This PDF file contains the front matter associated with SPIE Proceedings Volume 8579, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Interaction of subsequent laser pulses becomes important relevant with the use of high-repetition rate fs-laser systems for ophthalmic laser surgery. Therefore, we investigated the interaction of temporally separated laser pulses in water by time-resolved photography. With decreasing temporal separation of pulses the probability of laser-induced optical breakdown (LIOB) is firstly diminished by disturbed focusing into persisting gas bubbles. Finally, LIOB is totally impaired by the expanding or collapsing cavitation of the preceding pulse. Hence, laser-tissue interaction might be accompanied by a raised laser energy transmission. In conclusion, these results are of great interest for the prospective optimization of the ophthalmic surgical process with modern fs-lasers.

Nonlinear absorption of femtosecond laser pulses enables the induction of bubble cavities in the interior of eye cornea without affecting other parts of an eye, a phenomena utilized for flap formation in laser assisted corneal surgery. In the present study laser pulses were focused in the interior of the sections of bovine cornea. Tight focus of the laser pulses results in the plasma formation followed by its explosive expansion, which drives cavity formation. The morphology of the generated features as well as the nature of the physical mechanisms of the phenomenon as a function of process parameters is discussed. Numerical model is proposed to develop predictive capabilities for the feature size and shape and the results are compared against the experimental findings.

Infrared nerve stimulation (INS) is rapidly becoming an important tool for basic research and a promising new clinical technology to selectively activate nerves to restore function, map the nervous system, and perform diagnostic procedures. To the best of our understanding, the mechanism of stimulation is photothermal; thus, describing the laserinduced heat distribution is fundamental to determining the relationship between stimulation pulse and neural response. This work develops both a framework describing the time evolution of the heat distribution induced by optical fluence and a novel method to extract thermal criteria for neural activation. We are first concerned with the general problem of describing the temperature distribution in a homogenous medium. To this end, we determine a Green’s function solution to the heat diffusion equation and convolve it with the optical fluence. This provides a general solution to the thermal problem of interest in the form of a single integral over time. Other useful closed form solutions can be determined for interesting special cases. This pursuit also yields an expression for the thermal relaxation time, which provides a rigorous description of thermal confinement for INS applications. The insight we gain from this framework allows us to extract thermal criteria for neural activation from experimental data. Our work provides both insight into the mechanism for stimulation and understanding sufficient to aid in the development of INS devices. Thermal criteria values will prove useful for choosing parameters such as spot size, pulse width, stimulation spacing, and stimulation depth in future INS applications.

Infrared neural stimulation (INS) is a novel technique for stimulating neurons with infrared light, rather than the traditional electrical means. There has been significant discussion in the literature on the mechanisms behind INS, while recent work has shown that infrared light stimulates neurons by causing a reversible change in their membrane capacitance. Nevertheless, the effect of different laser parameters on neuronal responses is still not well understood. To better understand this and to assist in designing light delivery systems, modelling of spatial and temporal characteristics of light delivery during INS has been performed. Monte Carlo modelling of photon transport in tissue allows the spatial characteristics of light to be determined during INS and allows comparisons of varying geometries and fibre designs. Finite element analysis of heat conduction can then be used to reveal the behavior of different pulse durations and the resulting temperature decay. The combination of the two methods allows for further insights into the mechanisms of INS and assists in understanding different mechanisms which promote INS. The model suggests there may be two regimes of INS, namely temperature limited for pulses under 100 μs and temperature gradient limited for longer pulses. this is compatible with previously published data, but requires further experimentation for confirmation. The model also provides a tool for optimising the design of emitters and implants.

A computer-based model has been built that simulates the response of the retinal pigmented epithelial (RPE) cell to laser exposure in the photochemical (non-thermal) damage exposure range (≥ 100 s exposures). The modeling approach used is knowledge-based, modular, and hierarchical, allowing the explicit modeling of the cascades of intracellular events in response to laser application. Thus, the model can be used to both analyze existing in vitro data sets, as well as efficiently direct sampling strategies for future in vitro and in vivo studies. This model has been validated using laboratory data from several studies reported in the literature using blue light (413 nm and 458 nm) lasers with 100 s, 200 s, and 3600 s exposure durations. The model was able to predict the in vitro ED50 response curve from these studies, as well as the results for which we have no in vitro data (extrapolated based on irradiance reciprocity), within 1-6% for the shorter duration exposures. Based on exploration of this computer model using lethal vs. non-lethal laser exposure scenarios, the RPE cell’s oxidative stress response differs quantitatively very little with respect to typical oxidative stress sources such as superoxide and hydrogen peroxide. However, in the lethal exposure scenarios the model points to a potential tipping point in the oxidative stress response of the mitochondrial-based cellular energetics. Further studies are underway to explore issues related to the levels of ATP/ADP and GSH/GSSG that are predicted by the model in these lethal vs. non-lethal exposure scenarios.

As-synthesized, poly(4-styrenesulfonic acid) (PSS)-coated and SiO₂ coated gold nanorods were taken up by NG108-15 neuronal cells. Exposure to laser light at the plasmon resonance wavelength of gold nanorods was found to trigger the differentiation process in the nanoparticle treated cells. Results were assessed by measuring the maximum neurite length, the number of neurites per neuron and the percentage of neurons with neurites. When the intracellular Ca²⁺ signaling was monitored, evidence of photo-generated transients were recorded without altering other normal cell functions. These results open new opportunities for peripheral nerve regeneration treatments and for the process of infrared nerve stimulation.

To quantitatively investigate photosensitization reaction in vitro against myocardial cells with photosensitizer rich condition in solution using Talaporfin sodium in the well of a 96 well plate, we studied photosensitization reaction progress in this well. We have proposed non-thermal conduction block of myocardium tissue using the photosensitization reaction with laser irradiation shortly after Talaporfin sodium injection. In above situation, the photosensitizer is located outside the myocardial cells in high concentration. To understand interaction of the photosensitization reaction in which the photosensitizer distributes outside cells, the photosensitization reaction progress in the well was studied. Talaporfin sodium (799.69 MW) solution and a 663 nm diode laser were used. The photosensitizer solution concentrations of 12.5-37.5 μM were employed. The photosensitizer fluorescence with 0.29 W/cm² in irradiance, which was optimized in previous cell death study, was measured during the laser irradiation until 40 J/cm². The photosensitizer solution absorbance and dissolved oxygen pressure after the laser irradiation were also measured. We found that the photosensitization reaction progress had 2 distinctive phases of different reaction rate: rapid photosensitization reaction consuming dissolved oxygen and gentle photosensitization reaction with oxygen diffusion from the solution-air boundary. The dissolved oxygen pressure and photosensitizer solution absorbance were 30% and 80% of the initial values after the laser irradiation, respectively. Therefore, oxygen was rate-controlling factor of the photosensitization reaction in the well with the photosensitizer rich condition. In the oxygen diffusion phase, the oxygen pressure was maintained around 40 mmHg until the laser irradiation of 40 J/cm² and it is similar to that of myocardium tissue in vivo. We think that our 96 well plate in vitro system may simulate PDT in myocardial tissue with photosensitization reaction parameters mentioned above.

Oxidative stress (OS) is increasingly implicated as an underlying pathogenic mechanism in a wide range of diseases, resulting from an imbalance between the production of reactive oxygen species (ROS) and the system's ability to detoxify the reactive intermediates or repair the resulting damage. ROS can be difficult to detect directly; however, they can be detected indirectly from the effects on oxidative stress biomarkers (OSB), such as glutathione (GSH), 3-nitrotyrosine, homocysteine, and cysteine. Moreover the reaction of transition metals with thiol-containing amino acids (for example GSH) oxidized by ROS can yield reactive products that accumulate with time and contribute to aging and diseases. The study of the interaction between OSB using functionalized nanoparticles (fNPs) has attracted interest because of potential applications in bio-sensors and biomedical diagnostics. A goal of the present work is to use fNPs to detect and ultimately quantitate OS in retinal pigment epithelial (RPE) cells subjected to external stressors, e.g. nonionizing (light) and ionizing (gamma) radiation. Specifically, we are investigating the assembly of gold fNPs mediated by the oxidation of GSH in irradiated RPE cells. The dynamic interparticle interactions had been characterized in previously reported work by monitoring the evolution of the surface plasmon resonance band using spectroscopic analysis (UV-VIS absorption). Here we are comparing the dynamic evolution of fNP assembly using photoacoustic spectroscopy (PAS). We expect that PAS will provide a more sensitive measure allowing these fNP sensors to measure OS in cell-based models without the artifacts limiting the use of current methods, such as fluorescent indicators.

The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

Irradiation of porphyrins bound to proteins applies to photodynamic cancer therapy, photo-reduction of water, and the possibility of modifying proteins to impart new functions. Upon binding Protoporphyrin IX (PPIX) and hemin, respectively, to human albumin (HSA), the bound products’ response to low-dose irradiation at pH 7.4 is examined in this study. Spectroscopic data suggests that irradiation of PPIX when bound to HSA causes small secondary and tertiary protein conformational changes. Alternately, sizeable alterations are not seen when hemin bound to HSA is irradiated. This difference indicates a different photophysical mechanism for PPIX than for hemin.

Photodynamic therapy (PDT) is an alternative antimicrobial treatment method. Different wavelengths of light sources mostly in the visible spectrum have been investigated for antimicrobial Photodynamic Therapy. Even though the wavelengths in near infrared spectrum have the advantage of higher penetration capability in biological tissue, they have not been preferred for PDT because of their possible photothermal effect in biological tissues. In our previous studies, the desired PDT effect was achieved with 809-nm diode laser and indocyanine green (ICG) on drug resistant pathogens. In this study, it was aimed to investigate the influence of different output powers during PDT applications with 809-nm diode laser to clarify whether there is a photothermal effect to kill the pathogens or only the photochemical effect of photodynamic therapy. 4 different output powers (500 mW, 745 mW, 1000 mW, 1500 mW) were examined in Laseronly and PDT groups of P. aeruginosa ATCC 27853 in vitro. In the PDT groups, a non-phototoxic ICG concentration (50 μl/ml) has been chosen to eliminate the toxic effect of ICG and evaluate only the thermal effect of laser. Applied energy dose (252 J/cm²) was kept constant by increasing the exposure duration (300, 240, 180 and 120 seconds respectively). These output powers in Laser-only or PDT groups did not seem to cause photothermal effect. There was not any significant decrease or increase on bacterial load after the applications with different output powers. Higher output powers in PDT groups with the same ICG concentration did not cause any higher killing effect.

The present work discusses effect of infrared (IR) femtosecond laser irradiation on neoplasm of white mice with experimental cervical cancer- 5 (CC-5 on the 20th and 30th days after tumor transplantation). Tumor tissue was irradiated by femtosecond erbium doped fiber laser: the wavelength is 1.55 μm, average and peak powers are1,25 mW and 6kW, respectively, irradiation trials n=10. The average energy density (energy dose) on a tissue for two groups of animals was 0,24 J/cm² and 0,36 J/cm² for a single trial. Irradiation was followed by biochemical determination of LPO AOS parameters (“Lipid peroxidation-antioxidants” system): malondialdehyde (MDA), activity of superoxide dismutase (SOD), catalase and glutathione-reductase (GR), glutathione-S-transferase (GST). A subsequent morphological study of tumor tissue was performed. Mathematical analysis of data demonstrates a weak dependence of the studied parameters on energy dose. The latter implies the trigger effect of IR femtosecond laser irradiation on redox-dependent processes in neoplasm at experimental cervical cancer.

With the recent development in the field of micro displays, retinal display and emerging technologies the issue of laser safety of these new devices becomes more and more important. To tackle these problems a lot of basic research will be necessary in order to find appropriate laser safety standards, since the current standards are not fully suitable. In order to avoid animal experiments as far as possible and also aiming for a simulation tool which would assist the manufacturers in their safety considerations, we have developed a thermodynamic model of the whole human eye. Using the software Hypermesh© and Ansys Fluent© we created a Finite-Volume-Method model capable of simulating the behavior of all parts of the eye. I.e. the temperature distribution at any point of the eye can be predicted over time. The model also includes the blood flow within the choroid aiming for a realistic thermal behavior.

In recent years, several studies have been investigating the impact of thermal lensing in ocular media on the visual function. These studies have shown that when near-infrared (NIR) laser energy (1319 nm) is introduced to a human eye, the heating of the eye can be sufficient to alter the index of refraction of the media leading to transient changes in the visible wavefront through an effect known as thermal lensing, while remaining at a safe level. One of the main limitations of experimentation with human subjects, however, is the reliance on a subject’s description of the effect, which can vary greatly between individuals. Therefore, a computational model was needed that could accurately represent the changes of an image as a function of changes in the index of refraction. First, to model changes in the index of refraction throughout the eye, a computational thermal propagation model was used. These data were used to generate a comprehensive ray tracing model of the human eye using Zemax ( Radiant Zemax Inc, Redmond WA) via a gradient lens surface. Using this model, several different targets have been analyzed which made it possible to calculate real-world visual acuity so that the effect of changes in the index of refraction could be related back to changes in the image of a visual scene.

In oral and maxillofacial surgery, there is a need for tissue engineered constructs for dental implants, reconstructions due to trauma, oral cancer or congenital defects. A non-invasive quality monitoring of the fabrication of tissue engineered constructs during their production and implantation is a required component of any successful tissue engineering technique. We demonstrate the design and application of a Raman spectroscopic probe for rapid and noninvasive monitoring of Ex Vivo Produced Oral Mucosa Equivalent constructs (EVPOMEs). We conducted in vivo studies to identify Raman spectroscopic failure indicators for EVPOMEs (already developed in vitro), and found that Raman spectra of EVPOMEs exposed to thermal stress showed correlation of the band height ratio of CH₂ deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. This is the first step towards the ultimate goal to design a stand-alone system, which will be usable in a clinical setting, as the data processing and analysis will be performed with minimal user intervention, based on already established and tested Raman spectroscopic indicators for EVPOMEs.

Angular domain spectroscopic imaging (ADSI) is a hyperspectral imaging technology that combines both optical spectroscopy and optical imaging into a single platform. The technique employs an array of micro-channels to perform angular filtration, whereby quasi-ballistic photons traversing a turbid sample are accepted, and scattered photons (imagedegrading) are rejected. The aim of the work reported here was to evaluate the effectiveness of an ADSI system at identifying targets buried within tissue-mimicking phantoms. Principal component analysis (PCA) was applied to spectral data-cubes to extract the main spectral features from the images. Targets of various absorption levels (indocyanine green), depths beneath the phantom surface, and background scattering levels were evaluated. Principal components were analyzed with k-means clustering. The extracted features were grouped and classified. Then, the sensitivity and specificity of the ADSI system were estimated. Angular domain spectroscopic imaging with PCA provided clear separation of targets of different absorber concentration and depth. The results led us to conclude that the technique holds potential for characterizing tissue specimens obtained during surgery.

Frequency-modulated light scattering interferometry, which employs a frequency-modulated coherent light source and examines the intensity fluctuation of the resulting scattered light using a heterodyne detection scheme, was utilized to evaluate the optical properties of liquid phantoms made of Intralipid® and Indian ink. Based on the diffusion theory, nonlinear fits to the power spectrum of the heterodyne-detected light intensity are performed and discussed in detail, and the optical properties of liquid phantoms are consequently retrieved.

Clinician’s recommendations on wheelchair pressure reliefs in the context of the high prevalence of pressure ulcers that occur in people with spinal cord injury is not supported by strong experimental evidence. Some data indicates that altered tissue perfusion and oxygenation occurring under pressure loads, such as during sitting, induce various pathophysiologic changes that may lead to pressure ulcers. Pressure causes a cascade of responses, including initial tissue hypoxia, which leads to ischemia, vascular leakage, tissue acidification, compensatory angiogenesis, thrombosis, and hyperemia, all of which may lead to tissue damage. We have developed an advanced skin sensor that allows measurement of oxygenation in addition to perfusion, and can be safely used during sitting. The sensor consists of a set of fiber optics probes, spectroscopic and Laser Doppler techniques that are used to obtain parameters of interest. The overriding goal of this project is to develop the evidence base for clinical recommendations on pressure reliefs. In this paper we will illustrate the experimental apparatus as well as some preliminary results of a small clinical trial conducted at the National Rehabilitation Hospital.

Mild hypothermia (HT), in which the brain is cooled to 32-33°C, has been shown to be neuroprotective for neurological emergencies such as head trauma and neonatal asphyxia. Xenon (Xe), a scarce and expensive anesthetic gas, has also shown great promise as a neuroprotectant, particularly when combined with HT. The purpose of the present study was to investigate the combined effect of Xe and HT on the cerebral metabolic rate of oxygen (CMRO₂) and cerebral blood flow (CBF). A closed circuit re-breathing system was used to deliver the Xe in order to make the treatment efficient and economical. A bolus-tracking method using indocyanine green (ICG) as a flow tracer with time-resolved near-infrared (TR-NIR) technique was used to measure CBF and CMRO₂ in newborn piglets.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is an attractive method because of the shorter decay time of photosensitivity compared with PDT using other drugs. However, the optimum conditions to perform ALA-PDT, e.g., drug dose, wavelength, and irradiation dose have never been clarified. To evaluate the effectiveness of PDT using ALA and its dependence on drug dose, wavelength, and irradiation dose in the treatment of tumors, the usefulness of a tumor model prepared with tumor cells grown on the chorioallantoic membrane of chicken eggs was studied by measuring the optical properties of the tumor model. The optical properties of tumor model were measured with a double integrating sphere optical setup and inverse Monte Carlo technique in the wavelength range from 350 to 1000 nm. The spectra of absorption and reduced scattering coefficients of the tumor model grown in the chicken eggs were compared with those of the other tumor model grown in mice. The measured optical properties of the tumor model using chicken eggs were similar to those of the tumor model using mice. These results indicate that the tumor model using chicken eggs is a suitable system to investigate the effectiveness of ALA-PDT. This in vivo assay system for tumors has advantages for evaluating antitumor effect of ALA-PDT because of its convenience, rapidity, and inexpensiveness.

Angular Domain Imaging (ADI) is a technique that is capable of generating two dimensional shadowgrams of attenuating targets embedded in a scattering medium. In ADI, an angular filter array (AFA) is positioned between the sample and the detector to distinguish between quasi-ballistic photons and scattered photons. An AFA is a series of micro-channels with a high aspect ratio. Previous AFAs from our group were constructed by micro-machining the micro-channels into a silicon wafer, limiting the imaging area to a one dimensional line. Two dimensional images were acquired via scanning. The objective of this work was to extend the AFA design to two dimensions to allow for two dimensional imaging with minimal scanning. The second objective of this work was to perform an initial characterization of the imaging capabilities of the 2D AFA. Our approach was to use rapid 3D prototyping techniques to generate an array of micro-channels. The imaging capabilities were then evaluated by imaging a 0.9 mm graphite rod submerged in a scattering media. Contrast was observed to improve when a second angular filter array was placed in front of the sample to mask the incoming light.

In customary implementation of three-dimensional (3D) Monte Carlo (MC) numerical model of light transport in heterogeneous biological structures, the volume of interest is divided into voxels by a rectangular spatial grid. Each voxel is assumed to have homogeneous optical properties and curved boundaries between neighboring tissues inevitably become serrated. This raises some concerns over realism of the modeling results, especially with regard to reflection and refraction on such boundaries. In order to investigate the above concern, we have implemented an augmented 3D MC code, where tissue boundaries (e.g., blood vessel walls) are defined by analytical functions and thus maintain their shape regardless of grid discretization. Results of the customary and augmented model are compared for a few characteristic test geometries, mimicking a cutaneous blood vessel irradiated with a 532 nm laser beam of finite diameter. Our analysis shows that at specific locations inside the vessel, the amount of deposited laser energy can vary between the two models by up to 10%. Even physically relevant integral quantities, such as linear density of the energy absorbed by the vessel, can differ by as much as 30%. Moreover, the values obtained with the customary model vary strongly with discretization step and don’t disappear with ever finer discretization. Meanwhile, our augmented model shows no such behavior, indicating that the customary approach suffers from inherent inaccuracies arising from physically flawed treatment of tissue boundaries.

A method for non-destructive determination of absorption and transport (reduced) scattering coefficients of turbid media (biological tissue, first of all) is presented. It refers to the spatially resolved diffuse reflectance techniques with optical fiber probe. The method is based on a more accurate (in comparison with diffusion) two parameters kinetic light propagation model and a special two step non-analog Monte Carlo technique, it involves no additional parameters and uses no assumptions about spectral dependencies of the coefficients, allows application of monochrome sources and probes with minimally possible number of reading fibers (only 2), goes without calibration phantoms and measurements. Numerical and experimental testing have showed the measured coefficients provide a good prediction of both light reflection and penetration fields in semi-infinite homogeneous media with low-mid absorption.

Angular Domain Imaging (ADI) employs an angular filter to distinguish between quasi-ballistic and scattered photons based on trajectory. A 2D angular filter array was constructed using 3D printing technology to generate an array of micro-channels 500 μm x 500 μm with a length of 12 cm. The main barrier to 2D imaging with the 2D angular filter array was the shadows cast on the image by the 500 μm walls of the angular filter. The objective of this work was to perform a resolution analysis of the 2D angular filter array. The approach was to position the AFA with a two dimensional positioning stage to obtain images of areas normally obstructed by the walls of the AFA. A digital light processor was also incorporated to generate various light patterns to improve the contrast of the images. A resolution analysis was completed by imaging a knife edge submerged in various uniform scattering media (Intralipid® dilutions with water). The edge response functions obtained were then used to compute the line spread function and the theoretical resolution of the imaging system. The theoretical system resolution was measured to be between 110 μm - 180 μm when the scattering level was at or below 0.7% Intralipid®. The theoretical resolution was in agreement with a previous resolution analysis of a silicon-based angular filter with a similar aspect ratio. The measured resolution was also found to be smaller than the size of an individual channel, suggesting that the resolution of an AFA based ADI system is not dependent on the size of the micro-channel.