This PDF file contains the front matter associated with SPIE Proceedings Volume 6435, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

The process of irreversible thermal denaturation of macromolecules involves cooperative bond breakage. Many bonds must break at the same time to allow denaturation. Hence, molecules are stabilized against thermal damage. However, these multiple bonds enforce a structural order on the macromolecule. A review of the literature on the entropy &Dgr;S (J/(mole K)) and enthalpy &Dgr;H (J/mole) of various endpoints of irreversible thermal denaturation (eg., whitening, contraction, loss of birefringence, necrosis, onset of heat shock proteins) indicate that the ratio &Dgr;S/&Dgr;H is constant at a value of 31.47x10-4 K-1, or 1/Tcrit where Tcrit ≈ 44.6°C. The free energy of denaturation is &Dgr;G = &Dgr;H - T&Dgr;S. At temperatures below Tcrit, more cooperative bonds yield more stability because &Dgr;H dominates over T&Dgr;S, but at temperatures above Tcrit more bonds yield less stable structure because T&Dgr;S dominates over &Dgr;H. Only one free parameter describes the kinetics of irreversible denaturation of macromolecules involving simultaneous breakage of multiple cooperative bonds, the &Dgr;H of the transition.

An assessment of skin damage caused by near-IR laser exposures is reported. The damage from two distinct laser-tissue temporal regimes is compared at two wavelengths (1.3 &mgr;m and 1.5 &mgr;m). Skin damage caused by thermal effects from single laser pulses is compared to damage caused by LIB (laser induced breakdown) using histological examinations. Modeling applications are explored to determine crossover points between thermal and photomechanical damage thresholds.

The Air Force Research Lab has developed a configurable, two-dimensional, thermal model to predict laser-tissue interactions, and to aid in predictive studies for safe exposure limits. The model employs a finite-difference, time-dependent method to solve the two-dimensional cylindrical heat equation (radial and axial) in a biological system construct. Tissues are represented as multi-layer structures, with optical and thermal properties defined for each layer, are homogeneous throughout the layer. Multiple methods for computing the source term for the heat equation have been implemented, including simple linear absorption definitions and full beam propagation through finite-difference methods. The model predicts the occurrence of thermal damage sustained by the tissue, and can also determine damage thresholds for total optical power delivered to the tissue. Currently, the surface boundary conditions incorporate energy loss through free convection, surface radiation, and evaporative cooling. Implementing these boundary conditions is critical for correctly calculating the surface temperature of the tissue, and, therefore, damage thresholds. We present an analysis of the interplay between surface boundary conditions, ambient conditions, and blood perfusion within tissues.

We give a closed-form expression for the temperature change induced by exposure to a continuous wave Gaussian laser under the assumption that no heat transfer occurs in the biological tissue. An explicit dependence of temperature change on fluence is established.

To properly assess skin damage caused by photonic exposure, the mechanisms of photon attenuation and subsequent heat production are investigated. Currently, voids exist in frequency specific electromagnetic properties such as the complex dielectric permittivity and conductivity necessary to define refractive index and attenuation values. We investigate these properties in several tissues such as blood, bone, skin, vitreous humor, cornea, retina and many others. Inside these tissues, exponential decrease in photon energy occurs due to attenuation. Because photon energy absorbed in tissue is expressed as heat in many instances, it follows that the dielectric properties of the material will also change as a function of the heating patterns as well as with frequency or wavelength. Conversely, changes in tissue thermal properties should change photon behavior as dispersion properties change. In our case we are concerned with existing data and theoretically determining dispersion properties over a large range of frequencies or wavelengths.

A scaling Monte Carlo method has been developed to quickly calculate diffuse reflectance spectra from multi-layered media for a wide range of optical properties. This method employs the photon trajectory information generated from a single baseline Monte Carlo simulation for a homogeneous medium to scale the exit distance and exit weight of photons for the new optical properties in multi-layered media. The method was tested for simulating diffuse reflectance from both a two-layered and a three-layered epithelial tissue model when using a flat-tip fiber-optic probe with variable source-detector separations. The scaling method can reduce computation time by more than two orders of magnitude compared to independent Monte Carlo simulations. This scaling method is particularly suited to simulating diffuse reflectance spectra or creating a Monte Carlo database to extract optical properties of layered media, which may potentially help spread the use of Monte Carlo modeling in the spectroscopy research of layered tissues.

Computational physics methods are described for the evaluation of the role of propagation with regard to laser damage to tissues. Regions of the optical spectrum, where linear and non-linear propagation affects irradiance distributions within tissues, are examined. Effects described include group-velocity dispersion, aberrations, thermal lensing, and self-focusing. Implications to exposure limits within safety standards, incorporating these irradiance-altering effects, are addressed such that inherent trends agree over wide temporal and spectral ranges, with damage thresholds measured experimentally. We present current regions of interest to the standard-setting community and recent works showing how propagation effects may be playing a key role in assessing damage thresholds.

To support refinement of the Maximum Permissible Exposure (MPE) safety limits, a series of experiments were conducted in vivo on Dutch Belted rabbit corneas to determine corneal minimum visible lesion thresholds for 2.0 &mgr;m continuous-wave laser irradiation. Single pulse radiant exposures were made at specified pulse durations of 0.1 sec, 0.25 sec, 0.5 sec, 1.0 sec, 2.0 sec and 4.0 seconds for spot 1/e² diameters of 1.17 mm and 4.02 mm. Lesions were placed in rows without overlap on rabbit cornea. The effect of each irradiation was evaluated within one minute post exposure and the final determination of lesion formation was made using a slit lamp one hour post exposure. Threshold lesions were defined as the presence of a superficial surface whitening one hour after irradiation. Probit analysis was conducted to estimate the dose for 50% probability (ED50) of laser-induced damage. Approximately 20 different radiant exposures were made for each exposure duration-spot size combination. At the threshold level, the diameters of barely visible opaque white lesions were smaller than the Gaussian 1/e² beam diameter. In selected survival animals, most of the threshold lesions were still visible 24 hours after exposure. The average lesion radius was approximately 0.4 ± 0.12 mm diameter for the 1.17 mm spot size and 1.0 ± 0.20 mm diameter for the 4.02 mm spot size. The exposure duration dependence of threshold average radiant exposure was described by an empirical power law equation: Threshold radiant exposure[J/cm2] = a x exposure duration[s] ^b, experimentally derived coefficient a was 9.79 and b was 0.669 for the 1.17 mm spot diameter; values of a and b were 4.57 and 0.456 respectively for the 4.02 spot diameter. Based on the experimental data and the empirical power law, the safety factors which were defined as threshold radiant exposure divided by MPE values were predicted for the 2.0 &mgr;m wavelength at various exposure durations and spot diameters. The minimum limit of the safety factor was approximately a factor of four for both 4.02 mm and 1.17 mm spot diameters. Due to the very sharp boundary and small uncertainties of damage threshold determination, it is suggesting that a factor of 4 "padding" is adequate and safety standard may not need to be changed.

A tissue phantom of water with an absorbing dye, Allura Red, was used to observe the effects of thermal lensing in a thick sample exposed to a CW 532 nm Verdi laser. A collimated beam was sent through a sample 2.9 cm thick. Results from the collimated beam revealed qualitative information about thermal lensing in a liquid media. The studies presented here argue the relevance of incorporating oscillating factors such as convectional flow into higher order thermal lensing models in a fluid such as water.

Because of the unique properties with regard to the absorption in organic tissue, pulsed Er:YAG laser has found most interest for various application in medicine, such as dermatology, dentistry, and cosmetic surgery. However, consensus regarding the optimal parameters for clinical use of this tool has not been reached. In this paper, the laser ablation characteristics of soft tissue by Er:YAG laser irradiation was studied. Porcine skin tissue in vitro was used in the experiment. Laser fluences ranged from 25mJ/mm² to 200mJ/mm², repetition rates was 5Hz, spot sizes on the tissue surface was 2mm. The ablation effects were assessed by the means of optical microscope, ablation diameters and depths were measured with reading microscope. It was shown that the ablation of soft biotissue by pulsed Er:YAG laser was a threshold process. With appropriate choice of irradiation parameters, high quality ablation with clean, sharp cuts following closely the spatial contour of the incident beam can be achieved. The curves of ablation crater diameter and depth versus laser fluence were obtained, then the ablation threshold and ablation yield were calculated subsequently, and the influence of the number of pulses fired into a crater on ablation crater depth was also discussed.

Drilling of bone and tooth tissue belongs to recurrent medical procedures (screw- and pin-bores, bores for implant inserting, trepanation etc.). Small round bores can be in general quickly produced with mechanical drills. Problems arise however by angled drilling, by the necessity to fulfill the drilling without damaging of sensitive soft tissue beneath the bone, or by the attempt to mill precisely noncircular small cavities. We present investigations on laser hard tissue "milling", which can be advantageous for solving these problems. The "milling" is done with a CO2 laser (10.6 &mgr;m) with pulse duration of 50 - 100 &mgr;s, combined with a PC-controlled galvanic beam scanner and with a fine water-spray, which helps to avoid thermal side-effects. The damaging of underlying soft tissue can be prevented through control of the optical or acoustical ablation signal. The ablation of hard tissue is accompanied with a strong glowing, which is absent during the laser beam action on soft tissue. The acoustic signals from the diverse tissue types exhibit distinct differences in the spectral composition. Also computer image analysis could be a useful tool to control the operation. Laser "milling" of noncircular cavities with 1 - 4 mm width and about 10 mm depth is particularly interesting for dental implantology. In ex-vivo investigations we found conditions for fast laser "milling" of the cavities without thermal damage and with minimal tapering. It included exploration of different filling patterns (concentric rings, crosshatch, parallel lines and their combinations), definition of maximal pulse duration, repetition rate and laser power, optimal position of the spray. The optimized results give evidences for the applicability of the CO2 laser for biologically tolerable "milling" of deep cavities in the hard tissue.

Our photo thermal reaction heating architecture balloon realizes less than 10 s short term heating that can soften vessel wall collagen without damaging surrounding tissue thermally. New thermal balloon angioplasty, photo-thermo dynamic balloon angioplasty (PTDBA) has experimentally shown sufficient opening with 2 atm low pressure dilation and prevention of chronic phase restenosis and acute phase thrombus in vivo. Even though PTDBA has high therapeutic potential, the most efficient heating condition is still under study, because relationship of treatment and thermal dose to vessel wall is not clarified yet. To study and set the most efficient heating condition, we have been working on establishment of temperature history estimation method from our previous experimental results. Heating target of PTDBA, collagen, thermally denatures following rate process. Denaturation is able to be quantified with measured collagen birefringence value. To express the denaturation with equation of rate process, the following ex vivo experiments were performed. Porcine extracted carotid artery was soaked in two different temperature saline baths to enforce constant temperature heating. Higher temperature bath was set to 40 to 80 degree Celsius and soaking duration was 5 to 40 s. Samples were observed by a polarizing microscope and a scanning electron microscope. The birefringence was measured by polarizing microscopic system using Brace-Koehler compensator 1/30 wavelength. The measured birefringence showed temperature dependency and quite fit with the rate process equation. We think vessel wall temperature is able to be estimated using the birefringence changes due to thermal denaturation.

In order to seal air leak from lung dissection plane in thoracotomy, we studied diode laser irradiation (wavelength: 810nm) with surface stain of indocyanine green (ICG, peak absorption wavelength: 805nm) ex vivo. In general, this air leak is sealed by suturing with weak tension and large margin of parenchyma. This suturing requires surgeon's skill and takes long time. Moreover, lung ventilatory performance is significantly impaired. Since laser tissue welding is novel method to adhere living tissue with thin thermally denatured attachment layer, we propose to seal the lung dissection plane with laser irradiation. Our aim of this study is to investigate the sealing mechanism as well as optimum condition to develop reliable laser sealing method for dissected lung plane in surgery, using practical laser-dye combination. Compartment of extracted porcine lung was prepared as a lung model, which volume was approximately 60cm^3. ICG solution (2.5mg/ml) was applied to the dissection plane of this lung model with 1minute. The diode laser (power density: 8-40W/cm^2) irradiated to the plane, moving the laser spot with constant speed (v=1mm/s). The heat degeneration depth and smoothness of the laser irradiated surface were observed by a microscope. When power density was over 24W/cm^2, heat degeneration depth was over 1.5E-4 m. There were no pin holes on the surface and the air leak seemed to be sealed completely. We also evaluated the air leak by endotracheal pressure. In the case of above condition, the heat degeneration depth was the same that of previous reported result with CO2 laser.

An absorption characteristic and a thermal relaxation time of a target biomedical tissue is an important parameter for development of low-invasive treatment that considers of interaction between biomedical tissue and laser. Laser irradiations with a wavelength corresponding to the absorption characteristics of tissue enable selective treatment. Furthermore, the thermal effect to tissue can be controlled at the laser irradiation time which depends on the laser pulse width and reception rate. A free electron laser (FEL) can continuously vary the wavelength in the mid-infrared region, has a unique pulse structure; the structure at the Institute of the Free Electron Laser (iFEL) consist of train of macropulses with a 15 &mgr;s pulse width, and each macropulse contained a train of 300-400 ultrashort micropulse with a 5 ps pulse width. In a previous report, we have proposed a novel laser treatment such as soft tissue cutting, dental treatment and laser angioplasty using the tenability of the FEL. To investigate the thermal effect to the biomedical tissue, we developed a FEL pulse control system using an acousto-optic modulator (AOM). The AOM commonly are used the Q-switch for the pulse laser generation, has a high pulse control efficiency and good operationally. The system can control the FEL macropulse width from 200 ns. This system should be a novel tool for investigating the interaction between the FEL and biomedical tissue. In this report, the interaction between FEL pulse width and biomedical tissue will be discussed.

The reduced scattering coefficient, &mgr;_s, was determined using oblique angle illumination and imaging backscattered light intensity. The distance &Dgr;r between the point of light incidence (hot-spot) and the circular symmetric diffuse reflectance centre, is ∼1/&mgr;_s^'. Previously, &Dgr;r was obtained analyzing a 1D strip aligned with the laser beam. We improved this method by calculating a 2D intensity image with extended dynamic range by assessing camera linearity, superimposing images with multiple integration times, and compensating for lens vignetting. The hot-spot algorithm utilises several images to minimize speckle variations and account for laser beam shape. Diffuse centre position is obtained by filtering the superimposed image with decreasing thresholds using momentum analysis to determine circular symmetry. The method was evaluated on 18 optical liquid phantoms with &mgr;_s&egr;[1.5, 3.0] mm^-1 and &mgr;_s&egr;[0.01, 0.16] mm^-1. The 2D method had better linearity with &mgr;_s and smaller variations due to more stable hot-spot detection, than the 1D method. The anisotropy factor g was obtained by fitting measured and Monte Carlo simulated spatially resolved intensity decays and verified with a laser Doppler flowmetry technique. With an optimal compensation for the &mgr;_a dependence, the rms error in &mgr;_s^' estimation was 2.9%.

The absorption coefficient, the scattering coefficient and the anisotropy factor of a highly scattering medium are determined using the diffuse reflectance of an obliquely incident beam of circularly polarized light. This approach determines both the anisotropy factor and the cutoff size parameter for the fractal continuous scattering medium such as biological tissue and tissue phantoms from depolarization of the backscattered light.

A study of in-vivo classification of pigmented skin lesions using oblique-incidence diffuse reflectance spectroscopy is presented. Spatio-spectral data in the wavelength range from 455 to 765 nm are collected from 111 pigmented lesions including 10 histopathologically diagnosed as melanoma. The first 67 lesions are used for training the classifiers, and 44 lesions are used for testing. The first classifier separates (1) malignant melanoma and severe dysplastic nevi from (2) moderate and mild dysplastic nevi, common nevi, actinic and seborrheic keratoses. The second classifier next distinguishes between (a) moderate and mild dysplastic nevi, common nevi from (b) actinic and seborrheic keratoses. The third classifier further separates (I) moderate and mild dysplastic nevi from (II) common nevi. The first classifier performs with 100% sensitivity and 91% specificity with overall classification rates of 93% and 95 % for the training and testing sets, respectively. The second classifier has classification rates of 95% and 97 % for the training and testing sets, respectively, whereas the third classifier has classification rates of 98% and 94 % for the training and testing sets, respectively.

Angular Domain Imaging (ADI) within highly scattering media employs micromachined angular filter tunnels to detect nonscattered photons which pass through the tunnels unattenuated while scattered photons collide with the tunnel walls. Each tunnel is micromachined approximately 51 &mgr;m wide by 10 mm long in silicon, giving a maximum acceptance angle of 0.29 degrees. The ADI technique is inherently independent of wavelength, and thus multispectral laser sources can be incorporated. Previous ADI experiments employed a 488-514 nm Argon ion laser source. This paper describes the construction of a new imaging system utilizing a high-power (up to 0.5 W) laser diode at the 670 nm wavelength, along with an aspheric and cylindrical lens system for shaping the beam into a collimated line of light. ADI results of biological samples (i.e. chicken breast tissue) are also presented. Image resolution is 204 &mgr;m or better in compressed chicken breast tissue approximately 3.8 mm in thickness. Digital image processing techniques are employed to improve image contrast, definition, and detectability of test structures. Because silicon is 40% reflective, scattered light at up to three times the acceptance angle is not sufficiently absorbed by the angular filter tunnels and contributes significant background noise, thus decreasing image contrast and detectability. Roughening of the tunnel surface using a NH4OH etchant solution scatters light hitting the walls, thus allowing it to be absorbed. Images after roughening show dramatic reductions in background scattered light levels between tunnels, suggesting that further experiments will make progress towards improved contrast and detectability of structures.

The reduced outflow rate caused by the increased resistance through trabecular meshwork (TM) has been thought to be the main reason for elevated intraocular pressure (IOP). It has been demonstrated that femtosecond laser pulses tuned to 1.7 μm wavelength can create the partial thickness channel in the sclera in ex vivo human eyes [1] and aqueous outflow can be increased by these channels in porcine eyes [2]. It was also shown that the outflow rate is reduced over time in ex vivo human eyes [3]. Therefore, the control experiment without laser treatment at the same condition was conducted and showed that outflow was reduced by 1.5 ± 0.8 μl/min at 15mmHg and 1.8 ± 1.0 μl/min at 25mmHg. However, the outflow rate increased by 0.26 μl/min at 15mmHg and 0.15 μl/min at 25mmHg after the partial thickness channel was created, meaning the amount of increased outflow rate might be more than measured considering the outflow reduction in control experiment. We suggest that the femtosecond laser created partial thickness channel can increase the outflow rate and delay the progression of glaucoma.

An ultrasound-based method to detect and characterize the laser-induced microbubbles was developed. This method is based on temporal measurement of passive acoustic emission from cavity during laser-tissue interaction and simultaneous active pulse-echo ultrasound probing of the cavitation bubble. These measurements were used to estimate the location of the nanosecond laser induced cavity and to monitor the spatial and temporal behavior of the microbubble. The measurements agreed with estimates derived from a well-known Rayleigh model of the cavity collapse. Overall, the studies indicate that the developed ultrasound technique can be used to detect and accurately measure laser-induced microbubbles in tissue.

A series of experiments in a new animal model for retinal damage, cynomolgus monkeys (Macaca fascicularis), have been conducted to determine the damage threshold for 12.5-nanosecond laser exposures at 1064 nm. These results provide a direct comparison to threshold values obtained in rhesus monkey (Macaca mulatta), which is the model historically used in establishing retinal maximum permissible exposure (MPE) limits. In this study, the irradiance level of a collimated Gaussian laser beam of 2.5 mm diameter at the cornea was randomly varied to produce a rectangular grid of exposures on the retina. Exposures sites were fundoscopically evaluated at post-irradiance intervals of 1 hour and 24 hours. Probit analysis was performed on dose-response data to obtain probability of response curves. The 50% probability of damage (ED50) values for 1 and 24 hours post-exposure are 28.5(22.7-38.4) &mgr;J and 17.0(12.9-21.8) &mgr;J, respectively. These values compare favorably to data obtained with the rhesus model, 28.7(22.3-39.3) &mgr;J and 19.1(13.6-24.4) &mgr;J, suggesting that the cynomolgus monkey may be a suitable replacement for rhesus monkey in photoacoustic minimum visible lesion threshold studies.

Pulsed, mid-infrared lasers can evoke neural activity from motor as well as sensory neurons in vivo. Lasers allow more selective spatial resolution of stimulation than the conventional electrical stimulation. To date, few studies have examined pulsed, mid-infrared neural stimulation and very little of the available optical parameter space has been studied. We found that pulse durations as short as 20 ?s elicit a compound action potential from the gerbil cochlea. Moreover, stimulation thresholds are not a function of absolute energy or absolute power deposited. Compound action potential peak-to-peak amplitude remained constant over extended periods of stimulation. Stimulation occurred up six hours continuously and up to 50 Hz in repetition rate. Single fiber experiments were made using repetition rates of up to 1 kHz. Action potentials occurred 2.5-4 ms after the laser pulse. Maximum rates of discharge were up to 250 action potentials per second. With increasing stimulation rate (300 Hz), the action potentials did not respond strictly after the light pulse. The results from these experiments are important for designing the next generation of neuroprostheses, specifically cochlear implants.

Microsecond pulsed laser systems, like the Thulium, Holmium and Erbium laser are being used for a broad range of medical applications in a liquid environment. Usually, the tissue ablation mechanism of these lasers is based on the instant formation of water vapor. When used with fiber delivery systems, the refraction of the beam coming out of the fiber will change the moment the liquid boundary turns to vapor. This dynamic change can be used in a controlled way but can also have adverse effects if not appreciated. In this study, the effect of the vapor phase change was investigated for various fiber shapes regarding optical and mechanical properties using high speed imaging and ray-trace simulation. Fiber tips of various shapes (bare, angled, tapered, ball shaped) were imaged with high-resolution using 1 &mgr;s light flashes in a video sequence of delay times from 1 to 2000 &mgr;s during exposure with pulsed 2.1 &mgr;m Holmium and pulsed 2.9 &mgr;m Erbium laser pulses. The tip was position in water or near a tissue surface. The dynamics of the explosive vapor bubble changed due to angle of refraction at the silica/vapor interface depending of the shape of the fiber tip. Ball shaped fibers form focused and highly divergent beams, angled fibers become side firing and tapered tips more concentrated. The observations are supported by ray-trace simulation. Clinically this mechanism can be used e.g. to create tiny side firing fibers in root channels of teeth. However, a damaged fiber tip may become unexpectedly side-firing resulting in adverse effects e.g. during lithotripsy. Ball-shaped fibers may be more resistant for damage due to impact with tissue. Using microsecond pulsed laser systems, the change in optical action of the fiber tip in liquid can influence the effectiveness and safety of the procedure.

A variety of effects is attributed to the photo stimulation of tissues, such as improved healing of ulcers, analgesic and anti-inflammatory effects, stimulation of the proliferation of cells of different origins and stimulation of bone repair. Some investigations that make qualitative evaluations, like wound healing and evaluation of pain and edema, can be conducted in human subjects. However, deeper investigations on the mechanisms of action of the light stimulus and other quantitative works that requires biopsies or destructive analysis has to be carried out in animal models or in cell cultures. In this work, we propose the use of planarians as a model to study laser-tissue interaction. Contrasting with cell cultures and unicellular organisms, planarians are among the simplest organism having tissue layers, central nerve system, digestive and excretory system that might have been platforms for the evolution of the complex and highly organized tissues and organs found in higher organisms. For the present study, 685 nm laser radiation was employed. Planarians were cut transversally, in a plane posterior to the auricles. The body fragments were left to regenerate and the proliferation dynamics of stem cells was studied by using histological analysis. Maximum cell count was obtained for the laser treated group at the 4^th experimental day. At that experimental time, we also had the largest difference between the irradiated and the non-irradiated control group. We concluded that the studied flatworm could be an interesting animal model for in vivo studies of laser-tissue interactions.

The non-invasive methods of treatments have been studying for the improvement of quality of life (QOL) of patients undergoing treatment. A photodynamic therapy (PDT) is one of the non-invasive treatments. PDT is the methods of treatment using combination of a laser and a photosensitizer. PDT has few risks for patients. Furthermore, PDT enables function preservation of a disease part. PDT has been used for early cancer till now, but in late years it is applied for age-related macular degeneration (AMD). AMD is one of the causes of vision loss in older people. However, PDT for AMD does not produce the best improvement in visual acuity. The skin photosensivity by an absorption characteristic of a photosensitizer is avoided. We examined new PDT using combination of an ultra-short pulsed laser and indocyanine green (ICG).

Bubble formation is a well identified phenomenon within short (ns) and ultrashort (fs) laser pulses-aqueous media interactions. Bubble formation might be produced by three different mechanisms: (1) optical breakdown, (2) rarefraction wave and (3) overheating of the material. Experiments where transparent and scattering tissue models that mimic biological tissue were irradiated with a Q-switched, 532 nm, 5 nanosecond, Nd:YAG and Ti:sapphire femtosecond laser systems. The type of bubble (transient or permanent) and initial bubble diameter were characterized as a function of time as well as the number of pulses and repetition rate at which they were delivered. Threshold fluence for bubble formation in scattering tissue model was also studied. Two types of bubbles were identified depending on the number of pulses and the repetition rate at which they were delivered: transient (type 1) and permanent (type 2) bubbles. There is an insignificant difference in the fluence required to form a bubble in transparent tissue models regardless of the depth at which the beam was focused; in contrast, for scattering tissue models, the fluence required to form a bubble in deep positions is significantly higher than that of more superficial beam focus positions.

In this paper we report on our combined measurements of the visible lesion thresholds for porcine skin for wavelengths in the infrared from 810 nm at 44 fs to 1318 nm at pulse durations of 50 ns and 350&mgr;s to 1540 nm including pulse durations of 31 ns and 600 &mgr;s. We also measure thresholds for various spot sizes from less than 1 mm to 5 mm in diameter. All three wavelengths and five pulse durations are used extensively in research and the military. We compare these minimum visible lesion thresholds with ANSI standards set for maximum permissible exposures in the infrared wavelengths. We have measured non-linear effects at the laser-tissue interface for pulse durations below 1&mgr;s and determined that damage at these short pulse durations are usually not thermal effects. Damage at the skin surface may include acoustical effects, laser ablation and/or low-density plasma effects, depending on the wavelength and pulse duration. Also the damage effects may be short-lived and disappear within a few days or may last for much longer time periods including permanent discolorations. For femtosecond pulses at 810 nm, damage was almost instant and at 1 hour had an ED50 of 8.2 mJ of pulse energy. After 24 hours, most of the lesions disappeared and the ED50 increased by almost a factor of 3 to 21.3 mJ. There was a similar trend for the 1.318 &mgr; laser for spot sizes of 2 mm and 5 mm where the ED50 was larger after 24 hours. However, for the 1.54 &mgr; laser with a spot size of 5 mm, the ED50 actually decreased by a small amount; from 6.3 Jcm-2 to 6.1 Jcm-2 after 24 hours. Thresholds also decreased for the 1314 nm laser at 350 &mgr;s for spot sizes of 0.7 mm and 1.3 mm diameter after 24 hours. Different results were obtained for the 1540 nm laser at 600 &mgr;s pulse durations where the ED50 decreased for spot sizes 1 mm and below, but increased slightly for the 5 mm diameter spot size from 6.4 Jcm-2 to 7.4 Jcm-2

Historically, safety analyses for radio frequency emission and optical laser exposures have been designed to define the threshold level for tissue damage. To date, no experimental studies have documented damage thresholds to living tissues in the terahertz (THz) range of electromagnetic frequencies (0.1 - 10 THz). Exposure limits exist as extrapolated estimates at the extreme bounds of current occupational safety standards for lasers and radio frequency sources. Therefore, due to the lack of published data on the safety of terahertz emissions, an understanding of the bioeffects of tissue exposures to terahertz beams is necessary. The terahertz frequency band represents an intermediate range in which both optical and radiofrequency methods of theory and experimentation can be selectively employed and compared for consistency. We report on work recently completed to reconcile the theoretical methods of optical and radio-frequency radiative transport modeling, while additionally discussing preliminary theoretical estimates of damage thresholds to skin tissue from terahertz energy and work planned to validate these findings experimentally.

Many lasers have claimed the clinical efficacy on skin rejuvenation. Systematic and comparative studies are needed to compare different laser effects and probe into the mechanism of laser skin rejuvenation. We performed this study to compare collagen remodeling with different laser effects on mice model in vivo. After depilation, the back skin of KM mice was used for the study. The 595nm pulsed dye laser (10ms), 1320nm Nd:YAG laser(350 ?s), 1064nm Q-switched (5ns) and long-pulsed Nd:YAG(0.3ms) lasers were applied based on optimal tissue reaction fluence test to irradiate one side of the mice back and leave the other side as the control. Then the collagen remodelling was evaluated at 0, 1, 7, 21, 30 and 60 days, with biophysical parameters' measurements, histological and biochemical examination. All lasers applied showed a statistical improvement in skin elasticity, dermal thickness and synthesis of hydroxyproline compared with their own controls. The Q-switched 1064-nm laser resulted in greater improvement of skin elasticity, dermal thickness, and higher synthesis of hydroxyproline than the other lasers after two months of treatments, while there was no significant difference among the 595nm, 1320nm and long-pulsed 1064nm lasers. Collagen type III increased markedly after the Q-switched 1064-nm laser treatment whereas more collagen type I was elicited by the 1320-nm laser.

Acute (1-hr, 6-hr) and longer term (24-hr) effects of laser injury on retinal function and cellular responses have been studied in the Great Plains rat snake, Elaphe guttata emoryi. This animal is of interest for vision research because its eye has an all-cone retina. A linear array of 5 thermal lesions was placed in the retina of anesthetized animals, near the area centralis, using a Nd:VO₄ laser (532 nm), that delivered 50 mW per 10-msec pulse. Retinal function was assessed with the pattern electroretinogram (PERG), recorded before and after the placement of the lesions. PERGs were elicited with counterphased square-wave gratings, and were analyzed by Fourier analysis. The fate of lesioned cells was assessed by immunohistological staining for the transcription factor, NF-&kgr;B (which is activated by ionizing and nonionizing radiation), as well as for the apoptosis marker, caspase-9. The normal snake PERG had the maximum, real amplitude frequency component, determined by Fourier analysis, at the reversal frequency of the grating (i.e. shifts/sec). In the hour following the lesion-producing laser exposures, the PERG response exhibited frequency doubling, i.e. a new response waveform appeared at twice the reversal frequency. By 24-hr post exposure, many lesioned photoreceptors stained positively for both NF-&kgr;B and caspase 9. Because the PERG largely reflects retinal ganglion cell activity, the appearance of frequency doubling in the PERG suggests that complementary (push-pull) inputs to ganglion cells are disrupted by the laser lesions. The immunohistological results indicate that activation of NF- B is not necessarily associated with photoreceptor survival after a laser injury.

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

We delivered a therapeutic gene, hepatocyte growth factor (HGF), to skin grafts of rats using laser-induced stress waves (LISWs) with the objective of enhancing their adhesion. The density and uniformity of neovascularities were enhanced significantly in the grafted skins that were transfected using LISWs, suggesting the efficacy of this method to improve the outcome of skin transplantation.

Ocular laser exposures resulting in damage at the retina typically involve cellular alterations in the retinal pigment epithelial (RPE) layer. To provide guidelines for eye-safe exposure to lasers, the laser safety community has relied on damage assessment in nonhuman primate studies. Simple and reliable model systems for laser bioeffects that use cultured RPE cells, rather than animals, are thus desirable. We have characterized our artificially pigmented hTERT-RPE1 model by identifying ED₅₀ thresholds over a wide range of laser parameters and cell culture conditions. When summarized as action spectra and temporal action profiles (log threshold fluence versus log exposure duration), trends (pigment-dependent) in our cell model data are strikingly similar to the threshold trends reported for animal models (literature). In addition, the rapidity and flexibility (laser delivery) with which studies are performed in our culture model has benefited computational modeling efforts.

Although various methods for gene transfer have been investigated, a practical gene delivery system that fulfills the requirements for clinical application has not yet been developed. Gene transfer by the use of laser-induced stress waves (LISWs) is a physical method to facilitate gene transfer into cells with the effect of stress waves generated by irradiation an absorbing material with high-intensity laser pulses. This method has high spatial controllability and a potential for catheter-based gene transfer. We demonstrated selective high transfection efficiency in vivo. However, there remains a problem that transfection efficiency is limited to less than several percent in vitro. Thus we attempted to improve transfection efficiency by using plasmid DNA modified with cationic-liposome. Plasmid DNA coding for enhanced green fluorescent protein (EGFP) had been modified with Lipofectamine and it was added to a dish for NIH3T3 cell culture. A black rubber disk was placed on the upper side of the cells; the disk was irradiated with 532 nm, nanosecond laser pulses (spot diameter, 3 mm; fluence, 1.3 J/cm2; number of pulses, 20). 24 hours after application of LISWs, transfection efficiency was evaluated with a fluorescence microscope, where efficiency was defined as the ratio of the number of cells emitting fluorescence to the total number of cells. At a DNA concentration of 7.8 &mgr;g/ml, transfection efficiency with naked plasmid DNA was as low as 0.05%, while it was increased to 23.7% by using plasmid DNA modified with Lipofectamine. Since both of the naked plasmid DNA and cell membranes have negative charge, plasmid DNA concentration around cells should be low. Since DNA-Lipofectamine complexes carry positive charge, density of plasmids existing around cells should be increased, resulting in much improved transfection efficiency.

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