoration of damaged or diseased tissue is anticipated. Furthermore, our results obtained in this study may prove an excellent opportunity to gain insights into cross-talk between circadian rhythms and bone formation.

It has been studied the intravenous laser irradiation of blood (ILIB) influence with helium-neon laser (HNL) of 630 nm wavelength on some of lipid peroxidation (LPO) and antioxidant system (AOS) findings, aside-base status (ABS) and blood oxygen transport (BOT), state of dermal microhaemodynamics (MGD) in the intact rabbits and after modeling of local ischemia of brain (LIB). Depending on conditions of organism functioning (norm or brain ischaemia) ILIB has resulted in stimulating or normalizing effects on the whole metabolic and microhaemocirculation processes which had been studied during our investigation. It is discussed the mechanisms of pathogenetic directivity of ILIB influence in cerebral ischaemia

In this work, a Finite Element calculations based on diffusion approximation are compared with Monte Carlo transport data code in time-resolved reflectance simulations of light propagation in a three-layered head model, which can be seen as a very simplistic approximation of the adult head. We also address the effects caused by the cerebrospinal fluid (CSF), filling the space between the skull and the brain, on the accuracy of the diffusion approximation for different values of CSF reduced scattering coefficients μ_s' varying between 0.1 and 1 mm^-1. Significant differences between transport and diffusion calculations show that diffusion approximation fails to describe accurately light propagation in voidlike region such as the cerebrospinal fluid (CSF), in which absorption and scattering are very small compared to the surrounding media, whereas the Monte Carlo predictions are not greatly affected. However, It is shown that the diffusion equation should provide reasonable solutions with a CSF reduced scattering coefficient μ_s' = 0.3 mm^-1. The results indicate that a multi-layered model including CSF is more appropriate for the determination of the optical properties of the human head and to obtain accurate solutions of the forward problem with diffusion approximation.

A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes. Liposomes are known to be efficient and selective photosensitizer (PS) drug delivery vesicles, however, their chemical and physical effects on the photochemical properties of the photosensitizer have not been well characterized. The liposomes employed in this study (both blank and photosensitizer-complexed) were characterized to determine their: a) size distribution (dynamic light scattering), b) image (scanning electron microscope, confocal fluorescence microscopy), c) concentration of particles (flow cytometry), d) temperature-dependant phase transition behavior (differential scanning calorimetry, and e) spectrofluorescent spectrophotometric properties, e.g. aggregation, in the confined environment. The fluorescence decay behavior of two families of encapsulated photosensitizers, di-and tetrasulfonated metallophthalocyanines, and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function of the liposome's physical properties (size-scale, distribution and concentration of scatterer) and the impact of the photosensitizer spatial confinement determined. It is found that the achievable size range and distribution of the PS-liposomes is controlled by the chemical nature of the PS for large liposomes (1000 nm), and is PS independent for small PS-liposomes (~140nm). The lifetime decay behavior was studied for all three photosensitizer-liposome systems and compared before and after confinement. We found the nature of the decay to be similar before and after encapsulation for the sulfonated phthalocyanines containing ionic moieties (primarily monoexponential) but not for HPPH. In the latter, the decay transitioned from multi- to monoexponential decay upon localizing lypophilic HPPH to the liposomal membrane. This behavior was confirmed by obtaining a similar change in lifetime response with an independent timedomain system. We also varied the environment in temperature and oxygen content to examine the effects on the fluorescent lifetimes of the liposomal complexes. The fluorescence decay of all three PS-containing liposomes showed that the local spatial confinement of PS (dictated by the PS chemistry) into different domains within the liposome directly controls the temperature-response. Membrane-bound photosensitizers were less sensitive to temperature effects as illustrated by the decay dynamics observed in solu, that is, they developed a unique decay behavior that correlated with the phase transition of the membrane. The fluorescent lifetime of PS-encapsulated liposomes in deoxygenated environments, relevant to oxygen independent type I phototoxicity, was also probed in the frequency-domain revealing that liposome-confined PS display very different trends than those observed in solu.

This paper proposes to explore the pattern of lymphoblastic cell line K562 cells death, the effects on their cell cycle induced by 5,10,15,20-tetra-p-sulphonato-phenyl-porphyrin-based photodynamic therapy (TS₄PP-PDT). Flow cytometry combined with Annexin V-FITC/PI labeling was used to detect the pattern of K562 cells' death induced by TS₄PP-PDT. These effects frequently lead to induction of apoptosis by the mitochondrial pathway involving caspases. The transmission electron microscope (TEM) and confocal laser scanning microscopy (CLSM) were used to detect the localization and time-biodistribution of sensitizers in the cells. After 1 h of TS₄PP administration, the sensitizer shows a non-uniform distribution, following that after 4h of administration, the sensitizer to be localized in some cellular targets and an increased fluorescence intensity is being detected. After 8 h and 24 h post-administration, the sensitizer is released from the cells and the light-irradiation (He-Ne laser, &lgr;=632.8 nm) could start. Immediately after irradiation, many typical apoptotic bodies were seen in the cells treated. Most of the cells treated were necrotic at 24 hours following irradiation.

The new aqueous gel compositions based on 5-aminolevulinic acid (ALA) for fluorescent diagnostics and photodynamic therapy of superficial diseases have been elaborated. Biodegradable polymer was used as gel-forming component and some additives - solubilizer, emulgator etc - were used to improve distribution uniformity and penetration ability. These new compositions represent colorless and transparent gels which are long-term stable at the storage temperature of ≤5°C. The effectiveness of aqueous gels as ALA delivery systems is demonstrated. After topical administration of the gel at the skin with Ehrlich tumor inoculated subcutaneously, ALA effectively induces the synthesis of protoporphyrine IX (PPIX) in the skin and in the tumor. Intensity of ALA-induced PPIX fluorescence grows with increasing ALA concentration in the gel and time of gel application. The deepness of ALA penetration in the tumor reaches 5-6 mm after 4 h gel application. A distribution of ALA-induced PPIX in mouse tumor tissue after 4 h gel application depends on ALA concentration in gel. Intensity of PPIX fluorescence in deep-located part of tumor was substantially higher with gel containing 20% ALA than with ones containing 10% or 5% ALA.

Photodynamic therapy (PDT) has been used in the treatment of various skin diseases including non melanoma skin carcinomas (NMSC). However, until now there are no publications concerning the efficacy of PDT after topical application of mTHPC. Although topical photosensitizer application presents many advantages over systemic drug administration, ALA-induced protoporphyrin IX is the only sensitizer topically used so far. In the present study photodynamic efficacy of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation is investigated after topical application in hairless SKH-HR1 mice, bearing non melanoma skin carcinomas. The drug was applied topically for drug - light interval of 4 hours. The fluence rates were 100 and 50 mW/cm² and two total energy doses, 10 J/cm² and 100 J/cm² were studied in groups of 5 animals. Three PDT sessions were performed in each animal, once every 7 days. The final evaluation of PDT effects was performed 14 days after the 3rd PDT treatment by measuring the geometrical characteristics of tumors. The groups treated with 100 mW/cm² presented a higher complete tumor remission than the group of 50 mW/cm² but an unusual high mortality. In the group of 50 mW/cm² and 100 J/cm², although the complete tumor remission percentage is poor, the tumor growth rate was decreased. No lesion, papilloma, or tumor was observed in the treated area even six months after tumor remission. Furthermore tumours up to 7 mm were achieved to be treated, indicating that this novel mTHPC formulation could be used for deeper and not only superficial carcinomas or lesions.

The aim of this work is to study pharmacokinetics and photodynamic efficiency of aluminium phthalocyanine chloride (AlClPc) in dimethylsulfoxide/Tween 80/water solution, after topical application on hairless mice bearing non-melanoma skin carcinomas. The concentration of photosensitizer in normal skin and tumor biopsies 1-6 hours after application was assessed by fluorescence spectroscopy of chemical extractions. The concentration of photosensitizer was 40 times higher in tumor than in normal skin even 1 h after application. For photodynamic therapy (PDT) AlClPc was excited by a diode laser emitting at 670 nm, 1 h after application. Seven different combinations of therapeutic parameters were chosen. The efficiency was assessed as the percentage of complete tumor remission, the tumor growth retardation and the cosmetic outcomes. The highest complete remission 60% was achieved with the combination of 75 mW/cm² with 150 J/cm². No recurrence rate was observed in any treatment parameters group and the cosmetic outcome in all completely treated tumors was excellent. The results show that the effectiveness of PDT is highly dependent on fluence rate. In addition, they are promising for further investigation of this PDT scheme in preclinical studies mainly in non-melanoma skin carcinomas up to 7mm.

Crohn's disease is an inflammatory bowel disease originating from an overwhelming response of the mucosal immune system. Low dose photodynamic therapy (PDT) may modify the mucosal immune response and thus serve as a therapy for Crohn's disease. Most patients with Crohn's disease show inflammatory reactions in the terminal ileum or colon where PDT treatment is feasible by low-invasive endoscopic techniques. However, the tube like geometry of the colon, it's folding, and the presences of multiple foci of Crohn's lesions along the colon require the development of adequate light delivery techniques. We present a prototype light delivery system for endoscopic clinical PDT in patients with Crohn's disease. The system is based on a cylindrical light diffuser inserted into a diffusing balloon catheter. Homogenous irradiation is performed with a 4 W diode laser at 635 nm. Light dosimetry is performed using a calibrated integrating sphere. The system can be used with conventional colonoscopes and colonovideoscopes having a 3.8 mm diameter working channel. The feasibility of PDT in colon with our prototype was demonstrated in first clinical trials.

This work are devoted our experience with photodynamic therapy (PDT) with <<Photosens>> for patients with choroidal neovascularization (CNV). 18 patients with subfoveal CNV in age-related macular degeneration (AMD), 24 patients with subfoveal CNV in pathological myopia (PM) and 4 patients with subfoveal CNV associated with toxoplasmic retinochoroiditis were observed. CNV was 100% classic in all study patients. Standardized protocol refraction, visual acuity testing, ophthalmologic examinations, biomicroscopy, fluorescein angiography, and ultrasonography were performed before treatment and 1 month, 3 months, 6 months, and 1 year after treatment; were used to evaluate the results of photodynamic therapy with <<Photosens>> (0.02% solution of mixture sulfonated aluminium phtalocyanine 0.05 mg/kg, intravenously). A diode laser (<<Biospec>>, Inc, Moscow) was used operating in the range of 675 nm. Need for retreatment was based on fluorescein angiographic evidence of leakage at 3-month follow-up intervals. At 3, 6, 9 month 26 (56.5%) patients had significant improvement in the mean visual acuity. At the end of the 12-month minimal fluorescein leakage from choroidal neovascularization was seen in 12 (26.1%) patients and the mean visual acuity was slightly worse than 0.2 which was not statistically significant as compared with the baseline visual acuity. Patients with fluorescein leakage from CNV underwent repeated PDT with <<Photosens>>. 3D-mode ultrasound shown the decreasing thickness of chorioretinal complex in CNV area. Photodynamic therapy with <<Photosens>> can safely reduce the risk of severe vision loss in patients with predominantly classic subfoveal choroidal neovascularization secondary to AMD, PM and toxoplasmic retinochoroiditis.

14 patients with transional-cell bladder cancer in stage T1N0M0G2 after transurethral bladder resection were offered adjuvant treatment with PDT. Adjuvant PDT was performed 1-1.5 months after transurethral bladder resection for superficial bladder cancer. Prior to PDT conventional and fluorescent cystoscopy were performed. In the absence of inflammation and after full epitalisation of postoperative wound a session of therapy was performed. 24 hours prior to PDT-session photosensitizer Photosens was injected intravenously in the dose of 0.8 mg per kg of body weight. Prior to PDT local anesthesia of urethra with lidocain-gel was performed. Cystoscopy was carried out. PDT was performed with diode laser "Biospec" (675 nm). During the session the place of standing diffuser and the volume of a bladder were controlled. After 7 months of observation no tumor recidivists were observed. Registered side effects were not life-threatened. 5 patients had pain or discomfort in suprapubic area, ceasing spontaneously or requiring administration of analgetics. No systemic side-effects or allergic reactions were observed. The method can be used in out-patient practice. Absence of early recidivists shows efficiency of PDT in the treatment of superficial bladder cancer. Further study is necessary to estimate optimal regimen of PDT. The further controlling of condition on the patients in this group is required. At the laboratory animals' experiment, we conducted the explorations devoted to the influence of the photodynamic effect at the prostate's tissues.

Choroidal metastasis (CM) are more and more spreading type of eye's neoplasma. The frequency of CM is increasing with prolonging of cancer patients' life. And it makes worse the quality of their life because blindness. Photodynamic therapy (PDT) is very delicate modality, which can be used for this purpose. The aim of this work was to open the possibility and to determine the efficacy of photodynamic therapy (PDT) in the treatment of patients with CM. PDT was performed simultaneously with standard chemotherapy in 8 oncological patients with CM. We used photosensitizer Photosens in doses of 0.3 mg/kg and light doses 150 J/cm² (675 nm). PDT was performed in the some stances. Its are ranged from 7 to 10. Complete tumor regression was achieved in 6 cases. The high retina ablation was developed in one case. And in one case effect was not complete: tumor size reduced from 5 mm to 3 mm of thickness. We didn't notice any recurrence for 6-18 months follow-up. PDT is modality that could to be used in the in the combined treatment of the CM.

Diode laser welding is a technique proposed in ophthalmic surgery to induce immediate sealing of clear corneal wounds. The welding effect is achieved irradiating the area, previously treated with a chromophore, by the use of a low power diode laser: the resulting thermal effect induces structural modifications in the stromal collagen, that welds upon cooling. We present a study on the temperature dynamics developing during welding in a human eye. An infrared thermocamera was used to measure the temperature variations on the surface of the cornea during clinical penetrating keratoplasty (corneal transplant). The experimental data were used as a starting point for a theoretical investigation of the temperature rising inside the ocular structures: we developed a mathematical model based on the bio-heat equation and solved by the use of the Finite Element Method (FEM). The predictive accuracy was verified by comparing the temperature post-processing description with the results obtained from the thermographic data. The model was then used to study the temperature rise and heat propagation inside the eye. Experimental results and model analysis indicated the occurrence of heat confinement during the treatment procedure and a modest enhancement of the temperature (reaching about 55°C inside the laser treated wound), thus evidencing the safety of the procedure in clinical applications.

Studies on corneal surgery and flap processing on enucleated porcine eyes have been performed using a dedicated 100 kHz femtosecond laser source based on Ytterbium technology. IR (1035 nm) and green (517 nm) flap processing have been studied. Comparisons for ocular femtosecond laser surgery are discussed in terms of process efficiency and safety aspects. Flaps with a typical diameter of 6 mm and 150 μm thick have been performed in less than 2 min with both wavelengths. The transmittances of femtosecond laser pulses through the ocular media of porcine eyes have been measured for a collimated beam and during flap processing. More than 25% of energy is transmitted through the whole eye at the retina during IR pulses flap processing. Concerning green pulses, if less energy is necessary to perform the flap which is of prime interest, the transmission of green light is very high and could be an undeniable obstacle for the safety.

Selective retina treatment (SRT) is a new laser based method to treat retinal diseases associated with disorders of the retinal pigment epithelium (RPE). Applying microsecond laser pulses tissue damage spatially confined to the retinal pigment epithelium (RPE) is achieved. The RPE cell damage is caused by transient microbubbles emerging at the strongly absorbing melanin granules inside the RPE cells. Due to the spatial confinement to the RPE the photoreceptors can be spared and vision can be maintained in the treated retinal areas. A drawback for effective clinical SRT is that the laser induced lesions are ophthalmoscopically invisible. Therefore, a real-time feedback system for dosimetry is necessary in order to avoid undertreatment or unwanted collateral damage to the adjacent tissue. We develop a dosimetry system which uses optical interferometry for the detection of the transient microbubbles. The system is based on an optical fiber interferometer operated with a laser diode at 830nm. We present current results obtained with a laser slit lamp using porcine RPE explants in vitro and complete porcine eye globes ex vivo. The RPE cell damage is determined by Calcein fluorescence viability assays. With a threshold criterium for RPE cell death derived from the measured interferometric signal transients good agreement with the results of the viability assays is achieved.

Retinal photocoagulation is an established treatment of different retinal diseases. The treatment relies on a short, local heating of the tissue which induces a denaturation. The resulting scar formation may for example prevent the further detachment of the retina. The extent of the coagulation is besides other parameters mostly dependent on the induced temperature increase. However, until today a temperature based dosimetry for photocoagulation does not exist. The dosage is rather based on the experience of the treating physicians to achieve visible whitish lesions on the retina. In this work a technique is presented, which allows an online temperature monitoring during photocoagulation. If an absorbing material is irradiated with short laser pulses, a thermoelastic expansion of the absorber induces an acoustic wave. Its amplitude is dependent on the temperature of the absorber. For analyzing the applicability of the optoacoustic temperature determination for dosimetry, measurements were performed on enucleated porcine eye globes. The pressure transients are detected by an ultrasonic transducer, which is embedded in an ophthalmologic contact lens. As long as no strong lesions occur, the determined temperatures are almost proportional to the power of the treatment laser. Using a spot diameter of 200 μm and different laser powers, the temperature rise at the end of the 400 ms irradiation was found to be approximately 0.16 °C/mW. The onset of the denaturation was observed around 50°C. The far aim of this project is an automatic regulation of the treatment laser onto a desired temperature course.

Selective Retina Treatment (SRT) is a new method to treat eye diseases associated with disorders of the RPE. Selective RPE cell damage is achieved by applying a train of 1.7 μs laser pulses at 527 nm. The treatment of retinal diseases as e.g. diabetic maculopathy (DMP), is currently investigated within clinical studies, however 200 ns pulse durations are under investigation. Transient micro bubbles in the retinal pigment epithelium (RPE) are expected to be the origin of cell damage due to irradiation with laser pulses shorter than 50 μs. The bubbles emerge at the strongly absorbing RPE melanosomes. Cell membrane disruption caused by the transient associated volume increase is expected to be the origin of the angiographically observed RPE leakage. We investigate micro bubble formation and dynamics in porcine RPE using pulse durations of 150 ns. A laser interferometry system at 830 nm with the aim of an online dosimetry control for SRT was developed. Bubble formation was detected interferometrically and by fast flash photography. A correlation to cell damage observed with a vitality stain is found. A bubble detection algorithm is presented.

In most retinal laser treatments the therapeutic effect is initiated by a transient temperature increase at and around the retinal pigment epithelium (RPE). Especially in long exposure time treatments like Transpupillary Thermotherapy (TTT) choroidal perfusion has a strong influence on the realized temperature at the fundus. The fundus blood circulation and therefore the heat dissipation is influenced by the intraocular pressure (IOP), which is investigated in the study presented here. In order to reduce the choroidal perfusion, the IOP is increased by injection of physiological saline solution into the eye of anaesthetized rabbits. The fundus is irradiated with 3.64 W/cm² by means of a TTT-laser (λ = 810 nm) for t = 20 s causing a retinal temperature increase. Realtime temperature determination at the irradiated spot is achieved by a non invasive optoacoustic technique. Perfusion can be reduced by increasing IOP, which leads to different temperature increases when irradiating the retina. This should be considered for long time laser treatments.

Intraocular lens (IOL) implantation is the standard technique to treat cataract. Despite recent progress in surgical procedures, posterior capsule opacification is one of the sill remaining postoperative complications of cataract surgery. We present a novel strategy to reduce the incidence of posterior capsule opacification. A drug delivery polymer suitable for manufacturing intraocular lenses has been developed which enables repeated drug release in a non-invasive and controlled manner. The therapeutic molecules are attached through a UV light sensitive linkage to the polymer backbone which is mainly responsible for the optical properties of the intraocular lenses. However, UV light can not trigger the release of drug from the polymer due to the high absorption of the cornea. We developed linkers which enable drug release by two-photon absorption induced cleavage of the linker structure. Since the two-photon absorption requires high photon densities, this does not occur in ambient light conditions in daily life, but is easily triggered by focused laser beams from a pulsed laser. In this proof-of-principle study we have employed a cyclobutane type linker and investigated the properties of the therapeutic system with the approved drugs 5-fluorouracil and chlorambucil. The controlled drug delivery was successfully demonstrated in vitro and additional cell tests confirmed that the device itself shows no cytotoxicity until photochemical activation. This presented concept can provide a powerful method in ophthalmic drug delivery.

Typical postoperative complications in cataract surgery are that refractive power and curvature of the implanted intraocular lens (IOL) do not have optimum values, requiring the patient to wear viewing aids. This is mainly because biometric data relevant for calculation of the IOL's shape cannot be determined with the required precision. Hence, there is a need for methods to tune the focal length postoperatively in a non-invasive manner. We have developed polymers where we can induce a change in refractive index by linking or cleaving bonds between a su.ciently large number of side groups of the polymer main chain in a photoinduced cycloaddition or cycloreversion reaction, respectively. These photoreactions lead to a change in refractive index great enough to be interesting for the concept of in vivo tunable IOL's. The photochemical reaction can be triggered by a two-photon process (TPA) using a pulsed laser system, i.e. the energy required for bond breaking is provided by two photons in the visible range. This is important because light in the UV cannot induce undesired changes of the refractive index owing to the strong UV-absorption of the cornea. Undesired changes due to light in the visible range of the spectrum are unlikely to happen because photon density of sun light is much too low for TPA. Due to the excellent spatial resolution that can be achieved with two-photon processes one cannot only modify the refractive index of the entire lens but also selectively in well defined areas enabling to correct for aberrations such as astigmatism. Here, we present new polymers that do not only exhibit a photo induced change of refractive index great enough to induce a change of focal length of more than two diopters in a standard IOL. These new polymers have also significantly improved material properties with respect to the fabrication of the IOL and the TPA-sensitivities and the light energy required to induce the refractive index change.

According to Helmholtz' theory of accommodation one of the mayor reasons for the development of presbyopia is the increasing sclerosis of the lens. One concept to overcome this hardening of the lens is to regain its flexibility by inducing gliding planes inside the lens. Femtosecond laser pulses are a suitable tool for this treatment. Showing in former work that we could increase the flexibility of enucleated porcine (ex vivo) lenses up to 25%, we focused our recent work on human autopsy lenses. The age of the human donors ranged between 20 and 70 years. For an evaluation of the gain in flexibility the lens' thickness was measured undertaking the Fisher's spinning test before and after laser treatment. Depending on the age and the quality of applied cutting pattern the lens thickness increased after treatment up to 0.4 mm leading to an theoretical increase of several dioptres of optical power. The flexibility could be increased up to 70 % compared to the measurements before treatment. Since the age of the human donors had a broad range, leading to different degrees of lens hardening, the variance of the measured flexibility changes was up to 30%. An addition the influence of the laser treatment onto the lens on the accommodation amplitude will be shown in a three dimensional finite-element simulation.

Ultrafast femtosecond lasers are used increasingly for a wide range of medical purposes. The immediate tissue response to pulses above a certain threshold is optically or laser induced breakdown, which is often visible as gas-filled cavities that persist for some time. In the present study, we attempted to define the cavitation threshold in the human lens in vitro using multiphoton effects based on radiation from a femtosecond 800 nm Ti:Sapphire laser. Cavitations were observed from pulse energy densities exceeding 16 mJ/cm², but only after several minutes of exposure and not as a result of a single laser pulse. This suggests that cavitations were caused by a process which differs from the single-pulse cavitations observed at higher intensities. To evaluate whether the release of gas was caused by ionization and plasma formation or by thermal effects, we introduced pauses into the pulse train, which did not change the total exposure time needed to form a cavitation. This suggests that local heating did not play a significant role in producing the observed phenomenon, suggesting that photochemical reactions may be involved. These results demonstrate that there are several types of ultrafast laser effects in the lens that have a potential for therapeutic application and treatment of eye disease though further studies are needed to shed light on the nature of the formation of delayed cavitations.

Background/purpose: In vivo studies have demonstrated that phototherapy accelerates wound healing in the clinical environment; however the exact mechanism is still not completely understood. The main focus of this study was to use in vitro laboratory results to establish an effective treatment regimen that may be practical and applicable to the clinical environment. This in vitro study aimed to compare the cellular responses of wounded fibroblasts following a single exposure of 5 J/cm² or multiple exposures of low doses (2.5 J/cm² or 5 J/cm²) on one day of the week to a single application of a higher dose (16 J/cm²) on day 1 and day 4. Methodology: Cellular responses to Helium-Neon (632.8 nm) laser irradiation were evaluated by measuring changes in cell morphology, cell viability, cell proliferation, membrane integrity and DNA damage. Results: Wounded cells exposed to 5 J/cm² on day 1 and day 4 showed an increase in cell viability, increase in the release of bFGF, increase in cell density, decrease in ALP enzyme activity and decrease in caspase 3/7 activity indicating a stimulatory effect. Wounded cells exposed to three doses of 5 J/cm² on day 1 showed a decrease in cell viability and cell proliferation and an increase in LDH cytotoxicity and DNA damage indicating an inhibitory effect. Conclusion: Results indicate that cellular responses are influenced by the combination of dose administered, number of exposures and time between exposures. Single doses administered with sufficient time between exposures is more beneficial to restoring cell function than multiple doses within a short period. Although this work confirms previous reports on the cumulative effect of laser irradiation it provides essential information for the initiation of in vivo clinical studies.

We describe a new method for delivering macromolecules into the target cells based on light-absorbing cationic colloidal gold nanoparticles that are irradiated by focused femtosecond laser pulses. Cationic colloidal 15nm gold particles which were made by conjugation with poly-L-Lysine, were attached on the anionic sites, especially on the membrane, of CHO-K1 cells because of their strong positive charge at physiological pH. Target cells labeled with cationic gold nanoparticles were imaged under two-photon fluorescence microscopy, and lifetime images of the same targets were taken by TCSPC technique in order to verify the fluorescence of the marker and the luminescence of the gold particles. A macromolecular 10k Dalton fluorescein isothiocyanate dextran (FITC-D), was added into the sample and the focused femtosecond laser of two-photon fluorescence microscopy was employed to scan the target cells layer by layer. Typical laser power level used in biological imaging is about 3-5 mW. Here the laser power of scanning was below 5 mW in order to prevent photochemical damage of the fs-pulses alone and to localize effects to the nanoparticles on a nano-scale. After scanning the target cells under stack mode, macromolecular fluoresceins surrounding the cells was observed to cross the membrane and to diffuse in the cytoplasma. Comparing with the images before scanning, the two-photon fluorescence and fluorescence lifetime images revealed the delivery of FITC-D into target cells.

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.