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Oxidative stress has been implicated in several biological and pathological aspects. Reactive oxygen species (ROS) have been proposed to act as signal transduction molecules activating reactions leading to cell rescue or to cell apoptosis/necrosis. In the present study, oxidative stress was induced by photosensitization of zinc phthalocyanine (ZnPc) in human fibroblasts using a photodynamic dose that did not lead to apoptosis or necrosis. The induction of oxidative stress was performed at the microscope stage in preassigned time. The cascade of phenomena evoked was studied in real time and at the single cell level using confocal laser scanning microscopy. Using specific vital fluorescent probes, alterations induced by oxidative stress in mitochondria membrane potential, in intracellular pH and in calcium concentration were recorded. Image processing and analysis techniques were used to quantify the observed changes. Subcellular localization of the photosensitizer was studied in order to determine the primary and immediate ROS target. It was found that ZnPc is mainly localized in the mitochondria region.
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Metronomic photodynamic therapy (mPDT), a procedure in which both the photosensitizer and light are delivered continuously so that the individual doses overlap pharmacologically is introduced. The fundamental hypothesis in mPDT is that by providing therapy at low fluence over extended periods of time, there is potential for improved selectivity in tumor cell kill through non necrotic pathways. This is especially important in the treatment of malignant brain tumors, in which selectivity between damage to tumor cells versus normal brain tissue is critical. Previous studies have shown that low-dose PDT using aminolevulinic acid (ALA)-induced protoporphyrin IX (PiIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue nor apoptosis in the latter. However, in order to achieve tumor control, multiple PDT treatments, such as hyper fractionation or metronomic delivery, are required, where the frequency and duration of the treatment are determined by the levels of apoptosis achieved in relationship to tumor cell doubling times, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of interstitial devices for extended light delivery at a sufficiently high enough density to achieve low fluence exposure to the brain adjacent to tumor or the entire hemisphere. In a rat model we evaluated the feasibility of delivering sustained ALA administration via the drinking water for up to 10 days without loss of PPIX selectivity. Post mortem quantitative spectrofluorimetry of tumor, normal brain and other tissues demonstrates a 4 times higher PPIX concentration in the 9L gliosarcoma model without noticeable toxicity. Light sources and delivery devices based either on laser diode or light emitting diode (LED) coupled to an implanted optical fiber were shown to be feasible. The maximum permissible spacing of cylindrical isotropic emitters is determined using known apoptotic indices and the necrosis threshold value for white matter. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.
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We have applied a spectroscopic system capable of monitoring the fluorescence dynamics of photosensitiser at micron-scale locations within individual cells. This report shows that the accumulation of protoporphyrin IX (PpIX) within the nucleus of formalin-fixed keratinocytes, fibroblasts, and a metastatic squamous carcinoma cell line, following incubation with 5-aminolaevulinic acid (ALA), is dependent upon both incubation time and cell proliferation status. We
demonstrate that the process of photobleaching can be monitored via the depletion in PpIX fluorescence emission during exposure to 532 nm laser light. All spectra show a progressive reduction of the 634 nm PpIX peak - following a bi-exponential decay which is consistent with a singlet oxygen mediated process. The rate of photobleaching, when plotted as a function of light dose, increases with reduced incident laser power. The generation of the hydroxyaldehyde-chlorin photoproduct, as monitored by the increase in fluorescence emission centred on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, there is evidence of PpIX fluorescence recovery during the dark period, and an increase in bleaching rate at the onset of the second exposure. These results are in qualitative agreement with measurements performed in vivo which demonstrate that the photodynamic dose is dependent upon fluence-rate and oxygen status.
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In this project a technique to optically measure the temperature is evaluated. The measurement is to be performed through optical fibres during photodynamic laser treatments or laser thermo therapy of malignant tumours. For this technique Cr3+-doped crystals were used. The lifetime of the ions’ fluorescence were measured, since the fluorescence is strongly temperature dependent. A piece of a crystal was attached to the tip of an optical fibre. The crystal was excited at 635 nm, which is the wavelength most frequently used for photodynamic treatment.
An accuracy in the temperature measurement of ± 0.3 °C was obtained for Cr:LiSAF in the region 20 - 70 °C. This is well within the requirements for this application. Alexandrite and Cr:YAG were also evaluated in this study, also yielding a very good accuracy. A laser treatment was simulated using pork chop as tissue phantom and the temperature was measured.
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A novel photodynamic therapy system based on interstitial illumination using multiple fibres is under development. The aim with this system is to enable treatment of large tumour volumes and also to utilise real-time measurements to allow on-line dosimetry. Important dosimetric parameters to measure are light fluence rate, sensitizer fluorescence intensity and local blood oxygenation. A construction which allows all functions to be readily performed with a single system is presented. We believe that interstitial PDT utilising this technique may be attractive in many clinical situations.
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Spider leg veins represent an important aesthetic problem. This clinical study was performed to investigate the pulsed near-infrared diode laser (810 nm) treatment of spider leg veins. 35 female patients were enrolled in this study and treated twice with laser densities of energy (fluence) between 60 - 100 J/cm2 and a spot size of 50 mm2. Histological examinations were performed to investigate morphological and functional effects. Spectroscopic investigations were used as a non-invasive evaluation tool. After the first laser treatment 15 patients showed a complete disappearance (CR); in the remaining 20 patients a remarkable improvement (RI) was seen (N=35). After six months of follow-up
CR was noted in 6 patients, RI in 6, a stable situation in 9, and scar formation in one patient (N=21). The histological examination before and after laser treatment showed no cellular inflammatory reactions. The mean vascular areas were significantly reduced after the laser treatments. Vis-NIR spectroscopic investigations showed almost an immediate occurrence of the haemoglobin double peak and a prompt decrease of the visible remittance after laser treatment of
the skin. Pulsed diode laser therapy is a safe and effective option for the treatment of spider leg veins. Objective in vivo- monitoring by remission spectroscopy and the histology of biopsy specimens show the immediate and long-term laser effects of the irradiated skin in detail.
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ELAN is a new method for treating atherosclerotic vessels. Its purpose is to restore wall flexibility by removing arterial wall tissue from the outer arterial layer. This leads to expansion of the narrowed vessel resulting in increased blood flow. We present results of treatment of arteries in vitro. Tissue removal was done by excimer-laser ablation using ns-pulses of 193nm wavelength. We also discuss therapy control. OCT images and light diffusion measurements are presented.
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A one-dimensional finite difference model has been used to investigate the temperature distribution within thulium fibre
laser-irradiated tissue. Temperature-time and temperature-depth
profiles are presented for various laser stimulus parameters in
the 2 micron region. These current calculations are aimed at
determining theoretical temperature distributions in the application of relatively low power fibre lasers for thermal stimulation of cutaneous nerves in human pain processing. Theoretical skin surface temperatures are compared with those from thermal camera measurements during thulium fibre laser irradiation. The effectiveness of the thulium fibre laser for thermally stimulating cutaneous nerves is confirmed.
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We developed a new surgical procedure to improve the recurrence rate using an Er:YAG laser as dissection tool for the carpal ligament with the objective to ablate a small amount of the carpal ligament and to denaturate its ends. The Er:YAG Laser was transmitted to the applicator via a GeO fiber. With this system we proceeded 10 carpal ligament dissections without any complications in the follow-up period. All patients were free of pain and recurrence.
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Selective tissue damage on the cellular level can be achieved due to microbubble formation around laser heated intracellular pigments. To get an estimate on damage ranges we investigated microbubble dynamics (lifetime, size) around isolated melanosomes of the retinal pigment epithelium in aqueous suspensions by fast flash photography and probe laser transmission. For short pulsed laser irradiation (12ns@532nm) an increasing radiant exposure results in larger bubble sizes. Bubble diameters ranging from 1μm to 10μm were observed. Applying longer pulses (1.8μs@527nm), increasing radiant exposure leads to an earlier onset of bubble growth relative to the pulse. In this case, the diameter of the bubble is nearly independent of the radiant exposure, whereas the number of bubbles during the pulse increases towards higher radiant exposures.
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Irradiation of 15 nm gold particles with nano- and picosecond laser pulses can create locally temperatures beyond the critical point of water. Due to the short heating times the temperature is localised to the vicinity of the particles. Under irradiation with nano- and picosecond pulses an inactivation of the enzymes alkaline phosphatase and chymotrypsin which were bound to the surface of the particles was observed. As expected by strong temperature gradient caused by
the short irradiation time the protein inactivation is localized within a few tens of nanometers when picosecond pulses were used. This was shown by irradiating gold protein conjugates in which the protein was bound via two anitbodies to the particle.
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The potential of a new continuous wave Thulium YAG laser is investigated for tissue ablation and cutting focusing on applications in minimally invasive surgery. The laser emits at a wavelength of 2.01μm, which is well suited for tissue ablation due to its high absorption by water. The laser power can be tuned up to 60 W output through a 365 μm core diameter quartz fibre. For the ablation studies, the quartz fibre was placed in contact under various pressures (20 to 90mN) to porcine liver under saline solution in vitro at angles varying between 30° to 60°. The influence of different powers (10 to 60W) and cutting velocities (2 to 10mm/s) on the incision depth and coagulation zones of the tissue were investigated. A maximum incision depth of 3.3 mm was found with a power of 60W, a cutting velocity of 2mm/s and a fibre-tissue angle of 45°. The incisions were surrounded by coagulated tissue between 0.4 and 0.8mm in thickness, sometimes with an inner zone of carbonization of 0.2mm on average. In conclusion, the first experiments show that a cw Thulium laser is very well suited for tissue dissection as required in minimally invasive surgery.
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Small-size lesions to cerebral cortex induced by the CO2 laser have found applications in neurophysiology for anatomical-functional mapping of the brain functions by areas. The advantage of this new technique in comparison to traditional ones (mechanical, electrical and chemical methods) consists in the possibility of making well-controlled lesions of small and repeatable size to cerebral cortex. In this work, we employed numerical and physical modelling to study the effect of the CO2 laser beam diameter and power density on the ablation rate, size and shape of the laser-induced lesions. Our study showed that the existing linear mathematical model of the laser-induced ablation is sufficiently correct only for the case of relatively shallow lesions. For large penetration depths, the mathematical model of laser-tissue interaction that accounts for the exponential decay of laser power density with penetration depth gave better agreement with the experiment than the existing linear model.
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Irradiation of cell-layers with focussed 2.8 μm ir-laser allows to control the cell temperature from room temperature up to 100°C. Temperatures were calculated for a cell culture model and verified experimentally by thermal mapping of the cell-surrounding medium by means of thermochromic liquid crystals (TLC). Irradiation power and time were varied and associated biological effects like necrosis and apoptosis were observed with respect to the irradiation dosis.
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Theoretical and experimental studies of the influence of low-intensity laser radiation, on the velocity of microcirculation of the erythrocytes of patients with the cardiovascular disease “in vivo" are carried out. Dynamic light scattering techniques were used to monitor the variation in the perfusion of micro capillary blood flow during irradiation under "in vivo" conditions and compared to the change in average size of aggregates of the blood effects observed "in vitro" using static scattering of light. It is shown that the process of the fragmentation of erythrocytes depends on amount of energy absorbed by biological tissues. This conclusion is supported by the good qualitative agreement with the theoretical model, based on the heat transfer theory within the dermis.
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Diode pumped Nd:glass all-solid-state femtosecond laser is promising for next generation refractive surgery, with the advantages of excellent surgical precision, minimal tissue damage and improved system stability and compactness. The microscopic evaluation of the outcome of femtosecond laser surgery is crucial before clinical applications. By two-photon laser scanning microscopy and non-invasive second harmonic imaging, the three dimensional ultrastructure of the porcine cornea is visualized without requiring slicing or staining. The minimal-invasive corneal flap cutting and non-invasive intrastromal surgery are investigated. Femtosecond laser intrastromal surgery demonstrated high ablation precision and mimimal side effects. However, there are elongated filaments/streaks observed in the cornea stroma, most likely due to the focusing optics and self-focusing.
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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.
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Ultrafast lasers are suitable for different surgical procedures in the eye: As a main goal in our group, the application of fs-lasers in refractive surgery is studied. Therefore,experiments in living rabbits were conducted, with wound healing studies up to 120 ays after surgery. Moreover,highly precise cuts inside the cornea were used to do keratoplasty an lamellar keratoplasty. Further applications like a new technique to overcome presbyopia are shown.
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In the field of otolaryngology a precise contactless treatment of the bones in the middle ear is eligible. For this reason lasers are investigated for the use in this field. The main risk during laser surgery in the middle ear (e.g. stapedotomy) is the damage of hair cells in the inner ear due to heat diffusion or high pressure fluctuations. While the temperature problem has been resolved by shortening the pulse durations; the transfer of a recoil momentum due to the ablation process rises as another problem. To measure this momentum, special spring plates were designed as vibration disks for the mounting of the tissue. The probes were exposed to amplified Ti:Sapphire Laser pulses with a pulse length of 100fs and a power density up to 6,4*1013W/cm2. The beam of a Laser Doppler Vibrometer was focused on backside of the plate to monitor its motion. The results were compared to a damage threshold of hair cells in the inner ear calculated by a literature value for the Sound Pressure Level (SPL)-Threshold. The first results lead to SPL values below the critical value but measurements with a higher time resolution are necessary to verify this conclusion.
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Secondary cataracts are quite often observed after implantation of polymeric intraocular lenses. The reason for this complication is that lens epithelial cells remain in the capsular bag when the natural lens is removed. They begin proliferation and cause secondary cataracts. It is not desireable to add cell toxic agents at the time of the implantation because wound healing is negatively affected. We have developed polymeric intraocular lenses which are equipped with a drug depot which may be released non-invasively through photochemical treatment. In the example presented the drug is 5-fluoruracil (5FU) which is covalently bound to the polymer. Deliberation of 5FU from the polymer is done photochemically. Since light is transmitted permanently through the artificial intraocular lens and wearing of special glasses by the patient should be omitted conventional photochemistry is not a suitable tool for the drug release. The polymer-5FU linkage is designed in a way that it has a high two-photon absorption cross-section. Two-photon absorption is used to selectively release 5FU from the lens. The one-photon reaction is blocked since the cornea does absorb UV light. The principle shown here is not limited to 5FU but may be applied to other drugs also.
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An advanced model of blood vessel heating by laser radiation is proposed for tasks of laser skin surgery and therapy. Blood vessel is modeled by infinite circular cylinder situated in skin dermis. Heat conduction equation taking into account the inhomogeneous internal source function is calculated. The source function inside the blood vessel is calculated according to the theory of diffraction of electromagnetic radiation on infinite circular cylinder. Dynamics of the temperature fields inside the vessels as a function of vessel diameters and duration of irradiance is calculated for the wavelength of 0.532 μm. It is determined the irradiance conditions whereby the near-homogeneous heating along the perimeter of walls of blood vessels on minimum laser exposure to surrounding tissues is achieved.
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In the stent theory for the medical applications, especially in coronary heart disease, the metallic stent of high quality demands the ability for precision micromaterial processing. It possesses an inherent advantage of adequate radiopacity. This paper describes the current status as well as fabrication of such metallic stent of length 20 mm and dia. 2.1 mm with an annular tube thickness of 0.2 mm, by using the short pulse Nd-YAG laser. Fine structures with
slit width of 0.1 mm and pitch better than 0.2 mm are created with sharpness and low roughness in the cut surface. Some features on the reduced heat affected zone and dross removal process of the cut surfaces of the stent as well as a few techniques on preventing restenosis are well documented.
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The authors introduce the design of a compact phototherapy unit capable of fulfilling the recommendations of the clinical use of lasers and LEDs in phototherapy for the treatment of neonatal jaundice. The system keeps the duration of phototherapy to the minimum required for efficient treatment. Our leading clinical experience as well as the wavelength selection rules will be presented.
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The effects of 980-nm diode laser and electrolytic lesions in Wistar rat brain tissue were observed by immunohistochemical staining for CD68 marker and Hematoxylin-Eosin (H&E). Bilateral lesions; laser lesions (2W/2sec) in the right hemispheres, and electrolytic lesions (1.5mA/20sec) in the left hemispheres were done through in vivo stereotaxic neurosurgical procedure. Subjects were classified into three groups due to the recovery period. Subjects in Group I, II, and III were sacrificed after 0, 2 and 7 days of recovery period respectively. After saline perfusion their brains were dislocated, and paraffin embedded sections were taken. One section for H&E and one for CD68 were cut consecutively in 3μm thickness by examining the lesion in every 30-μm thickness. CD68 was found more efficient marker than H&E in observing the after-effects of both types of lesions. The total damage of laser was smaller than that of electrosurgical unit. The shape of the ablated area in laser induced lesions was more spherical than that of electrosurgical unit. The number of macrophages increased as the recovery period increased for all subjects. Group III showed the highest number of macrophages in three, and the number of macrophages around electrolytic lesion is nearly 1.5 times higher than that of laser lesion. The remarkable ablating ability, the damage zone created and the healing of nearby tissue clearly showed that the 980-nm diode laser is an effective and useful alternative to electrosurgical unit in neurosurgery.
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Use of intravenous laser irradiation of blood (ILIB) is considered to be the most effective method of laser therapy and its application is expedient pathogenetically in the ischemic disturbances. The aim of this study is to investigate ILIB influence with infrared laser (IL) with 860 nm wavelength on hemostasis, acid-base status (ABS) of blood in normal rabbits and after modeling of local ischemia of brain (LIB). Experimental cerebral ischemia is characterized by
development of hypercoagulation syndrom and metabolic acidosis. ILIB with infrared radiation of 2.0 mW power provokes hypocoagulation in intact animals. Application of ILIB in rabbits after LIB contributes for hemostasis and acid-base status normalizing compared to operated animals. IL radiation with 8,5 mW power results in marked hemostatic
activation in all animals. Therefore, beneficial effect of low power laser radiation (LPLR) manifests in narrow power diapason in experimental brain ischemia.
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Photodynamic therapy (PDT) wit h total photobleaching of a photosensitizer (PS) was studied theoretically. The equations for spatial-temporal distribution of photochemical dose (PD) under various irradiations were obtained. Both surface irradiation and interstitial irradiation were considered. The possibilities of having constant therapeutic dose within a large extent of a tissue and even of obtaining the maximal dose at a particular depth were shown.
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Photodynamic Therapy, PDT, promises to be an effective and minimally invasive modality for the treatment of this early stage disease. Typically PDT of solid tumours is performed using an array of interstitial fibreoptic light sources implanted in the tumour, illuminated continuously with light from a single laser until the required lethal dose of light is delivered. The literature teaches that PDT is dose rate independent so that the illumination intensity of the sources will not influence the therapeutic outcome. Recent work, however, suggests that this teaching should be qualified. PDT will saturate when the cellular oxygen is depleted, consumed by the photo-oxidation of the photosensitizer. This implies that time fractionated light delivery should increase the lethality of treatment.
In this paper we describe a computer controlled time fractionated fibre optic light delivery/detection system that may be used to synchronize treatment and tissue perfusion. Two laser diodes are each coupled to a computer controlled fibreoptic switch. The switch couples the laser sequentially to an array of interstitial fibre optic light sources. The dose delivered to each source is time moderated so that a non-uniform dose distribution may be delivered across the tumour.
We describe preliminary results of this light delivery system and the photo-toxin QLT0074 for the treatment of rats with AT an -H flank tumours. We also compare tissue response to both time-fractionated and continuous light delivery.
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The quantification of the fluorescence is important to design optical equipment for Photodynamic Diagnosis. The fluorescence intensity of Protoporphyrin IX in solution and induced in cells, cultivated in the hen's eggs model, have been measured photometrically. With an optimized CCD-camera-systems fluorescent tumor areas were detected before and after PDT. The ratio of dead cells was detected by staining with trypan blue after PDT. The measurements were carried out various energy densities at the time of maximal PpIX-enrichement.
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