This PDF file contains the front matter associated with SPIE Proceedings Volume 8092, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Pulsed laser irradiated gold nanoparticles can mediate cell membrane permeabilization, cell elimination and protein inactivation by mechanical effects of nanocavitation. Besides therapeutic applications irradiated gold nanoparticles are investigated as contrast agent in photoacoustic imaging. Especially for imaging of structures in deeper tissue the use of irradiation wavelengths in the range between 700 and 1100 nm is necessary, because of lower tissue absorption and scattering. Depending on their shape the nanorods absorption band can be shifted into this near infrared range. Thus, it can be expected to mediate stronger effects with nanorods irradiated at this band. In contrast to spherical particles nanorods irradiated with nanosecond laser pulses tuned to the wavelength of their maximum absorption are not suitable to cause expected effects. We found that an expanding vapor bubble causes a rapid change in refractive index of the surrounding medium and results changes of the nanorods optical properties. These changes remain transient for a stable particle shape and transcent into permanent change, when melting occurs. Thus, for the purpose of cell killing or enhanced contrast in photoacoustic imaging higher photothermal stability is required. We show here by means of calculations and experiments, that a porous silica coating stabilizes the wavelength position of the longitudinal plasmon resonance of irradiated nanorods. These silica shelled gold-nanorods retained their optical properties and showed increased photothermal stability under nanosecond pulsed laser irradiation.

During optical therapies, several types of interaction between the optical radiation and the target tissue can occur. The application of different power densities and the variation of the exposure time can cause from photochemical reactions to photodisruption. Photothermal therapy (PTT) is based in the thermal interactions, where the biological injury is provoked by a given increase of their temperature during the exposition to the optical source. Another treatment option very extended in several clinical fields due to its promising results is Photodynamic Therapy. This treatment modality is based in photochemical reactions where it is also required oxygen and the administration of a photosensitive substance known as photosensitizer. The use of nanotechnology in optical therapeutic techniques, constitutes a novel promising treatment strategy. Specifically, gold nanoparticles can improve different issues related to the transport of photosensitizers or the light energy absorption and the subsequent heat generation. This work focuses in the effects that can produce the use of gold nanoparticles in Photothermal and Photodynamic Therapies applied to skin diseases commonly treated by means of these techniques. We present a thermal model that permits to calculate the temperature distribution in different kinds of pathological dermatological tissues depending on the optical power provided by the optical source. The results obtained permit to compare the thermal injury produced depending on not only the provided power but also the type of pathology and the incorporation or not of gold nanoparticles in the target tissue.

Selective caries treatment has been anticipated as an essential application of dentistry. In clinic, some lasers have already realized the optical drilling of dental hard tissue. However, conventional lasers lack the selectivity, and still depend on the dentist's ability. Based on the absorption property of carious dentin, 6 μm wavelength range shows specific absorptions and promising characteristics for excavation. The objective of this study is to develop a selective excavation of carious dentin by using the laser ablation with 6 μm wavelength range. A mid-infrared tunable pulsed laser was obtained by difference-frequency generation technique. The wavelength was tuned around the absorption bands called amide 1 and amide 2. In the wavelength range from 5.75 to 6.60 μm, the difference of ablation depth between demineralized and normal dentin was observed. The wavelength at 6.02 μm and the average power density of 15 W/cm², demineralized dentin was removed selectively with less-invasive effect on normal dentin. The wavelength at 6.42 μm required the increase of average power density, but also showed the possibility of selective ablation. This study provided a valuable insight into a wavelength choice for a novel dental laser device under development for minimal intervention dentistry.

Nanosecond long laser pulses are used in medical applications where precise tissue ablation with minimal thermal and mechanical collateral damage is required. When a laser pulse is incident on a material, optical energy will be absorbed by a combination of linear and nonlinear absorption according to both: laser light irradiance and material properties. In the case of water or gels, the first results in heat generation and thermoelastic expansion; while the second results in an expanding plasma formation that launches a shock wave and a cavitation/boiling bubble. Plasma formation due to nonlinear absorption of nanosecond laser pulses is originated by a combination of multiphoton ionization and thermionic emission of free electrons, which is enhanced when the material has high linear absorption coefficient. In this work, we present three experimental approaches to study pressure transients originated when 6 ns laser pulses are incident on agar gels and water with varying linear absorption coefficient, using laser radiant exposures above and below threshold for bubble formation: (a) PVDF sensors, (b) Time-resolved shadowgraphy and (c) Time-resolved interferometry. The underlying hypothesis is that pressure transients are composed of the superposition of both: shock wave originated by hot expanding plasma resulting from nonlinear absorption of optical energy and, thermoelastic expansion originated by heat generation due to linear absorption of optical energy. The objective of this study is to carry out a comprehensive experimental analysis of the mechanical effects that result when tissue models are irradiated with nanosecond laser pulses to elucidate the relative contribution of linear and nonlinear absorption to bubble formation. Furthermore, we investigate cavitation bubble formation with temperature increments as low as 3 °C.

Enhanced visualization of tissue contrast and morphological boundaries is demonstrated by analyzing OCT volume data in two distinct wavelength regions. The extension of this so-called simultaneous dual-band method to three dimensions is realized by a custom-built 3D spectral domain OCT system imaging in the 800 und 1200 nm wavelength domain. Color representations of the spectral differences of high resolution OCT volume data significantly simplify the discrimination of different tissue structures beyond the capabilities of cross-sectional spectroscopic OCT approaches.

We present the use of fiber optic microendoscopy to image bacterial infection in the skin and lungs using an animal model. The contact probe microendoscope we have constructed has a 4 μm resolution, a 750 μm field of view, and a 1 mm outer diameter. Well resolved regions of bacterial infection were imaged for subcutaneous inocula of 10⁶ to 10¹ CFU and intra-tracheal inocula of 10⁸ to 10⁶ CFU. Results reveal a linear relationship between average fluorescence and CFU, suggesting potential for using this device for quantitative analysis. Detection limits of 10⁴ CFU for skin samples and 10⁷ CFU for lung tissue were determined. In addition, bacteria were also qualitatively visible in lung tissue down to 10⁶ CFU. Confocal imaging was used to confirm the presence of bacteria in tissue samples.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique which gives access to the local environment of fluorophores in living cells. However, to correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently a long laser exposure time which is not compatible with the observation of dynamic molecular events and which induces cellular stress phenomena. For reducing this exposure time, we have developed an original approach to statistically inflate the number of collected photon. This approach called Adaptive Monte Carlo Data Inflation (AMDI) combines the well-known bootstrap technique with an adaptive Parzen kernel. We have evaluated its potential on experimental FLIM data in vivo. We have demonstrated that our robust method allows estimating precisely fluorescence lifetime with exposure time reduced up to 50 times for mono-exponential (corresponding to a minimum of 20 photons/pixel) and 10 times for bi-exponential decays (corresponding to a minimum of 5000 photons/pixel) in comparison with the standard fitting method. Furthermore, thanks to AMDI, we demonstrate that it becomes possible to estimate accurately all fitting parameters in FRET experiments without constraining any parameter. An additional benefit of our technique is that it improves the spatial resolution of the FLIM images by reducing the commonly used spatial binning factor.

Imaging of cells is an interesting and challenging problem as they do not appreciably change the amplitude of the electromagnetic radiation interacting with them. Phase contrast techniques can be used to overcome this hurdle. Interferometric phase contrast techniques like digital holography can be used for quantitative phase contrast microscopic imaging of transparent objects and it yields the three dimensional profile of the object under investigation. These methods also have advantage of numerical focusing, allowing one to focus on to any desired object plane. But most of the interferometric quantitative phase contrast techniques require two beams as well as the adjustment of the beams for high fringe contrast, requiring stringent optical conditions. Here we present a single beam phase retrieval technique for quantitative phase contrast microscopy of cells. The phase information of the object is obtained by sampling the volume speckle field generated by the object at several axial planes. These intensity patterns are used iteratively in the diffraction integral to retrieve the phase information about the object. The advantages of this technique include compactness, immunity to external vibrations as well as the prospect of usage of low coherent sources.

Photosensitizer fluorescence photobleaching and Singlet Oxygen (1O2) Luminescence Dosimetry (SOLD) are being studied as potential dosimetric tools for ALA-PDT of skin diseases. However, the correlation of both SOLD data and PpIX fluorescence to ¹O₂ distribution is difficult to interpret because of the temporal and spatial variations of the PDT parameters (light fluence rate, photosensitizer concentration and oxygen concentration). This work used our dynamic model to investigate both dosimetry approaches for varied PpIX concentration and distribution, and three commonly used treatment wavelengths. The results show that SOLD is much less dependent upon the treatment parameters, which implies it has better potential as a "gold standard" dosimetric tool for clinical PDT.

Objective: Provide preclinical data on the feasibility of 5-aminolevulinic acid (5-ALA) -based photodetection (PD) and Photodynamic Therapy (PDT) of early childhood tumors. Methods: Hepatoblastoma (HuH6), neuroblastoma (MHH-NB11) and N1-fibroblast cell lines were tested for their relative capacities to synthesize Protoporphyrin IX (PpIX) from 5-ALA and for their susceptibility to PDT in vitro. HuH6-cells were also inoculated in the peritoneum of rats. The pharmacokinetics of porphyrin accumulation was measured in 9 rats by laparoscopic spectroscopy. 5-ALA was applied by i.p. injection of 500 mg/kg bw. In another 21 animals, tumors (n=20), liver (n=5) and peritoneum (n=4) were treated by PDT laparoscopically. 48 h after irradiation, animals were again incubated with 5-ALA and then sacrificed and tissues were removed for further investigation. Results: Both tumor cell lines showed higher levels of porphyrin fluorescence than the fibroblasts. Cell viability testing proved the HuH6 cells to be most susceptible to PDT. Pharmacokinetic measurements of PpIX in xenografted tumors showed a peak at 80-200 min after i.p. injection of 5-ALA. Irradiation resulted in pronounced photobleaching at all irradiated sites and necrosis of tumor and liver tissue, whereas peritoneum appeared to remain unaffected. Necrosis induced by PDT could be seen in fluorescence microscopy due to the lack of porphyrin synthesis in necrotic tissue after the re-incubation with 5-ALA.

The use of LED devices for phototherapy has been expanding in the last decade. This technology provides a safer emission spectrum in large target tissue areas when compared to laser emissions. For enhancing the phototherapeutic effects of red light emitted by LEDs, a simple optical concentrator capable of efficient light concentration and homogenization was developed. The LEDs wavelength of 660 nm is coincident with an absorption peak of the mitochondrial photoreceptor molecule cytochrome c oxidase. The prototype was optimized by non-sequential ray-tracing software ZEMAX, attaining both excellent light uniformity and 50mW/cm² irradiance at the concentrator output end. Heat emanated from the LEDs source is effectively dissipated by the side walls of the concentrator, ensuring a nearly constant temperature environment for tissue treatment. The prototype was tested on cutaneous hyperpigmented marks caused by cupping in two healthy volunteers. Marks were irradiated by LEDs radiations with or without the use of concentrator respectively. Equal exposure durations and light fluences were tested. The use of the concentrator-coupled LEDs source revealed an activation of blood movement immediately after LEDs exposure, an effect not attainable by the LEDs source without the concentrator even at extended exposure time. Promising futures for the treatment of inflammation, tissue repair and skin rejuvenation could be expected by adopting this simple technique.

Sentinel lymph node biopsy is the gold standard method to detect a metastatic invasion from the primary breast cancer. This method can avoid patients to be submitted to full axillary chain dissection. In this study we present and compare two near-infrared optical probes for the sentinel lymph node detection, based on the recording of scattered photons. The two setups were developed to improve the detection of the dye injected in clinical routine: the Patent Blue V dye. Herein, we present results regarding clinical ex-vivo detection of sentinel lymph node after different volume injections. We have previously published results obtained with a two-wavelength probe on phantom and animal models. However this first generation device did not completely account for the optical absorption variations from biological tissue. Thus, a second generation probe has been equipped with four wavelengths. The dye concentration computation is then more robust to measurement and tissue property fluctuations. The detection threshold of the second setup was estimated at 8.10^-3μmol/L, which is about 37 times lower than the eye visibility threshold. We present here the preliminary results and demonstrate the advantages of using four wavelengths compared to two on phantom suspensions simulating the optical properties of breast tissues.

Photo-bleaching of in-vivo skin autofluorescence intensity under continuous low power laser irradiation has been studied. Temporal behavior of single-spot fluorescence and spectral fluorescent images have been studied at continuous 405 nm, 473 nm and 532 nm laser excitation and/or pre-irradiation, with power densities well below the laser-skin safety limits. Skin autofluorescence photo-memory effects (laser signatures) have been observed and analyzed, as well.

Optical methods allow investigating biological tissue noninvasively without ionizing radiations. Moreover, considering low absorption processes in the tissue in the near-infrared wavelengths range, biological tissue can be deeply investigated. In this field, we studied the resolution limits of the detection of one and two tumour-like heterogeneities embedded in the middle plane of a slab that mimics a breast enclosed between two transparent plates. We used the diffusion equation in order to model the photons propagation in such slab. It is solved in the time-domain by means of a finite element method. We computed time-resolved trans-illumination data based on lateral scan of the slab. The timedependent transmitted light, received at the opposite of the source, was transformed in the frequency-domain and the modulation and phase-shift of the signal are then obtained. The resulting phase-shift considering the embedded objects was analyzed versus the distance between the objects. Then, the resolution limits were estimated considering different modulation frequencies and a noise level. The overall combinations took into account a set of optical properties that mimics realistic optical properties for healthy breast tissue and tumours.

This paper is devoted to the development and testing of accelerated-time calculation for the spatially resolved reflectance in multiple layer turbid medium that facilitates the use of Monte Carlo simulation (MC) in medical physics applications. To mitigate the inconveniences associated to long execution times, the MC code has been speeded up by using efficient computational hybrid technique computing on graphics processing units (GPU). This method effectively reduces the simulation time by a factor of 8 compared to the stand-alone GPU-based MC code.

We developed a method to determine the optical properties of biological tissue from the results of integrating sphere measurements. Our Monte Carlo model developed for this purpose considered the geometry of the investigated sample as well as the features of the integrating sphere setup. To improve the accuracy of our results, we incorporated the wavelength dependence of the anisotropy factor of the investigated tissue into the Monte Carlo model. To determine the anisotropy factor, we performed goniometric measurements on six porcine dura mater tissue samples and quantified the phase function at a wavelength of 650 nm. The averaged anisotropy factor was taken into account in the Monte Carlo simulations. The result of these simulations were combined in a table lookup. We used this table lookup to interpret the results from the integrating sphere measurements. We present as a result of our measurements the absorption coefficient and the reduced scattering coefficient of porcine dura mater tissue in the wavelength range of 450 to 650 nm.

Long term in vivo observations at large penetration depths and minimum sample disturbance are some of the key factors that have enabled the study of different cellular and tissue mechanisms. The continuous optimization of these aspects is the main driving force for the development of advanced microscopy techniques such as those based on nonlinear effects. Its wide implementation for general biomedical applications is however, limited as the currently used nonlinear microscopes are based on bulky, maintenance-intensive and expensive excitation sources such as Ti:sapphire ultrafast lasers. We present the suitability of a portable (140x240x70 mm) ultrafast semiconductor disk laser (SDL) source, to be used in nonlinear microscopy. The SDL is modelocked by a quantum-dot semiconductor saturable absorber mirror (SESAM). This enables the source to deliver an average output power of 287 mW with 1.5 ps pulses at 500 MHz, corresponding to a peak power of 0.4 kW. The laser center wavelength (965 nm) virtually matches the two-photon absorption cross-section of the widely used Green Fluorescent Protein (GFP). This property greatly relaxes the required peak powers, thus maximizing sample viability. This is demonstrated by presenting two-photon excited fluorescence images of GFP labeled neurons and second-harmonic generation images of pharyngeal muscles in living C. elegans nematodes. Our results also demonstrate that this compact laser is well suited for efficiently exciting different biological dyes. Importantly this non expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices, facilitating its widespread adoption in biomedical applications.

In this work optical tweezers with elliptical beam profiles have been developed in order to examine the effect of optical force on fresh red blood cells (RBC) in isotonic, hypertonic and hypotonic buffer solutions. Considering that the optical force depends essentially on the cell surface and the cytoplasmic refractive index, it is obvious that biochemical modifications associated with different states of the cell will influence its behaviour in the optical trap. Line optical tweezers were used to manipulate simultaneously more than one red blood cell. After we have been manipulated a RBC with an elliptical laser beam profile in an isotonic or hypertonic buffer, we noticed that it rotates by itself when gets trapped by optical tweezers and undergoes folding. Further shape deformations can be observed attributed to the competition between alignment and rotational torque which are transferred by laser light to the cell. In hypotonic buffer RBCs become spherical and do not rotate or fold since the resultant force due to rays emerging from diametrically opposite points of the cell leads to zero torque. Manipulation of fresh red blood cells in isotonic solution by line optical tweezers leads to folding and elongation of trapped RBCs. Membrane elasticity properties such as bending modulus can be estimated by measuring RBC's folding time in function with laser power.

The objective of this study was to examine the in vitro effect of a single or a multiple doses of low-level laser irradiation (LLLI) on proliferation of the human osteosarcoma cell line, SAOS-2. SAOS-2 cells were divided in five groups and exposed to LLLI (659 nm diode laser; 11 mW power output): group I as a control (dark), group II exposed to a single laser dose of 1 J/cm², group III irradiated with a single dose of 3 J/cm², and group IV and V exposed for three consecutive days to 1 or 3 J/cm², respectively. Cellular proliferation was assessed daily up to 7 days of culturing. The obtained results showed an increase in proliferative capacity of SAOS-2 cells during the first 96 h of culturing time in once-irradiated cells, as compared to control cells. Furthermore, a significantly higher proliferation in the group IV and V was detected if compared to a single dose or to control group after 96 h and 7 days. In conclusion, the effect of the single dose on cell proliferation was transitory and repeated irradiations were necessary to observe a strong enhancement of SAOS-2 growth. As a future perspective, we would like to determine the potential of LLLI as a new approach for promoting bone regeneration onto biomaterials.

Low-level laser irradiation (LLLI) increases ATP production and energy supply to the cell which could increase sperm motility, acrossomal reaction and consequently the fertilizing potential. The aim of this study was to characterize the biochemical and topological changes induced by low power laser irradiation on bull sperm cells. Post-thawing sperm were irradiated with a 633nm laser with fluence rates of 30, 150 and 300mJ.cm^-2 (power of 5mW for 1, 5 and 10minutes, respectively); 45, 230, and 450mJ.cm^-2 (7.5mW for 1, 5 and 10 minutes); and 60, 300 and 600mJ.cm^-2 (10mW for 1, 5 and 10 minutes). Biochemical and metabolical changes were analyzed by FTIR and flow cytometry; oxygen reactive species production was assessed by TBARS and the morphological changes were evaluated by AFM. Motility had no difference among times or powers of irradiation. Increasing in ROS generation was observed with power of 5mW compared to 7.5 and 10mW, and with 10min of irradiation in comparison with 5 and 1min of irradiation. This higher ROS generation was related to an increase in acrossomal and plasma membrane damage. FTIR results showed that the amount of lipids was inversely proportional to the quantity of ROS generated. AFM images showed morphological differences in plasma/acrossomal membrane, mainly on the equatorial region. We conclude that LLLI is an effective method to induce changes on sperm cell metabolism but more studies are necessary to establish an optimal dose to increase the fertility potential of these cells.

The multiple scattering of light can increase efficiency of laser therapy of inflammatory diseases enlarging the treated area. The light absorption is essential for treatment while scattering dominates. Multiple scattering effects must be introduced using the Monte Carlo method for modeling light transport in tissue and finally to calculate the optical parameters. Diffuse reflectance measurements were made on high concentrated live leukocyte suspensions in similar conditions as in-vivo measurements. The results were compared with the values determined by MC calculations, and the latter have been adjusted to match the specified values of diffuse reflectance. The principal idea of MC simulations applied to absorption and scattering phenomena is to follow the optical path of a photon through the turbid medium. The concentrated live cell solution is a compromise between homogeneous layer as in MC model and light-live cell interaction as in-vivo experiments. In this way MC simulation allow us to compute the absorption coefficient. The values of optical parameters, derived from simulation by best fitting of measured reflectance, were used to determine the effective cross section. Thus we can compute the absorbed radiation dose at cellular level.

We have sought for non-invasive diagnosis of blood during the extracorporeal circulation support. To achieve the goal, we have newly developed a photon-cell interactive Monte Carlo (pciMC) model for optical propagation through blood. The pciMC actually describes the interaction of photons with 3-dimentional biconcave RBCs. The scattering is described by micro-scopical RBC boundary condition based on geometric optics. By using pciMC, we modeled the RBCs inside the extracorporeal circuit will be oriented by the blood flow. The RBCs' orientation was defined as their long axis being directed to the center of the circulation tube. Simultaneously the RBCs were allowed to randomly rotate about the long axis direction. As a result, as flow rate increased, the orientation rate increased and converged to approximately 22% at 0.5 L/min flow rate and above. And finally, by using this model, the pciMC non-invasively and absolutely predicted Hct and hemoglobin with the accuracies of 0.84±0.82 [HCT%] and 0.42±0.28 [g/dL] respectively against measurements by a blood gas analyzer.

This paper presents a discussion about the potential of photoacoustics with regard to its application in surgical assistance during minimally invasive, laser assisted interventions. Aim of the work is the detection of obscured large blood vessels in order to prevent unintentional dissection. Based on spectroscopic investigations of the target tissue (liver), a wavelength for the photoacoustic excitation laser was chosen with respect to a high absorption contrast between the vessel and the surrounding liver tissue. An experimental setup featuring a simple liver model is created. Preliminary results show, that vessels with a diameter of 2 mm can be detected up to a distance of 1 mm from the treatment fibre. It is shown, that detection of acoustic waves induced inside liver is feasible over distances higher than 10 cm.

Introduction: Warm ischemia and bleeding during laparoscopic partial nephrectomy place technical constraints on surgeons. Therefore it was the aim to develop a safe and effective laser assisted partial nephrectomy technique without need for ischemia. Patients and methods: A diode laser emitting light at 1318nm in cw mode was coupled into a bare fibre (core diameter 600 μm) thus able to transfer up to 100W to the tissue. After dry lab experience, a total of 8 patients suffering from kidney malformations underwent laparoscopic/retroperitoneoscopic partial nephrectomy. Clinically, postoperative renal function and serum c-reactive protein (CRP) were monitored. Laser induced coagulation depth and effects on resection margins were evaluated. Demographic, clinical and follow-up data are presented. Results: Overall interventions, the mean operative time was 116,5 minutes (range 60-175min) with mean blood loss of 238ml (range 50-600ml) while laser assisted resection of the kidney tissue took max 15min. After extirpation of the tumours all patients showed clinical favourable outcome during follow up period. The tumour size was measured to be 1.8 to 5cm. With respect to clinical safety and due to blood loos, two warm ischemia (19 and 24min) must be performed. Immediate postoperative serum creatinine and CRP were elevated within 0.1 to 0.6 mg/dl (mean 0.18 mg/dl) and 2.1-10 mg/dl (mean 6.24 mg/dl), respectively. The depth of the coagulation on the removed tissue ranged between <1 to 2mm without effect on histopathological evaluation of tumours or resection margin. As the surface of the remaining kidney surface was laser assisted coagulated after removal. The sealing of the surface was induced by a slightly larger coagulation margin, but could not measured so far. Conclusion: This prospective in-vivo feasibility study shows that 1318nm-diode laser assisted partial nephrectomy seems to be a safe and promising medical technique which could be provided either during open surgery as well as laparascopically. This application showed good haemostasis and minimal parenchymal damage. Oncological safety appears to be warranted by the use of diode laser. Further investigations and development are needed for on-line detection of the remain coagulation margin, optimisation of the treatment equipment, and finally to train the application technique.

Dissection of liver tissue can be performed by different techniques (ultrasound, mono and bipolar dissection, water jet dissection and by stapler). In this animal study the potential of a Thulium fiber laser system was investigated for open parenchyma dissection. Based on a cw Thulium fiber laser (IPG laser GmbH, Burbach, Germany), emitting a wavelength at 1.9 μm and a maximal power at 50 W, a surgical dissection device was developed at the Medical Laser Centre Luebeck. Cw laser radiation (40 Watt) was transmitted via a 365 μm fiber with a polished distal fiber tip. Procedure was performed in contact mode; irradiance at the distal fiber tip was 38.2 kW/cm². After general anesthesia and a median laparotomy an atypical laser resection of the liver was performed in 3 pigs. Healing process was controlled after 2-3 weeks by histological analysis (H&E staining). The final evaluation data included total resection time, blood loss, bile leakage and mass of dissected tissue. All animals treated in this study were cared for in accordance to the European convention on animal care. In general the dissection with the 1.9 μm laser radiation was easily performed. Hemostasis was highly sufficient so blood loss and bile leakage was negligible. Total resection time including hemostasis of the remaining tissue was 26 ± 12 min. Weight of resected tissue was 17 ± 8 g. During survival period no complications (bleeding or inflammation) occurred. After 2 weeks histology showed ongoing scar formation about 1 - 2 mm in depth of the dissected area.

Laser assisted vascular repair is a new optimized technique based on the use of ICG-infused chitosan patch to close a vessel wound, with or even without few supporting single stitches. We present an in vivo experimental study on an innovative end-to-end laser assisted vascular anastomotic (LAVA) technique, performed with the application of ICGinfused chitosan patches. The photostability and the mechanical properties of ICG-infused chitosan films were preliminary measured. The in vivo study was performed in 10 New Zealand rabbits. After anesthesia, a 3-cm segment of the right common carotid artery was exposed, thus clamped proximally and distally. The artery was then interrupted by means of a full thickness cut. Three single microsutures were used to approximate the two vessel edges. The ICG-infused chitosan patch was rolled all over the anastomotic site and welded by the use of a diode laser emitting at 810 nm and equipped with a 300 μm diameter optical fiber. Welding was obtained by delivering single laser spots to induce local patch/tissue adhesion. The result was an immediate closure of the anastomosis, with no bleeding at clamps release. Thus animals underwent different follow-up periods, in order to evaluate the welded vessels over time. At follow-up examinations, all the anastomoses were patent and no bleeding signs were documented. Samples of welded vessels underwent histological examinations. Results showed that this technique offer several advantages over conventional suturing methods: simplification of the surgical procedure, shortening of the operative time, better re-endothelization and optimal vascular healing process.

Pulsed ArF excimer laser radiation at 6.4 eV, at fluence exceeding the ablation threshold, will debride burn eschar and other dry necrotic erosions of the skin. Debridement will cease when sufficiently moist viable tissue is exposed, due to absorption by aqueous chloride ions (Cl^-) through the non-thermal process of electron photodetachment, thereby inhibiting collateral damage to the viable tissue. ArF excimer laser radiation debrides/ablates ~1 micron of tissue with each pulse. While this provides great precision in controlling the depth of debridement, the process is relatively time-consuming. In contrast, XeCl excimer laser radiation debrides ~8 microns of tissue with each pulse. However the 4.0 eV photon energy of the XeCl excimer laser is insufficient to photodetach an electron from a Cl^- ion, so blood or saline will not inhibit debridement. Consequently, a practical laser debridement system should incorporate both lasers, used in sequence. First, the XeCl excimer laser would be used for accelerated debridement. When the necrotic tissue is thinned to a predetermined thickness, the ArF excimer laser would be used for very precise and well-controlled debridement, removing ultra-thin layers of material with each pulse. Clearly, the use of the ArF laser is very desirable when debriding very close to the interface between necrotic tissue and viable tissue, where the overall speed of debridement need not be so rapid and collateral damage to viable tissue is undesirable. Such tissue will be sterile and ready for further treatment, such as a wound dressing and/or a skin graft.

The goal of this research is to use the information contained in the mechanisms occurring during laser tattoo removal process. We employed a fast laser beam deflection probe (BDP) to measure the cracking sound that originates from the dye explosions in the process known as selective photothermolysis. The experiments were performed in vitro (skin phantoms), ex vivo (marking tattoos on pig skin) and in vivo (professional and amateur decorative tattoos on several patients). The signal includes the information about the energy released during the interaction, specific for different skin and tattoo conditions.

Histological slices of skin samples with the subcutaneous adipose tissue after laser irradiation at different doses are analyzed. These data may be used at carrying out of the analysis of histological slices of skin samples with the subcutaneous adipose tissue after photodynamic therapy. The obtained data are important for safe layer-by-layer dosimetry of laser irradiation used in the treatment of obesity and cellulite.

While using a laser to process hard tissue, it is difficult to guarantee, that the laser beam is always perpendicular to the tissue surface. Therefore, it is necessary to know the dependence of ablation depth on angle of incidence for preoperative planning. Considering the propagation of the ablation front, an Addition Model is developed in this work. It indicates that the shape of a crater ablated by a single pulse with non-zero angle of incidence can be regarded as the sum of the original tissue surface and a symmetric profile, which is corresponding to the shape of a crater ablated by perpendicularly incident beam. Meanwhile, the ablation depth at a point P is defined as the distance from P to the original tissue surface along the optical axis of the incident beam. In the context of this definition, the dependence is experimentally studied. The results of the experiments were unexpected: the ablation depth is independent of angle of incidence up to ca. 55°. Possible reasons for these results are discussed.

The optical properties of the cornea have been a research subject of great interest for many years. Several early theories have been put forward to explain with more or less success the optical transparency of this tissue, but it was not until Maurice demonstrated in a very elegant way during the 50s that this optical transparency could be explained by the regular ultrastructure of the cornea. When becoming edematous, the cornea's ultrastructure is perturbed and the tissue becomes a strongly scattering medium. With the emergence of ophthalmologic surgery by ultrashort pulse lasers in recent years, a regain of interest in the subject of corneal transparency arose. However, relatively little and no recent data of transparency spectra measurements covering a large wavelength range is available in the literature. The purpose of this study is to provide quantitative values for light scattering and its relation to the degree of edema by measuring the spectrum of transmitted light through corneas presenting different degrees of edema. This paper focus on the comparison of laboratory measurements published earlier with a new simple method we propose We also for eye banks to quantitatively measure the degree of transparency of corneal grafts by measuring the modulation transfer function of a Siemens star viewed through a corneal graft. Indeed, there is no current method to determine the transparency of corneal graft but the subjectivity of the laboratory technician or the ophthalmic surgeon.

Retinal photocoagulation is an established treatment for various retinal diseases. The temperature development during a treatment can be monitored by applying short laser pulses in addition to the treatment laser light. The laser pulses induce optoacoustic pressure waves that can be detected at the cornea. Aim of this work is the investigation of the accuracy of the determined temperatures during a treatment. To calibrate the temperature dependency of the measured pressure, whole enucleated porcine eyes were heated using an infrared laser beam, while probing the retina optoacoustically. The temperatures and the optoacoustic pressure waves were measured simultaneously using thermocouples and a piezoelectric element, respectively. From the deviation of the individual measurements an error of less than 15% in the calibration regime between 37 °C to 55 °C was found. Furthermore, the spatial and temporal temperature course was investigated. Calculations were performed to simulate the temporal and spatial temperature development during photocoagulation. A theoretical model to determine the peak temperature of the irradiated tissue from the mean temperature measured by optoacoustics was developed. The validity of the model was experimentally examined by heating the retina of porcine eyes with a laser beam diameter of 500 μm while successively measuring the temperature optoacoustically with a probe beam diameter of 500 μm and 100 μm at the center of the heated area, respectively. The deviation of the theoretical model and the experimental results were found to be less than 7%.

A high power, diode-pumped Er:YAG laser platform is presented, which has been integrated into devices for soft as well as hard tissue applications. The highly efficient side pumping by qcw laser diodes allows easy power scalability and miniaturization proven by a portable fractional ablative laser system based on a 2 W laser. The high repetition rate of up to 1 kHz combined with low energy pulses generates high thermal impact and consequently strong skin rejuvenation. Furthermore a laser for hard tissue applications with up to 15 W average output power at repetition rates up to 2 kHz is presented. The good beam quality allows coupling to 200 μm fibers and the variable pulse duration of 1 to 200 μs ensures precise and fast treatments.

We report oral cavity specific hard tissue ablation experiments at different fluence values using femtosecond laser. The set-up was composed by a high energy femtolaser, optical and mechanical equipment for focusing and displacement of the beam on the sample surface. Using a lens to focus the beam we have obtained fluence range between 75 J/cm² and 0.21 J/cm². Samples were human extracted teeth and mandible bone. Created structures were rows. Characterization of ablated structures was made by scanning electron microscope and optical microscope. Ablation areas images show crystalline and regular structures. There are not evidences of material burning under 75 J/cm². Generated structures are reproducible, dependent on tissue quality and surface roughness. Dimensions of structures are of tens microns, dependent on beam fluence and material hardness. We appreciate the potential of the method to about 1 micron precision. The results are positive considering the advantages of the method: ablation precision and no collateral damage.

Femtosecond laser induced optical breakdown allows for high-precision cutting of transparent materials with low energy deposit and little peripheral damage for applications in micromachining and minimally invasive medical surgery. Little peripheral damage is especially important for laser incisions in the posterior eye due to the vicinity to the retina. When applying laser pulses through the anterior eye, aberrations are introduced to the wave front, which cause a distortion of the focal volume and an increase in required pulse energy for tissue manipulation through photodisruption. To decrease the pulse energy, aberrations need to be corrected to restore a diffraction limited focus. In this work, the influence of an aberration correction using adaptive optics on the required pulse energy for an optical breakdown was investigated. The aberrations were introduced in an eye model using HEMA as eye tissue substitute and corrected in an optical setup including a deformable mirror and a Hartmann-Shack-Sensor. The laser pulses were focused by a plano-convex lens and the induced impact was compared for the aberrated and the corrected case. The pulse energy required to obtain an effect was reduced when correcting for aberrations. Therefore, adaptive optics can reduce the risk for potential peripheral damage during ophthalmic surgery.

Femtosecond laser surgery in the volume of corneal tissue is typically performed wavelengths of about 1 μm, which gives excellent results on transparent corneas. However, the outcome is much worse in the case of oedematous or pathological corneas as the laser beam propagation is disturbed by optical scattering. Our studies suggest that this phenomenon can be greatly reduced by using a better suited laser wavelength. Best results are obtained at 1.65 μm. Currently, no compact femtosecond laser at this wavelength is commercially available. We have developed a new simple, compact and stable laser source consisting of a non linear crystal pumped by a compact commercial solid-state laser emitting at 1.03 μm in a configuration of an Optical Parametric Generation (OPG). The output wavelength of this system can be tuned in the spectral range of 1.45 - 1.8 μm. A series of ex vivo penetrating incisions using energies of the order of a few microjoules on corneal tissues have been performed while varying the wavelengths from 1.45 μm to 1.7 μm. The results have been compared to experiments performed at 0.8 μm and 1 μm. The use of longer infrared wavelengths around 1.65 μm for femtosecond laser keratoplasty significantly improves the quality and the penetration depth of incision in case of pathological tissues, without inducing any additional side effects.

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.

Endovenous laser ablation (EVLA) is a common treatment method for varicose vein. However, the precise irradiation dose for EVLA is not understood quantitatively. The objective of this study is to evaluate EVLA quantitatively based on optical properties of the varicose vein tissue, and compare the efficacy and the safety at wavelengths of 980 nm and 1470 nm. A human varicose vein tissue was used as a sample. The samples were irradiated by using the 980 nm and 1470 nm laser diodes in various irradiation parameters. The power density was varied from 260 to 1710 W/cm² and the irradiation time was varied from 3 to 10 s. The optical properties of samples were determined by using a double integrating sphere and an inverse Monte Carlo method. The optical penetration depth of samples was estimated from the optical properties. In the 980 nm laser irradiation, the initial shrinkage of the tissue was observed during laser irradiation conducted at the average energy density of 3630 J/cm² (1210 W/cm², 3 s). In the 1470 nm laser irradiation, the initial shrinkage of the tissue was observed during laser irradiation conducted at the average energy density of 2600 J/cm² (260 W/cm², 10 s). Penetration depth of the vein wall at the wavelength of 980 nm and 1470 nm were 1.3 mm and 0.22 mm, respectively. The sample irradiated with the 1470 nm laser diode showed vein shrinkage in lower energy density than the 980 nm laser irradiation. The penetration depth at the wavelength of 1470 nm was smaller than the sample thickness about 0.8 mm. These data indicate that EVLA with the 1470 nm laser diode may be more effective and safer than EVLA with the 980 nm laser diode.

Aiming at studying solar photocoagulation in biological tissue with both low energy and high energy portions of solar spectrum, a simple color separation technique is proposed. The chromatic aberration characteristic of Fresnel lens is exploited to achieve color separation by a plane mirror with a large central elliptical hole, reflecting the solar radiation above 600nm to one fused silica light guide, while allowing the passage of the remaining radiation to another guide. ZEMAX™ ray-tracing code is used to optimize the performance of each optical component. To attain a stable solar coagulation, the prototype is tested on a two-axis solar tracker. The ex vivo measurement is performed on chicken breasts at the solar power level of 30W and the exposure time of 60 seconds, attaining a uniform coagulation over a large area of 15mm x 15mm. A strong dependence of the penetration depth on wavelength is observed. Our cost effective solar photocoagulation prototype produces the same type and extent of tissue coagulation ordinarily achieved with surgical laser equipment.

Acetaminophen (APAP) is the famous drug in global, and taking overdose Acetaminophen will intake hepatic cell injure. Desptie substantial progress in our understanding of the mechanism of hepatocellular injury during the last 40 years, many aspects of the pathophysiology are still unknown or controversial.1 In this study, mice are injected APAP overdose to damage hepatocyte. APAP deplete glutathione and ATP of cell, N-Acetyl Cysteine (NAC) plays an important role to protect hepatocytes be injury. N-Acetyl Cysteine provides mitochondrial to produce glutathione to release drug effect hepatocyte. By 6-carboxyfluorescein diacetate (6-CFDA) metabolism in vivo, glutathione keep depleting to observe the hepatocyte morphology in time. Without NAC, cell necrosis increase to plasma membrane damage to release 6-CFDA, that's rupture. After 6-CFDA injection, fluorescence will be retained in hepatocyte. For cell retain with NAC and without NAC are almost the same. With NAC, the number of cell rupture decreases about 75%.

The enhancement of the electromagnetic field in the surroundings of nanoparticles via surface plasmon resonance offers promising possibilities for biomedical applications. Here we report on the selective triggering of antibacterial activity using a new type of silver nanoparticles coated with silica, Ag@silica, irradiated at their surface plasmon frequency. The nanoparticles are able to bind readily to the surface of bacterial cells, although this does not affect bacterial growing since the silica shell largely attenuates the intrinsic toxicity of silver. However, upon simultaneous exposure to light corresponding to the absorption band of the nanoparticles, bacterial death is triggered selectively on the irradiated zone. Because of the low power density used in the treatments, we discard thermal effects as the cause of cell killing. Instead, we propose that the switched toxicity is due to the enhanced electromagnetic field in the proximity of the nanoparticles, which either directly (through membrane perturbation) or indirectly (through induced photochemical reactions) is able to cause cell death.

Optical tweezers is a powerful tool which is used to capture and manipulate microscopic particles such as dielectric microspheres and cells. In the single optical trap the beam is strongly focused to a diffraction limited spot by a high numerical aperture objective. Resently a new version of optical trap was demonstrated with optical fibers. Compared with the common optical tweezers which required high power microscope objective and carefully adjusted optical path, the fiber optical tweezers are compact in size and less expensive. Moreover, they have also a working distance not necessarily close to the objective as for a typical optical tweezers. In this work we present the development of a single beam optical fiber trapping system integrated with an optical fiber ablation system for micromanipulation of biological objects. The fiber trap was formed using a continuous wave He-Ne laser operating at 632.8 nm. The fiber ablation system was formed using a free-running Er:YAG laser operating at 2.94 μm with pulse duration of 80 μm. The ablation beam was coupled into the front end of a fluoride glass optical fiber via a focusing lens of 100 mm and a pinhole of 50 μm. We evaluated the fluoride glass optical fiber as far as attenuation and as far as the spatial distribution of the energy output is concerned. We verified that optical trapping and the micromanipulation of micro objects were easily achieved, by a focused laser beam, emerging from optical fiber inclined at 42 degrees to the sample.

Laser light is nowadays routinely used in the aesthetic treatments of facial skin, such as in laser rejuvenation, scar removal etc. The induced thermal damage may be varied by setting different laser parameters, in order to obtain a particular aesthetic result. In this work, it is proposed a theoretical study on the induced thermal damage in the deep tissue, by considering different laser pulse duration. The study is based on the Finite Element Method (FEM): a bidimensional model of the facial skin is depicted in axial symmetry, considering the different skin structures and their different optical and thermal parameters; the conversion of laser light into thermal energy is modeled by the bio-heat equation. The light source is a CO₂ laser, with different pulse durations. The model enabled to study the thermal damage induced into the skin, by calculating the Arrhenius integral. The post-processing results enabled to study in space and time the temperature dynamics induced in the facial skin, to study the eventual cumulative effects of subsequent laser pulses and to optimize the procedure for applications in dermatological surgery. The calculated data where then validated in an experimental measurement session, performed in a sheep animal model. Histological analyses were performed on the treated tissues, evidencing the spatial distribution and the entity of the thermal damage in the collageneous tissue. Modeling and experimental results were in good agreement, and they were used to design a new optimized laser based skin resurfacing procedure.

Optical parametric oscillators (OPOs) are attractive tools for research on tissue ablation upon infrared irradiation. Here, we report on the performance of several mid-infrared nonlinear crystals, namely type I and type II AgGaS₂ (AGS) and type I CdSiP₂ (CSP), used in synchronously-pumped OPOs tuned to a wavelength of 6.45 μm, coinciding with the amide II absorption band of proteins. CSP-based OPOs clearly exhibit better performance in comparison to AGS: First, the oscillation threshold with CSP is three (five) times lower than type II (type I) AGS. Second, the idler conversion efficiency is more favourable for CSP and allows reaching 27.5 mW of idler average power, while 13 and 6 mW are obtained with type II and type I AGS, respectively. Such performance makes CSP suitable for high power 6.45 μm surgical applications. Preliminary ablation experiments on liver tissues with our CSP-based OPO highlight the promising future of CSP in medical applications.

Phototherapy is able to modulate cellular metabolism of bone tissue and consequently accelerate the repair. The aim of this study was to evaluate the effect of this therapy in repair of bone monocortical defects in femurs of thirty male Wistar rats. The animals were divided into six groups (five animals for group), including three controls and three irradiated groups with different experimental times (14, 21, and 28 days after surgery). LED was used for the irradiation, emitting non-coherent light in the spectral range of 945±20 nm and output power of 48 mW, on one point of irradiation for four minutes. Seven treatment sessions were performed with 48 hours between sessions. For analysis on the bone repair, qualitative and quantitative assessments of Ca and P contents were done by micro x-ray fluorescence spectroscopy (μXRF) and the morphological structure was carried out using Scanning Electron Microscopy (SEM). The results showed the efficiency of infrared LED therapy, because the amount of mineral components analyzed by μXRF and the morphological features of cortical and trabecular bones, demonstrated by the SEM images, showed enhanced bone repair in the irradiated groups when compared to their corresponding control groups at all stages.

Photodynamic Therapy (PDT) is a technique for destroying tumor cells with little harm to surrounding healthy tissue. However, the light wavelength has limited penetration in the tissue, making the association of a surgical procedure needed for larger lesions. Electrosurgery (ES) is a recommended excision technique, but the optical properties of the tissue damaged by ES and its influence on PDT procedure are unknown. Twelve rats (Wistar) composed the animal model of four groups (ES, PDT, ES+PS+Light, PS+ES+Light), evaluating different orders of conjugation via fluorescence, imaging and necrosis depth. First histopathological analysis has shown a highly modified surface of tissue (integral structure loss and dehydration shrinkage), protein denaturation, accompanied by bleeding and inflammatory damage. Fluorescence imaging showed strong scattering of light at the surface of modified tissue, which may cause higher losses of light on the surface. Fluorescence spectra showed different photosensitizer emissions for distinct operation modes. The different tissue composition can also induce changes on absorption and scattering properties, influencing the light penetration. The study showed significant necrosis formation beyond the limits of electrosurgery damage, making possible the conjugate use of ES and PDT.