PDT may be an effective treatment for certain immune-mediated disorders. The immunomodulatory action of PDT is likely a consequence of effects exerted at a number of levels including stimulation of specific cell signaling pathways, selective depletion of activated immune cells, alteration of receptor expression by immune and non-immune cells, and the modulation of cytokine availability. QLT0074, a potent photosensitizer that exhibits rapid clearance kinetics in vivo, is in development for the treatment of immune disorders. In comparison to the well-characterized and structurally related photosensitizer verteporfin, lower concentrations of QLT0074 were required to induce apoptosis in human blood T cells and keratinocytes using blue light for photoactivation. Both photosensitizers triggered the stress activated protein kinase (SAPK) and p38 (HOG1) pathways but not extracellularly regulated kinase (ERK) activity in mouse Pam212 keratinocytes. In cell signaling responses, QLT0074 was active at lower concentrations than verteporfin. For all in vitro test systems, the stronger photodynamic activity of QLT0074 was associated with a greater cell uptake of this photosensitize than verteporfin. In mouse immune models, sub-erythemogenic doses of QLT0074 in combination with whole body blue light irradiation inhibited the contact hypersensitivity response and limited the development of adjuvant-induced arthritis. QLT0074 exhibits activities that indicate it may be a favorable agent for the photodynamic treatment of human immune disease.

Due to inflammatory/immune responses elicited by photodynamic therapy (PDT), this modality is particularly suitable in combination with various forms of immunotherapy for an improved therapeutic gain. A wide variety of approaches that may be applicable in this context include those focusing on amplifying the activity of particular immune cell types (neutrophils, macrophages, dendritic cells, natural killer cells, helper or cytotoxic T lymphocytes). Another type of approach is to focus on a specific phase of immune response development, which comprises the activation of non-specific inflammatory immune effectors, immune recognition, immune memory, immune rejection, or blocking of immune suppression. These different strategies call for a variety of immunotherapeutic protocols to be employed in combination with PDT. These include treatments such as: (1) non-specific immunoactivators (e.g. bacterial vaccines), (2) specific immune agents (cytokines, or other activating factors), (3) adoptive immunotherapy treatments (transfer of dendritic cells, tumor-sensitized T lymphocytes or natural killer cells), or (4) their combinations. Techniques of gene therapy employed in some of these protocols offer novel opportunities for securing a potent and persistent immune activity. Using PDT and immunotherapy represents an attractive combination for cancer therapy that is capable of eradicating both localized and disseminated malignant lesions.

The ideal cancer treatment modalities should not only cause tumor regression and eradication but also induce a systemic anti-tumor immunity. This is essential for control of metastatic tumors and for long-term tumor resistance. Laser immunotherapy using a laser, a laser-absorbing dye and an immunoadjuvant has induced such a long-term immunity in treatment of a mammary metastatic tumor. The successfully treated rats established total resistance to multiple subsequent tumor challenges. For further mechanistic studies of the antitumor immunity induced by this novel treatment modality, passive adoptive transfer was performed using splenocytes as immune cells. The spleen cells harvested from successfully treated tumor-bearing rats provided 100% immunity in the naive recipients. The passively protected first cohort rats were immune to tumor challenge with an increased tumor dose; their splenocytes also prevented the establishment of tumor in the second cohort of naive recipient rats. This immunity transfer was accomplished without the usually required T-cell suppression in recipients.

Photodynamic therapy can provide a reliable method of tumor destruction when the appropriate dosimetry is applied. Current dosimetry practice involves quantification of the drug and light doses applied to the tumor, but it would be desirable to monitor in vivo light and drug levels to provide the most accurate determination of dosimetry. In vivo measurements can be used to minimize variations in treatment response due to inter-animal variability, by providing animal-specific or patient-specific treatment planning. This study reports on the development of a micro-sampling method to measure fluorescence from tissue, which is not significantly affected by the tissue optical properties. The system measures fluorescence from the surface of a tissue, using a fiber bundle composed of individual 100 micron fibers which ar all spaced apart by 700 microns from one another at the tissue contact end. This design provides sampling of the fluorescence at multiple sites to increase the signal intensity, while maintaining a micro- sampling of the tissue volume just below the surface. The calibration studies here indicate that the 1/e sampling depth is near 60 microns when measured in optical phantoms, which are similar to typical tissue properties. The probe fluorescence signal is independent of blood concentration up to a maximum of 10% blood by volume, which is similar to most tumor tissue. Animal tests indicate that the sensitivity to drug concentration is essentially the same in when measured in murine liver and muscle tissues, both in vivo and ex vivo. These preliminary calibration results suggest that the probe can be used to measure photosensitizer uptake in vivo non- invasively and rapidly via conversion of fluorescence intensity to photosensitizer concentration.

In rats, the Harderian Gland secret Protoporphirin IX which is retained at acinar lumina. Since this photosensitizer is important for PDT of malignant tumors, we propose to study this gland as a model to help understanding PDT with endogenous photosensitizers. Twenty Wistar SPF adult rats were submitted to surgical exposure of both Harderian glands, revealing red fluorescence upon UV, characterizing the protoporphirin IX presence. After that, one gland of each pair (one kept as control) was irradiated with an 8 mW HeNe (6328 angstrom) for 45 minutes, delivering about 2.7 joules/mm². After 24 hours a group of 10 animals were scarified and the glands removed for histological analysis. The remaining animals were subjected to the same procedure but the glands were removed immediately after laser treatment. Histological and fluorescence analysis immediately after laser irradiation showed cell fragmentation with loss of acinar architecture with diffusion of protoporphirin in the cytoplasm of damaged cells, as well as interstitial edema. After 24 hours these alterations were more pronounced with accentuated loss of intraluminal protoporphirin and beginning of leukocytic demarcation of necrotic areas. The innate Harderian glands of rats, exposed to HeNe laser, showed a similar behavior as tumor tissue under PDT.

Infrared cameras have been used to monitor the thermal response of tissue to pulsed and continuous wave laser irradiation. A computer model has been developed previously to predict radiometric temperature estimations and demonstrate potential discrepancies between surface and radiometric temperatures. To quantitatively verify the modeling, experiments were performed in which gelatin phantoms (approximately 98% water) were irradiated with low-radiant- exposure (e.g. subablative) CO₂ laser pulses. Radiometric temperatures were estimated using a 3 - 5 micrometer band- limited thermal camera and compared to computer model predictions of the measured temperatures. By fitting model calculations to measured data, theoretical surface temperatures were determined as a function of time and the onset of nonlinear changes in the thermal response of tissue identified.

When applying noxious heat stimuli to human skin in the study of the pain system, one of the main problems is not to cause permanent damage. A better understanding of the temperature distribution and the propagation of heat, i.e. heat flux, in human skin is thus needed. In order to investigate these problems thoroughly, we have developed a 3-dimensional finite element model (FEM) 4-layer of human skin. The model is kept simple for better understanding of the boundary problems. The water content in each layer is used for determining the thermal properties. It is therefore not a homogenous structure. In this model the stratum corneum has been included with lower water content than in the epidermis. Simulations shows that the surface temperature reaches high levels whereas the temperature in the deeper structure is much lower. Thermal and optical constants found in the literature was applied. Heat propagation downwards and outwards from the source has been investigated to understand of the accumulation of energy in the boundary between two layers. Prediction of the heat flux at boundary between the epidermis and dermis shows that for repetitive stimulation there is a risk of exceeding the threshold temperature of 65 degrees Celsius for irreversible damage.

Previously we have developed a free boundary model for local thermal coagulation induced by laser light absorption when the tissue region affected directly by laser light is sufficiently small and heat diffusion into the surrounding tissue governs the necrosis growth. In the present paper keeping in mind the obtained results we state the point of view on the necrosis formation under these conditions as the basis of an individual layer therapy mode exhibiting specific properties. In particular, roughly speaking, the size of the resulting necrosis domain is determined by the physical characteristics of the tissue and its response to local heating, and by the applicator form rather than the treatment duration and the irradiation power.

In this study, we examined (1) the effect of partial denaturation and (2) repetitive irradiation on porcine septal cartilage during Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm², 5 mm spot size) exposure. Diffuse reflectance from a HeNe probe laser and internal stress were measured in mechanically deformed cartilage specimens (2 X 10 X 25 mm) during Nd:YAG laser irradiation. Specimens were first partially denaturation in heated saline water baths at selected temperatures (50 degrees Celsius, 70 degrees Celsius, and 100 degrees Celsius all for 30 minutes). Native and water bath heated specimens were sequentially irradiated three times (pulse duration varying from 5 - 12 seconds, determined by noting the onset of accelerated stress relaxation) with a 5 minute cooling interval between pulses. After the first laser pulse, diffuse reflectance and internal stress changes occurred synchronously (coupled); the peak in internal stress occurred within less than 1 second following observation of the peak in diffuse reflectance. With repeated laser irradiation, this time interval lengthens with eventual decoupling of the diffuse reflectance and internal stress. With decoupling, internal stress increases during laser heating, and abruptly decreases when irradiation is terminated. Decoupling occurs with greater frequency in specimens pre-heated in the water bath. With the first laser exposure, only 15% of control, 8% of 50 degree Celsius heated, 0% of 70 degree Celsius heated, and 8% of 100 degree Celsius specimens exhibited decoupling. However, after two laser exposures, decoupling was observed in 83% and 60% of specimens heated in water baths at 70 degrees Celsius (N equals 12) and 100 degrees Celsius (N equals 12), respectively; in contrast, decoupling was observed in less than 20% of the native (N equals 24) and 50 degrees Celsius (N equals 12) water bath pre-heated specimens with two laser irradiations. The effect of partial denaturation using water bath immersion mimics findings observed with sequential laser irradiation. Hence cartilage likely undergoes partial denaturation during laser reshaping, and that sustained laser irradiation may result in irreversible changes in the tissue matrix.

The effects of laser irradiation on molecular mass and conformation of pure chondroitin sulphate dissolved in phosphate buffered saline were investigated using size exclusion chromatography/multi-angle light scattering (SEC/MALS) and sedimentation velocity in the analytical ultracentrifuge. In addition, cartilage pieces immersed in buffer were irradiated with a laser in order to study whether cartilage components may diffuse away from the matrix and into the surrounding aqueous medium as a result of laser treatment. Size exclusion chromatography/multi-angle light scattering and sedimentation velocity measurements showed that (a) laser irradiation decreases the molecular mass of chondroitin sulphate and (b) laser irradiation of cartilage induces diffusion of macromolecules into the medium. The results obtained allow us to understand the mechanism of stress relaxation and structural alterations in cartilage under non- destructive laser radiation.

Responses of tissue to laser stimulation are crucial in both disease diagnostics and treatment. In general, when tissue absorbs laser energy photothermal interaction occurs. The most important signature of the photothermal reaction is the tissue temperature change during and after the laser irradiation. Experimentally, the tissue reaction to laser irradiation can be measured by numerous methods including direct temperature measurement and measurement of perfusion change. In this study, a multiple-channel temperature probe was used to measure tissue temperature change during irradiation of lasers with different wavelengths at different power settings. Tissue temperature in chicken breast tissue as well as skin and breast tumor of rats was measured during irradiation of an 805-nm diode laser. The vertical profiles of temperature were obtained using simultaneous measurement at several different locations. The absorption of laser energy by tissue was enhanced by injecting laser-absorbing dye into the tissue. A Nd:YAG laser of 1064-nm wavelength was also used to irradiate turkey breast tissue. Our results showed that both laser penetration ability and photothermal reaction depended on the wavelength of lasers. In the case of 805-nm laser, the temperature increased rapidly only in the region close to the laser source and the thermal equilibrium could be reached within a short time period. The laser absorbing dye drastically enhanced the thermal reaction, resulting in approximately 4-fold temperature increase. On the contrary, the laser beam with 1064-nm wavelength penetrated deeply into tissue and the tissue temperature continued increasing even after a 10-minute laser irradiation.

In this work we have used for the first time 1.56 micrometer fiber laser to study mechanisms of IR laser induced stress relaxation in cartilage. We have applied several in-situ monitoring techniques: local temperature measurements (IR radiometry and thermocouple), IR-light absorption, direct stress measurements, micro-balancing, visible light scattering and optical coherent tomography. We have measured temporal behavior of 1.56 micrometer laser light transmission through the cartilage sample at different intensities with synchronous temperature and stress monitoring. The observed bleaching effect (self-induced transparency) is caused by water release from irradiating zone, water evaporation from the cartilage surface and, also, by temperature shift and decrease of intensity of water absorption bands.

The ablation of porcine skin tissue has been investigated using nanosecond (ns) laser pulses at the wavelengths of 1064, 532 and 266 nm. The ablation probability has been measured near the threshold through detection of the secondary radiation from the tissue sample surface at different wavelengths. Experimental results have indicated that the ablation of the skin tissue in the wide range of ablating wavelength is caused by optical breakdown induced by the strong electromagnetic field of the nanosecond pulses. Furthermore, we conclude that the initial seed electrons acquire ionization energy from the incident optical field mainly through a momentum-relaxing drift mechanism.

To investigate the optimum irradiation conditions for the transmyocardial laser revascularization (TMLR), ablation characteristics have been explored in vitro with porcine myocardium tissues. With a nanosecond optical parametric oscillator (OPO), the ablation depth and the thickness of thermally damaged tissue were measured at a constant peak intensity or fluence in the ultraviolet spectral region of 230 - 400 nm. It was found that at a peak intensity of 80 MW/cm², the ablation depth steeply increased for < 300 nm, while the thickness of thermally damaged tissue decreased with decreasing the wavelength. To understand the wavelength dependence of the ablation characteristics, we measured the optical properties of the tissue. This showed that the total attenuation coefficient largely increased with decreasing the wavelength for < 300 nm. Therefore, it is considered that at the shorter wavelengths the optical energy density deposited in the tissue would be high enough to ablate the whole region of the light-penetrated tissue. However, the wavelength dependence might be changed at some higher intensities or fluences. But our experiment using the 3rd and 4th of a Q- switched Nd:YAG laser, the shorter wavelength (266 nm) still gave the deeper ablation for up to 2 - 5 J/cm². The influence of photochemical effects on the ablation mechanism is also discussed.

We measured thermal emission from cornea surface during and just after ArF excimer laser pulse with 65 MHz bandwidth, corresponding to 15 ns rise-time. The rise-time of the thermal emission measurement should be the same order of the heating pulse duration (ArF excimer laser pulse duration of 25 ns) to obtain the rapid temperature change of cornea surface. To acquire the available rise-time, we employed at 150 MHz cut off frequency photovoltaic HgCdTe detector with 100 MHz preamplifiers. We measured the peak temperature of 175 degrees Celsius at the fluence of 160 mJ/cm². The irradiated cornea temperature decreased rapidly with 280 ns in time constant. We measured the highest temperature elevation during the ArF excimer laser ablation of the cornea comparing with previous reports using our high speed temperature measurement system.

Craniotomy using a drill and saw frequently results in fragmentation of the skull plate. Lasers have the potential to remove the skull plate intact. TE CO₂ lasers operating at the peak absorption wavelength of bone ((lambda) equals 9.6 micrometer) and with pulse durations of 5 - 10 microseconds, approximately the thermal relaxation time in hard tissue, produced high ablation rates and minimal peripheral thermal damage. Both thick (2 mm) and thin (250 micrometer) bovine skull samples were perforated and the ablation rates calculated. Results were compared with Q-switched and free- running Er:YAG lasers ((lambda) equals 2.94 micrometer, (tau) _p equals 150 ns and 150 microseconds). The CO₂ laser perforated thick sections at ablation rates of 10 - 15 micrometer per pulse and fluences of approximately 6 J/cm². There was no discernible thermal damage and no need for water irrigation during ablation. Pulse durations >= 20 microseconds resulted in significant tissue charring which increased with the pulse duration. Although the Er:YAG laser produced ablation rates of approximately 100 micrometer per pulse, fluences > 30 J/cm² were required to perforate thick samples, and thermal damage measured 25 - 40 micrometer. In summary, the novel 5 - 10 microsecond pulse length of the TE CO₂ laser is long enough to avoid a marked reduction in the ablation rate due to plasma formation and short enough to avoid peripheral thermal damage through thermal diffusion during the laser pulse. Further studies with the TE CO₂ laser are warranted for potential clinical application craniotomy procedures.

An investigation into the interaction of a fiber deliverable, long pulse, xenon chloride (308 nm) excimer laser with hard biotissue has been carried out. The laser produces pulses of 200+ ns as opposed to around 10 - 20 ns for most of the previously reported data. The threshold of ablation and the maximum ablation depth (AD) in human molar dentine were found to be 0.30 +/- 0.05 J/cm² and 1.57 +/- 0.04 micrometer/pulse respectively. The threshold for enamel was found to be above the achievable fluence with the available optics. The ablation process was investigated as a function of fluence (approximately 0.1 - 6 J/cm²), pulse repetition rate (PRR) (5 - 25 Hz) and number of pulses (500 - 4000). Each variable was altered independently of the other two. At a constant number of pulses, ablation depth per pulse was found to increase linearly as a function of fluence, up to a saturation fluence of approximately 4 J/cm². Variation of the PRR alone was found to affect both the ablation threshold and the AD. For constant fluence and PRR, AD decreases non- linearly with an increasing number of pulses. This could be because at high pulse numbers the craters are deep, the walls of the crater absorb more energy and as it is increasingly difficult for the debris to escape, shielding of the tissue occurs. Shielding may also be due to absorption in a luminescent plume. At high fluence and PRR, sharp holes were formed in the dentine although charring was sometimes found around the edges. High PRR also induced considerable mechanical damage.

Previously we found that Ho:YAG laser (2120 nm) lithotripsy of uric acid stones produced cyanide, a known thermal breakdown product of uric acid. We now report that alloxan, another thermal breakdown product, is also likely produced. Uric acid stones (approximately 98% pure) of human origin were placed in distilled water and subjected to one of the following experimental treatments: unexposed control, exposed to Ho:YAG laser, Nd:YAG laser, or mechanically crushed. Samples were then processed for HPLC analysis with UV detection. Peaks were identified by comparison to authentic standards. All samples contained uric acid, with retention time (RT) about 6 min. All of the laser-exposed samples contained a peak that eluted at 2.5 min, identical to the RT of authentic alloxan. Ho:YAG laser irradiation, however, produced a larger presumed alloxan peak than did the Nd:YAG laser. The peak at 2.5 min, as well as unidentified later-eluting peaks, were present in the laser-exposed, but not the unexposed or mechanically crushed, samples. These results confirm the thermal nature of lithotripsy performed with long-pulse IR lasers.

The generation of shock waves and bubbles has been experimentally observed due to absorption of sub-nanosecond laser pulses by melanosomes, which are found in retinal pigment epithelium cells. Both the shock waves and bubbles may be the cause of retinal damage at threshold fluence levels. The theoretical modeling of shock wave parameters such as amplitude, and bubble size, is a complicated problem due to the non-linearity of the phenomena. We have used two different approaches for treating pressure variations in water: the Tait Equation and a full Equation Of State (EOS). The Tait Equation has the advantage of being developed specifically to model pressure variations in water and is therefore simpler, quicker computationally, and allows the liquid to sustain negative pressures. Its disadvantage is that it does not allow for a change of phase, which prevents modeling of bubbles and leads to non-physical behavior such as the sustaining of ridiculously large negative pressures. The full EOS treatment includes more of the true thermodynamic behavior, such as phase changes that produce bubbles and avoids the generation of large negative pressures. Its disadvantage is that the usual stable equilibrium EOS allows for no negative pressures at all, since tensile stress is unstable with respect to a transition to the vapor phase. In addition, the EOS treatment requires longer computational times. In this paper, we compare shock wave generation for various laser pulses using the two different mathematical approaches and determine the laser pulse regime for which the simpler Tait Equation can be used with confidence. We also present results of our full EOS treatment in which both shock waves and bubbles are simultaneously modeled.

IR laser ablation of skin is accompanied by acoustic signals the characteristics of which are closely linked to the ablation dynamics. A discrimination between different tissue layers, for example necrotic and vital tissue during laser burn debridement, is therefore possible by an analysis of the acoustic signal. We were able to discriminate tissue layers by evaluating the acoustic energy. To get a better understanding of the tissue specificity of the ablation noise, we investigated the correlation between sample water content, ablation dynamics, and characteristics of the acoustic signal. A free running Er:YAG laser with a maximum pulse energy of 2 J and a spot diameter of 5 mm was used to ablate gelatin samples with different water content. The ablation noise in air was detected using a piezoelectric transducer with a bandwidth of 1 MHz, and the acoustic signal generated inside the ablated sample was measured simultaneously ba a piezoelectric transducer in contact with the sample. Laser flash Schlieren photography was used to investigate the expansion velocity of the vapor plume and the velocity of the ejected material. We observed large differences between the ablation dynamics and material ejection velocity for gelatin samples with 70% and 90% water content. These differences cannot be explained by the small change of the gelatin absorption coefficient, but are largely related to differences of the mechanical properties of the sample. The different ablation dynamics are responsible for an increase of the acoustic energy by a factor of 10 for the sample with the higher water content.

A Mach-Zehnder interferometer was used for analysis of pressure waves generated by ultrashort laser pulse ablation of water. It was found that the shock wave generated by plasma formation rapidly decays to an acoustic wave. Both experimental and theoretical studies demonstrated that the energy transfer to the mechanical shock was less than 1%.

Optically generated acoustic waves have been used to temporarily permeate biological cells. This technique may be useful for enhancing transfection of DNA into cells or enhancing the absorption of locally delivered drugs. A diode- pumped frequency-doubled Nd:YAG laser operating a kHz repetition rates was used to produce a series of acoustic pulses. An acoustic wave was formed via thermoelastic expansion by depositing laser radiation into an absorbing dye. Generated pressures were measured with a PVDF hydrophone. The acoustic waves were transmitted to culture and plated cells. The cell media contained a selection of normally-impermeable fluorescent-labeled dextran dyes. Following treatment with the opto-acoustic technique, cellular incorporation of dyes, up to 40,000 Molecular Weight, was noted. Control cells that did not receive opto-acoustic treatment had unremarkable dye incorporation. Uptake of dye was quantified via fluorescent microscopic analysis. Trypan Blue membrane exclusion assays and fluorescent labeling assays confirmed the vitality of cells following treatment. This method of enhanced drug delivery has the potential to dramatically reduce required drug dosages and associated side effects and enable revolutionary therapies.

Penetration of anti-cancer drugs (especially macromolecular agents) from blood in tumor cells is limited due to the presence of physiological barriers: tumor capillary wall, slow diffusion in the interstitium, and cancer cell membrane. Interaction of exogenous nano- or microparticles with laser or ultrasonic radiation may enhance drug delivery in tumor cells due to laser- or ultrasound-induced cavitation. Our previous studies demonstrated enhanced delivery of model macromolecular anti-cancer drugs in tissues in vitro when laser or ultrasonic radiation is applied. In this paper, we studied laser-induced cavitation in suspension of strongly absorbing particles and laser-enhanced drug delivery in human colon tumors of nude mice in vivo. Cavitation kinetics and thresholds were measured for carbon and colored polystyrene particle suspensions. Histological examination of control and irradiated tumors with fluorescent microscopy demonstrated that Q-switched Nd:YAG laser irradiation enhances delivery of a model macromolecular drug (FITC-dextran) in tumor blood vessel and interstitium. Enhanced delivery of an anti-cancer drug (5-FU) that is currently used in clinics resulted in tumor necrosis and inhibited tumor growth. Results of our studies suggest that the drug delivery enhancement is due to cavitation produced by local heating of particles with pulsed laser radiation.

The potential application of an Erbium:YAG (Er:YAG) laser (Q_o equals 50 mJ/pulse; (tau) _p equals 275 microsecond(s) ; rep. rate equals 2, 10 Hz) with a sapphire delivery fiber for intracorporeal laser lithotripsy was explored. Preliminary measurements on calculus mass-loss and fragmentation efficiency were conducted and results were compared with that of Ho:YAG laser lithotripsy. Laser induced bubble and lithotripsy dynamics were investigated to assess the mechanism(s) involved in the fragmentation process. Results showed that the fragmentation efficiency (mass-loss/H_o - g.micrometers ²/J) in Er:YAG laser lithotripsy was about 2.4 times that of Ho:YAG laser lithotripsy (used: Q_o equals 500 mJ/pulse; (tau) _p equals 250 microsecond(s) ; rep. rate equals 10 Hz). Acoustic transients were found to have minimal effect during Er:YAG laser lithotripsy. Schlieren flash images suggested a predominantly photothermal mechanism due to direct laser energy absorption, which resulted in recrystallization and plume formation. These events indicated melting and chemical decomposition of the calculus composition. Another observation led to the possibility of a plasma-mediated photothermal mechanism. The 'Moses effect' facilitating pulsed mid-infrared laser delivery appeared more efficient for the Er:YAG laser than for the Ho:YAG laser. With the sapphire fiber, experimental results suggested the potential of an improved treatment modality by the Er:YAG laser for intracorporeal laser lithotripsy.

Laser induced optical breakdown (LIOB) in fluids produces a localized plasma, an expanding radial shock wave front, heat transfer from the plasma to the fluid, and the formation of cavitation bubbles. Collectively these phenomena are referred to as photodisruption. Subjecting photodisruptively produced cavitation bubble nuclei to an ultrasonic field can result in strong cavitation and local cellular destruction. The ability of ultrafast lasers to produce spatially localized photodisruptions with microJoule pulse energies in combination with the possibility of larger scale tissue destruction using ultrasound presents an attractive and novel technique for selective and non-invasive tissue modification, referred to as photodisruptively nucleated ultrasonic cavitation (PNUC). Optimization of PNUC parameters in a confocal laser and ultrasound geometry is presented. The cavitation signal as measured with an ultrasound receiver was maximized to determine optimal laser and ultrasound spatial overlap in water. A flow chamber was used to evaluate the effect of the laser and ultrasound parameters on the lysis of whole canine red blood cells in saline. Parameters evaluated included laser pulse energy and ultrasound pressure amplitude.

The purpose of this study is to evaluate the influence on the intervertebral disc cells after laser irradiation using three- dimensional culture system and to clarify the optimum Ho:YAG laser irradiation condition on percutaneous laser disc decompression (PLDD) for lumbar disc herniation. Since the Ho:YAG laser ablation is characterized by water-vapor bubble dynamics, not only thermal effect but also acoustic effect on cell metabolism might occur in the intervertebral disc. We studied the disc cell reaction from the metabolic point of view to investigate photothermal and photoacoustic effects on three-dimensional cultured disc cell. Intervertebral discs were obtained from female 30 Japanese white rabbits weighing about 1 kg. A pulsed Ho:YAG laser (wavelength: 2.1 micrometer, pulse width: about 200 microseconds) was delivered through a 200 micrometer-core diameter single silica glass fiber. We used the Ho:YAG laser irradiation fluence ranging from 60 to approximately 800 J/cm² at the fiber end. To investigate acoustic effect, the acoustic transducer constructed with polyvinylidene fluoride (PVdF) film and acoustic absorber was used to detect the stress wave. Thermocouple and thermography were used to investigate thermal effect. Concerning damage of plasma membrane and ability of matrix synthesis, thermal effect might mainly affect cell reaction in total energy of 54 J (closed to practically used condition), but in 27 J, acoustic effect might contribute to it. We found that total energy was key parameter among the optimum condition, so that temperature and/or stress wave may influence Ho:YAG laser-disc cell interactions.

Mechanisms of tissue damage are investigated for skin and cornea exposures from 1540 nm ('eye safe') laser single pulses of 0.8 milli-seconds. New skin model data point out the advantages of using the Yucatan mini-pig versus the Yorkshire pig for in-vivo skin laser exposures. Major advantages found include similarities in thickness and melanin content when compared with human skin. Histology from Yucatan mini-pig skin exposures and the calculation of an initial ED₅₀ threshold indicate that the main photon tissue interaction may not be solely due to water absorption. In-vitro corneal equivalents compared well with in-vivo rabbit cornea exposure under similar laser conditions. In-vivo and in-vitro histology show that initial energy deposition leading to damage occurs intrastromally, while epithelial cells show no direct injury due to laser light absorption.

The Vanderbilt University free-electron laser (FEL) provides a continuously tunable ((lambda) equals 2 - 10 micrometer) source of pulsed IR radiation with a pulse structure unlike those of conventional lasers (a macropulse of 5 microseconds consisting of a train of 1 ps micropulses at a frequency of 3 GHz). A numerical hydrodynamic code at Lawrence Livermore National Laboratory, known as LASTIS3D, was used to model the ablation of tissue using the FEL. This study investigates the role of the FEL pulse structure by comparing the results from simulations using a time-averaged energy deposition and a pulsetrain energy deposition.

We describe the results of preliminary studies on the interaction of continuous wave (cw), mid-infrared fiber laser light and soft biological tissues. An Er, Pr:ZBLAN fiber laser operating at a wavelength of 2.71 micrometer was used at 800 mW output power, a Tm-doped silica fiber laser at 1.98 micrometer provided up to 5 W output power, and a Yb:Er-doped silica fiber laser at 1.5 micrometer was used at 800 mW output power. Surface changes in tissue samples are described qualitatively and quantitatively, ablation velocity in tissue is measured, where observed, at the 800 mW power level, and sample sections are described with reference to histologically recognizable markers of thermal damage. The basic science described prepares the way toward ultimate clinical evaluations and applications of these new laser sources.

We studied coagulation layer controlled incision with newly developed continuous wave 2 micrometer, 3 micrometer cascade oscillation fiber laser in vitro. Since this laser device simultaneously oscillates 2 micrometer and 3 micrometer radiation, we could change tissue interaction by arranging power ratio of 2 micrometer to 3 micrometer radiation. About one watt of total irradiation power with various power ratios was focused to extracted fresh porcine myocardium or anesthetized rabbit on an automatic moving stage to obtain line incision. Macro photograph and microscopic histology were used to observe tissue interaction phenomenon. The incised specimen showed that precise cutting groove with thin coagulation layer was attained by a 3 micrometer based radiation, meanwhile addition of 2 micrometer radiation to 3 micrometer radiation made coagulation layer thicker. A heat conduction simulator using finite-element method was used to qualitatively explain obtained coagulation layer thickness. This precise incision with controllable side coagulation layer may effective to control bleeding during incision, for instance, for skin, liver, and kidney incisions. Pure continuous wave radiation of 2 micrometer and 3 micrometer may eliminate stress wave induced tissue damage which is frequently found in Ho:YAG and/or Er:YAG tissue interactions. Moreover, sapphire fiber might offer flexible power delivery to this new laser to establish endoscopic application and/or to improved beam handling.

Focusing light into a turbid medium was studied with Monte Carlo simulations. Focusing was found to have a significant impact on the absorption distribution in turbid media when the depth of the focal point (the distance between the focal point and the surface of the turbid media) was less than or comparable with the transport mean free path. Focusing could significantly increase the peak absorption and narrow the absorption distribution. As the depth of the focal point increased, the peak absorption decreased, and the depth of peak absorption increased initially but quickly reached a plateau that was less than the transport mean free path. A refractive-index-mismatched boundary between the ambient medium and the turbid medium deteriorated the focusing effect, increased the absorption near the boundary, lowered the peak absorption, and broadened the absorption distribution.

In Monte Carlo simulations of light propagating in biological tissues, photons propagating in the media are described as classic particles being scattered and absorbed randomly in the media, and their path are tracked individually. To obtain any statistically significant results, however, a large number of photons is needed in the simulations and the calculations are time consuming and sometime impossible with existing computing resource, especially when considering the inhomogeneous boundary conditions. To overcome this difficulty, we have implemented a parallel computing technique into our Monte Carlo simulations. And this moment is well justified due to the nature of the Monte Carlo simulation. Utilizing the PVM (Parallel Virtual Machine, a parallel computing software package), parallel codes in both C and Fortran have been developed on the massive parallel computer of Cray T3E and a local PC-network running Unix/Sun Solaris. Our results show that parallel computing can significantly reduce the running time and make efficient usage of low cost personal computers. In this report, we present a numerical study of light propagation in a slab phantom of skin tissue using the parallel computing technique.

An Inverse Monte Carlo program was developed based on a scaleable Monte Carlo algorithm. This program determines the skin optical ((mu) _a and (mu) _s) properties in vivo using reflectance and thermal measurements as inputs from different skin types, very light to very dark. Some basic assumptions are made: (1) epidermal thickness is close to 100 micrometer, (2) the scattering in the epidermis is the same or similar to the dermal scattering, (3) the dermal absorption and scattering coefficients are similar between individuals. Experimental measurements of reflectance and temperature were taken. These were input in a pair of Inverse Monte Carlo programs that generated the optical properties for the different skin types. A single layer Inverse Monte Carlo model was employed to determine the optical properties of the dermis. A 2-layer Inverse Monte Carlo program was used to determine the epidermal optical properties.

The optical properties of porcine nasal cartilage have been determined at a wavelength of 632.8 nm using two different techniques. A single integrating sphere method was used to measure diffuse reflectance and diffuse transmittance, while total attenuation was determined from separate measurements of collimated transmission. Optical properties (absorption and scattering coefficients, and scattering anisotropy) were obtained by comparing measured results with predictions of Monte Carlo computer simulations. An estimate of the validity of the optical properties was obtained by measuring the optical penetration depth in bulk cartilage tissue. The optical properties of porcine nasal cartilage are characterized by low absorption (0.14 plus or minus 0.05 cm^-1) and high scatter (304 plus or minus 47 cm^-1). The scattered light is highly forward peaked as indicated by the mean cosine of the scattering angle (0.973 plus or minus 0.005). Based on these results, the calculated optical penetration depth was in good agreement with that measured in bulk tissue. The results presented here are compared to those obtained in mammalian cartilage tissue by other investigators.

We present results on the retrieval of skin optical properties obtained by fitting of measurements of the diffuse reflectance of human skin. Reflectance spectra are simulated using an analytical model based on the diffusion approximation. This model is implemented in a simplex fit routine. The skin optical model used consists of five layers representing epidermis, capillary blood plexus, dermis, deep blood plexus and hypodermis. The optical properties of each layer are assumed homogeneously distributed. The main optical absorbers included are melanin in epidermis and blood. The experimental setup consists of a HP photospectrometer equipped with a remote fiber head. Total reflectance spectra were measured in the 400 - 820 nm wavelength range on the volar underarm of 19 volunteers under various conditions influencing the blood content and oxygenation degree. Changes in the reflectance spectra were observed. Using the fit routine changes in blood content in the capillary blood plexus and in the deep blood plexus could be quantified. These showed different influences on the total reflectance. The method can be helpful to quantitatively assess changes in skin color appearance such as occurs in the treatment of port wine stains, blanching, skin irritation and tanning.

Laser Doppler Imaging (LDI) has become an established technique for the two dimensional measurement of tissue perfusion but the uncertainty of photon penetration depth leads to ambiguous interpretation of what fraction of the tissue microcirculation is being sampled. This study investigates a diffuse reflectance technique for measuring tissue optical properties during LDI perfusion measurement for the simultaneous determination of photon penetration depth and tissue metabolic state. LDI and diffuse reflectance spectroscopy measurements were made on surgically exposed ligaments in pregnant and non-pregnant rabbits. Photon penetration depths are reported. It was observed that anisotropic scattering occurs due to the ordered alignment of collagen fibers within ligament. Tissue perfusion in the ligaments of pregnant animals was significantly lower than in non-pregnant animals. Tissue hemoglobin concentration and oxygenation, and percent vascularization are also reported showing no statistical difference between the ligaments in pregnant and non-pregnant rabbits. A significant difference was observed in the photon scattering coefficient between the pregnant and non-pregnant groups suggesting a change in fibril spacing and/or orientation, most likely caused by an increased laxity in the ligaments of the pregnant animals. These investigations compare well with previous biochemical and biomechanical information obtained on ligaments.

A multiple wavelength diffuse reflectance instrument was evaluated for the in-situ measurement of tissue optical properties. A diffusion dipole model with a non-linear least- squares fitting procedure was used to generate optical properties from the radial profiles of diffuse reflectance measurements. The diffusion model was compared with Monte Carlo simulations showing acceptable agrement for a range of distances far enough away from the source point. Instrumentation was evaluated by experimental measurement of tissue simulating phantoms where measured optical properties were compared to those obtained using a collimated transmission setup. The results showed agreement to within 5 - 10%. The optical properties of rabbit ligament and tendon are reported where anisotropic scattering due to collagen alignment was observed. This study represents the first known measurement of rabbit ligament and tendon optical properties and the use of diffuse reflectance to measure the 'lightguide' effects of collagen alignment. To evaluate instrument response to changes in tissue oxygenation levels the optical properties of human skin were measured before and during arterial occlusion. During arterial occlusion absorption at 633 nm increased while absorption at 810 nm remained relatively constant. These results are consistent with the deoxygenation of hemoglobin that occurs during occlusion.

The rate of randomization of linearly polarized light as a collimated polarized beam passes through scattering media was characterized by a diffusivity ((chi) ) (radians²/mean free path) in the angle space ((theta) ) describing orientation of the linear polarization: probability of orientation at an angle p((theta) ) equals exp[-(theta) ²/(2(sigma) ²)]/[(sigma) sqrt((pi) /2)] where (sigma) equals (chi) ^(tau ) and (tau) is the mean free path ((mu) _sL) where (mu) _s is the scattering coefficient (cm^-1) and L is sample thickness (cm). The media studied were polystyrene microsphere solutions, liver, muscle, and skin. The (chi) for microspheres ranged from 0.80 - 0.03 for 300 - 6000 nm diameter, and was 0.28 for skin, 0.06 for muscle, and 0.003 for liver.

We demonstrate the measurement of path-length-resolved optical phase space distributions as a new framework for exploring the evolution of optical coherence in a turbid medium. This method measures joint transverse position and momentum (i.e., angle) distributions of the optical field, resolved by optical path length in the medium. The measured distributions are related to the Wigner phase space distribution function of the optical field, and can provide complete characterization of the optical coherence in multiple scattering media. Optical phase space distributions are obtained as contour plots which enable a visual as well as quantitative method of characterizing the spatial coherence properties and wavefront curvature of the input and scattered fields. By using a broad-band source in a heterodyne detection scheme, we observe transmission and backscatter resolved by path length in the random medium, effectively providing timing resolution. New two-window heterodyne detection methods permit independent control of position and momentum resolution with a variance product that surpasses the uncertainty limit associated with Fourier transform pairs. Hence, high position and angular resolution can be simultaneously achieved. These techniques may provide new venues for using optical coherence in medical imaging.

The transverse spatial coherence of light evolves as the light traverses a random, multiple-scattering medium. For near- forward scattering, the wave-transport process can be described by a wave-transport equation for the spatial-angular Wigner function of the light, which is related to the spatial coherence function. Using a novel variable-shear Sagnac interferometer, we measured the Wigner function of initially coherent light after propagation through a multiple-scattering medium. We find good agreement between the wave-transport theory and the experimental results.

We have developed a new theoretical description of the optical coherence tomography (OCT) geometry for imaging in highly scattering tissue. The new model is based on the extended Huygens-Fresnel principle, and it is valid in the single and multiple scattering regimes. The so-called shower curtain effect, which manifests itself in standard OCT systems, is an inherent property of the extended Huygens-Fresnel model. We compare the theoretical analysis with experiments carried out on samples consisting of aqueous suspensions of microspheres and solid phantoms. We calculate the signal-to-noise ratio, and provide an estimation of the maximum attainable probing depth for shot-noise limited detection. Furthermore, we investigate the focusing of the Gaussian probe beam in the tissue using Monte Carlo simulations, and compare it to the extended Huygens-Fresnel model. Finally, we simulate the operation of the OCT system using a specially adapted Monte Carlo simulation code.

Different techniques for diagnostics and visualization of the inhomogeneous scattering media by means of the statistical analysis of spatial-temporal fluctuations of scattered light are considered: (1) polarization diagnostics and imaging based on the application of the polarization degree as visualization parameter; (2) imaging techniques on the basis of measurements of the contrast of multiply scattered speckles induced by partially coherent light scattering in the probed object; (3) modification of the speckle imaging technique based on the statistical analysis of time-integrated electronic images of the speckle patterns under coherent light illumination. Comparison of these methods with traditional approaches to the diagnostics and imaging of macroscopically inhomogeneous multiply scattering objects is made. Experimental results obtained with phantom scattering media and illustrating the potentialities of the discussed approaches are presented.

A confocal microscopy imaging system was devised to selectively detect second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.

Optical radar (LIDAR) is being used to remotely probe the atmosphere. Quantities that can be sensed on a path resolved basis include temperature, pressure, number density for specific molecules and atmospheric winds. We believe that the techniques used can be scaled down and used to analyze tissues in medical optics applications. As our first project using atmospheric optics technique, we are building a Heterodyne, Optical, Coherent tomography (HOCT) system for imaging tissue. This system will be described.

We will present a brief introduction to Mueller matrix imaging (MMI) from cradle to adolescence and then show how it can be effectively used for detection of objects embedded in a highly scattering medium when ordinary radiance imaging might fail. We will show which elements and combination of elements are important for gaining the highest contrast against the background continuum. The mapping of certain combinations of Mueller matrix elements into an equivalent human visual system will also be discussed.

We present a theoretical analysis on the use of polarized light in the detection of a model target in a scattering and absorbing turbid medium. Monte Carlo numerical simulations are used in the calculation of the effective Mueller matrix which describes the scattering process. Contrasts between various parts of the target and background are analyzed in the images created by ordinary radiance, by elements of the Mueller matrix and by the depolarization index. It is shown that the application of polarized light has distinct advantages in target detection and characterization when compared to the use of unpolarized light.

We have studied experimentally the problem of cell viability during laser-tissue interaction: how the cell viability can be regulated under certain laser regime. Many modern laser therapeutic techniques assume that cells should survive laser therapy or even to become activated (laser stimulation of immune system). An influence of ion balance was studied for human Red Blood Cells (RBC) during in vitro measurements of single RBC viability after laser pulse. Single cell viability was measured optically with Laser Viability Test which is based upon interpretation of cell photothermal response to pump pulse (532 nm, 10 ns, 10 - 50 (mu) J at 12 micrometer diameter). According to our experiments laser-induced cell damage occurs at different laser energy levels within one cell population (heterogeneity of cell viability) and besides is donor-dependent. The latter result means that laser therapeutic dose should be adjusted individually. For solutions of the ions Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ we have found strong dependence of cell viability upon the process of ion transport through the cell membrane which was regulated by variation of concentration and composition of above mentioned ions. An increase of Na⁺ or K⁺ concentration causes a decrease of RBC laser viability.

A non-stationary two-dimensional computer code for modelling of radiation and heat transport in heterogeneous biological tissues irradiated with laser is presented. Radiation transport is considered in the kinetic model, radiation transport equation is solved by the Monte Carlo method. Heat transport is considered in the heat conduction model, the heat equation is solved by a combination of Galerkin and the finite element methods. The code has passed a number of tests including comparisons with analytical solutions, numerical calculations of other authors, and with experimental results. The code can be used for working out and designing of laser surgical and therapeutic procedures. As well it can be used in inverse problem of experimental determination of optical and thermal parameters of biological tissues.

The main significance about the refractive index of bio- tissue, its measurement methods and its thermal properties in particular are presented in this paper. Most of bio-tissue can be regarded as random dispersed medium which inhomogeneous dimension is about the order of micrometers. In order to describe the optical behaviors of bio-tissue, we have to use the imaginary scattering and absorption properties. Meanwhile, an apparent index of refraction, or average refractive index has to be introduced to solve various boundary and temporal problems. Two possible measurement methods in which the experimental creativity is reflected are provided. We applied one of experimental setups to study the thermal response of refractive index of bio-tissue. Experimental results suggest that the index be temperature dependent on the process of heating. The tissues used in these experiments were porcine muscles. These measurements were taken at the 632.8 nm. The index of refraction keeps stable levels (1.364 +/- 0.001) below 36 Celsius degrees and (1.387 +/- 0.005) above 60 Celsius degrees, respectively, but increases with an increase in temperature from 36 to 60 Celsius degrees. The heating and cooling procedures are irreversible in optical property of tissue.

Knowledge of the optical properties of human whole blood has always been of great interest for medical applications. The aim of this study was to provide the dispersive relations of refractive index of human whole blood with different types in the visible and near-infrared ranges and other conditions. In order to overcome the scattering effect, we applied an unusual method based on total internal reflection. A focused light, a semicylindrical lens in contact with tissues and a linear CCD camera are used in the experimental apparatus. The critical angle and therefore the refractive index can be obtained from the spacial distribution of internal reflective light. A monochromator is chosen as the light source, the chromatic dispersion curve of materials can be determined directly and quickly. A set of values has been presented that relates the refractive index to wavelength and types of whole, undiluted blood. Our results suggest that the refractive dispersions be almost the same in the visible and near-infrared ranges no matter which blood type it belongs to. In addition, the relationship can be described by Cauchy's formula.

We suggest that the optical model of a tissue is constructed by different size particles and the corresponding density in a liquid just like some phantoms of tissues. Due to the fact that the nonhomogeneous dimension is about the order of several and hundred micrometers, the Fraunhofer diffraction and process are applicable to biological tissue. We define n(r) as the number of particles per unit volume in tissue with radiuses between r and r + d r, i.e. n(r) is an unnormalized probability density function. We have verified that all of scattering properties of tissue can be obtained as long as the equivalent particle size distribution function n(r) is measured. In particular, the relation between (mu) _s and S((theta) ) is dependent on the n(r). The equivalent particle size distribution is feasible for experimental methods and techniques, as well as necessary for scattering principle. Furthermore, the equivalent particle size distribution provide not only a new understanding about the scattering properties of tissue but also a new approach to obtain the scattering coefficient and phase function. In addition, this scheme is helpful to design more precision tissue-simulating phantoms.

The experimental evidences for photochemical mechanism of light activation of immune system are presented. The experiments were made in vitro using serums from normal blood, from blood of people with secondary immunodeficiency containing low-avidity IgG (up to 75 - 90%), and pure IgG buffer solutions obtained from the same serums. LED arrays and halogen lamps with narrow spectral filters from UV to IR were used as optical monochromatic sources. We have discovered that light can activate immune system by IgG transformation from low-avidity state to high-avidity one. This change has multistage irreversible character and depends on light wavelength and intensity. The estimations of optical effects were made by determination of IgG functional activity and quaternary antibody structure through the number of accessible functional protein residues in the IgG buffer solutions. Both methods showed very correlated results. Each IgG avidity transformation stage correlated with definite change of antibody spatial structure that, to our opinion, corresponds to the phenomenon pass through the several intermediate metastable forms, which are maintained by different intermolecular bonds. The bioaction spectrum of discovered effects is also presented.

The mechanisms of therapeutic action of laser radiation with and without photosensitizers were studied experimentally at cell level for living leukocytes. Single cell response to action of pathogens and to laser-based therapeutic processes was measured with photothermal (PT) image microscope. We have monitored cell functional properties during phagocytosis of bacteria S.aureus by human leukocytes. Laser therapy was applied in vitro to cell suspense and to the mixture of cells and microbes in three regimes: (1) low-power (30 - 50 mW/cm²) CW-laser radiation at 633 nm and at 670 nm; (2) photoactivated with 670 nm laser radiation Chlorin E6 at 0.2 mg/l (PDT); (3) photoactivated with 670 nm laser radiation aluminum disulfonated phthalocyanine (Photosens) at 0.2 mg/l (PDT). For all three regimes an increase of bactericidal effect was found only when the cells are involved into interaction with microbes. No bactericidal effect in the wide range of drug doses was found during direct application of PDT to bacteria in vitro. The best effect was found when the cells are treated with laser radiation only. Also laser radiation restored cell properties. As a result we suggest that immune stimulation depends more upon physical factors (laser) rather than upon pharmaceutical (photosensitizers).

It has been shown that NO is released under the exposure of the aqueous solutions of S-nitrosocompounds as well as blood plasma proteins and whole blood of healthy donors to UV and visible light. The NO release from degrading S- nitrosocompounds was monitored both spectrophotometrically (by nitrosohemoglobin formation) and using the quenching of pyrene fluorescence by nitric oxide. In addition to NO, thyil radicals which dismutate to disulfides, were formed under anaerobic conditions. In the presence of oxygen, peroxide compounds, cysteine acid derivatives and S-nitrocompounds are formed apart from disulfides, and NO is mainly converted to NO₂^-. It is suggested that NO releasing under the actin of UV and visible light from physiological depots induces vascular relaxation, which enhances the blood flow.

Traditional therapies of autografts and allogeneic banked bone can promote reasonable clinical outcome to repair damaged bone. However, under certain conditions the success of these traditional approaches plummets, providing the incentive for researchers to develop clinical alternatives. The evolving field of tissue engineering in the musculoskeletal system attempts to mimic many of the components from the intact, healthy subject. Those components consist of a biologic scaffold, cells, extracellular matrix, and signaling molecules. The bone biomimetic, i.e., an engineered matrix, provides a porous structural architecture for the regeneration and ingrowth of osseous tissue at the site of injury. To further enhance the regenerative cascade, our strategy has involved porous biodegradable scaffolds containing and releasing signaling molecules and providing a suitable environment for cell attachment, growth and differentiation. In addition, the inclusion of genetically modified osteogenic precursor cells has brought the technology closer to developing a tissue-engineered equivalent. The presentation will describe various formulations and the methods utilized to evaluate the clinical utility of these biomimetics.

With minimally invasive approaches the visual path to guide instruments becomes constricted. Often one is unable to visualize adequately interaction of the instrument with tissue. We have incorporated 1.2-mm diameter 10,000 pixel fiberoptic endoscopes into instruments for spinal surgery. With these instruments one has a direct view of the instrument's interaction with the surgical anatomy. We have studied a variety of endoscopic instruments including malleable forceps, retractors and punches in over 40 cases of lateral disc herniations, migrated disc fragments and spinal stenosis. The instruments provided excellent visualization of spinal structures. The size and effect of the pathologic process could be readily evaluated, as could neural decompression. Operative times were not significantly increased and there were no complications attributable to the instruments. This preliminary work documents that 'seeing instruments' can be safely used and add to our appreciation of operative anatomy. It is suggested that these instruments may provide more complete decompression through a more limited, less invasive, access. Further study of these instruments may provide better understanding of their overall utility.

Optical techniques can assess the heterogeneity and structural layers of biomaterial and implants. Such assessment can assist engineering of tissue patches and implants by assessing implant structure, monitoring the implant fabrication process, controlling the machining of the implant, and monitoring in vivo the body's host response to the implant. Optical scattering can quantify the granularity of a biomaterial on the scale of 0.1 - 10 micrometer. Optical coherence tomography can map heterogeneity on the scale of 2 - 20 micrometer. Optoacoustic imaging can image absorbing heterogeneities on the scale of 20 micrometer - 10 mm (or more). Diffusion optical tomography can image absorbing and scattering heterogeneities on the scale of 5 mm - 5 cm (or more). The opportunities for optical techniques in preparing biomaterials and implants are discussed.

The engineering of new biomaterials requires an in-depth understanding of the structure and function of the native tissues. Optical coherence tomography (OCT) is a non- destructive technique that allows the visualization of the microstructure of biological tissues. The aim of this study was to determine if OCT could be used to identify geometric properties of tendons such as crimp pattern. Freshly harvested tendon tissue was imaged with OCT during the application of incremental axial strain. Loads were simultaneously recorded during this imaging. Results revealed that birefringent banding perpendicular to the collagen fiber axis could be visualized and measured. This crimp banding disappeared at a very low strain. Birefringent banding parallel to the collagen fiber axis was also seen and it was observed that the number of parallel bands increased as strain increased. Stress-strain data was calculated and found to lie within the expected range. These results indicate that OCT may prove to be a useful tool for the non-destructive analysis of tissue microstructure.

Dental health care and research workers require a means of imaging the structures within teeth in vivo. For example, there is a need to image the margins of a restoration for the detection of poor bonding or voids between the restorative material and the dentin. With conventional x-ray techniques, it is difficult to detect cracks and to visualize interfaces between hard media. This due to the x-ray providing only a 2 dimensional projection of the internal structure (i.e. a silhouette). In addition, a high resolution imaging modality is needed to detect tooth decay in its early stages. If decay can be detected early enough, the process can be monitored and interventional procedures, such as fluoride washes and controlled diet, can be initiated which can help the tooth to re-mineralize itself. Currently employed x-ray imaging is incapable of detecting decay at a stage early enough to avoid invasive cavity preparation followed by a restoration with a synthetic material. Other clinical applications include the visualization of periodontal defects, the localization of intraosseous lesions, and determining the degree of osseointegration between a dental implant and the surrounding bone. A means of assessing the internal structure of the tooth based upon use of high frequency, highly localized ultrasound (acoustic waves) generated by a laser pulse is discussed. Optical interferometric detection of ultrasound provides a complementary technique with a very small detection footprint. Initial results using laser-based ultrasound for assessment of dental structures are presented. Discussion will center on the adaptability of this technique to clinical applications.

The structural information carried by the evanescent components of the scattered field is discussed for the case of a single homogeneous plane wave incident on a weakly scattering three-dimensional medium. It is shown that, unlike the homogeneous components of the scattered field, the evanescent components are related to the three-dimensional Fourier transform of the dielectric susceptibility through a generalized Radon transform. The region of the three- dimensional Fourier space that is accessible from evanescent wave measurements is discussed, as well as the spatial resolution attainable in a typical multiple-view scattering experiment.

Images of light distribution in biological soft tissue we used to study the optical characteristics of tissue. The light distribution image was taken under a microscope with light injected through a pinhole close to the edge of the top surface. Images taken on skin, fat, and muscle tissues were compared to study the effect of cellular structure and temperature on the light intensity distribution. Monte Carlo simulation with the same conditions was also performed to simulate the light intensity distribution in tissue for comparison. The anisotropy scattering of light in tissue is affected by the tissue microscopic structure, such as the direction of muscle tissue fibers. The change in optical properties of fat and muscle tissue with temperature was observed. The two-dimensional light distribution images offer more information than general reflectance and transmission measurements. By matching the simulated light intensity distribution with the light distribution image, the optical properties of biological tissue could be estimated. This method might be applied in tissue engineering as an economic way for evaluating the microscopic structure of tissue.

In this paper characterization of the biomechanical properties of human skin in vivo is studied both experimentally and by numerical modeling. These properties can be important in the evaluation of skin condition (e.g. aging) as well as skin disorders. Methods: In this study we focus on the static behavior of the dermis. Important features are stress-strain non-linearity and anisotropy; both are mainly determined by the collagen fiber network present in the dermis. A suitable constitutive model was developed by Lanir. An experimental set-up was developed and used to stretch the skin in vivo. Two pads are attached to the skin which are driven apart during the experiment. The forces and displacements of the pads are measured. A field of markers (6 X 12) is applied to the skin's surface between the pads. The displacement history of the markers can be determined by image analysis. Both measured forces and displacement histories are input that is used to estimate the unknown material parameters in Lanir's skin model. A numerical simulation model of the experiment (finite element method) is combined with an estimation algorithm (constrained sequential maximum-likelihood approach) to determine estimates of the material parameters. Results: Estimates of the skin parameters could be determined. However the procedure also shows that the skin model applied exhibits modelling errors.

A novel, imaged laser speckle strain gauge is described for directly measuring strain rates in biological tissues. Cortical bone samples were tested in tension in a custom- designed microtensile testing machine. Strain rates were evaluated simultaneously with both the laser speckle strain gauge and contact strain gauges and extensometers. Young's modulus values of the bone samples were estimated using the strain data acquired by all methods. The strain rates and modulus estimates determined through all the methods compared favorably with each other, with the modulus estimates calculated using the speckle data slightly higher than by the other methods (mean of 16.88 GPa for the speckle data vs. 13.4 GPa for the contacting methods). The speckle strain gauge has a strain resolution at least on the order of single microstrain and should prove to be useful in the mechanical evaluation of both native and engineered tissues.

Laser speckle techniques are well known in the non-destructive evaluation community. One particular application is to infer strain by monitoring the motion of the speckle pattern that results from coherently illuminating the object. Typically a reference image of the speckle pattern is acquired before deformation of the object. Motion (with respect to this reference image) of subsequent speckle patterns, which occurs when the object is stressed, are used to infer the resulting strain. A problem experienced in using this technique for measurements of hydrated tissues is the rapid decorrelation of the speckle patterns. Thus, application of speckle techniques to assessment of strain in biological tissues relies on rapid sampling of the speckle patterns and the use of processing algorithms that are aimed at inferring strain rates rather than absolute strains. We discuss a number of approaches to estimating strain rates based on sequential speckle patterns. Maximum likelihood methods are shown to be especially useful.

Mechanically deformed morphologic cartilage grafts undergo a temperature dependent phase transformation during sustained laser irradiation that results in reshaping of the specimen. While thermal, optical, and mechanical properties of cartilage undergoing laser heating have been previously investigated, the viability of these irradiated grafts has yet to be examined closely until now. In this study, chondrocyte viability following laser irradiation was determined by measuring the incorporation of radiolabelled sulfate (Na ³⁵SO₄^-2) into proteoglycan (PTG) macromolecules. Proteoglycans are highly sulfated and are the principal molecular constituents of cartilage matrix. Their synthesis directly reflects chondrocyte viability. By measuring the scintillation counts of ³⁵SO₄^-2 uptake and normalizing the value by the total protein content of each specimen we can determine the level of PTG synthesis rates following laser reshaping. Regional baseline PTG synthesis rates as a function of location was determined by dividing each specimen into six regions. All regions except the most cephalic are demonstrated similar PTG synthesis rates. The most cephalic region exhibited a significantly greater PTG synthesis rates. In order to establish a positive control for this study, specimens were immersed in boiling saline water for approximately 40 minutes. The boiled specimens demonstrated a fivefold increase in normalized radioisotope uptake and suggest that the non-specific uptake of radioactive Na³⁵SO₄^-2 is caused by structural alterations in the collagen matrix caused by extensive thermal exposure. To avoid this thermal artifact, another positive control was established using nitric oxide was to induce apoptosis of the chondrocytes, resulting in significantly lower PTG synthesis compared to untreated tissue. Cartilage specimens (25 X 10 X 2 mm) were irradiated with light emitted from an Nd:YAG laser (25 W/cm², (lambda) equals 1.32 micrometer) while radiometric surface temperature, internal stress, and backscattered light were simultaneously recorded. Individual specimens underwent either one, two, or three sequential laser exposures with the duration of each exposure determined in real-time from observation of characteristic changes in integrated backscattered light intensity that correlate with thermal mediated stress relaxation. A five-minute time interval between each irradiation was given to allow the cartilage to return to thermal equilibrium. Average laser exposure for each irradiation sequence was recorded (5, 8.3, 12.2 sec). PTG synthesis decreased with increasing laser exposure, but was noted to remain above baseline levels for NO treated tissue. To further refine these results and minimize the effect of regional tissue variations, 7 mm diameter discs excised from the most cephalic portions and a middle region of the pig nasal septal cartilages were irradiated. A reduction of PTG synthesis rates was noted with each successive irradiation, suggesting that laser mediated cartilage reshaping acutely does not eliminate the population of viable chondrocytes. The degree of reduction in PTG synthesis is dependent upon the time-temperature dependent heating profile created during laser irradiation, and carefully monitored dosimetry is necessary to ensure chondrocyte viability.

The selective microphotocoagulation is a new technique to damage the retinal pigment epithelium (RPE), which is desired for treatment of several retinal diseases. By applying a train of microsecond(s) laser pulses it is possible to selectively destroy these cells and simultaneously spare the adjoining photoreceptor and neural tissue. We applied microsecond laser pulses of a Nd:YLF laser (527 nm), at a repetition rate of 500 Hz to porcine RPE. The light is absorbed in the RPE and by thermoelastic expansion, an optoacoustic (OA) signal will be generated which could be measured by an ultrasonic transducer. With this setup, the baseline temperature increase at the RPE, during irradiation can be determined, since the optoacoustic pressure signal depends on the temperature of the irradiated RPE. We found a linear dependence of the OA amplitude to the RPE sample temperature. At higher irradiance we proved the formation of microbubbles and bubble collapse in the RPE with OA techniques.

In wave-based remote sensing of distant objects embedded in a random medium a high-frequency electromagnetic wave is scattered by object discontinuities, and portions of the scattered radiation can traverse the same random inhomogeneities as the initial incident field, leading to an anomalous intensity distribution. Here, we present a possible realization for the resolving properties of an object using the double-passage effects and construct the intensity response at the image plane of an optical system, resulting from backward reflection from a target having discontinuities. The object plane -- image plane relations are formulated and manageable algorithms are obtained by using the random propagators of the Stochastic Geometrical Theory of Diffraction. The resolving properties of periodic spatial objects are investigated.

Imaging techniques involving transillumination require detailed knowledge of radiation path(s) between source and detector. When imaging with near infra-red in tissue this is particularly problematic due to the high scattering cross section. The population of 'direct path' photons is so small that information must be gathered from the much larger (but still small) population of 'snaked path' photons. Path Integral (PI) models set out to find the most likely of these paths, not by random sampling as in Monte Carlo based techniques, but directly: A cost function (Lagrangian) is constructed based on the physics of the scattering processes/absorption and integrated along the photon path to generate a total cost (Action). This is minimized using variational calculus to extract the most likely path. Whilst the PI approach is not new, the work presented here is novel in constructing the Lagrangian using local path descriptors. This allows explicit inclusion of an absorption term and also lends itself to arbitrary numbers of constraints on intermediate 'visit' points, path directions, and overall path length. Scaling symmetries are used to further reduce the computational expense of the method.

In this paper we present a theoretical study of focused beam wave pulse propagation and diffusion in highly scattering discrete random media. By using Wigner distributions, we calculate an explicit closed-form expression for the reduced intensity of focused beam waves. From this analysis, we find that the extent to which the reduced intensity focuses depends upon the attenuation it experiences from scattering and absorption. We then solve the diffusion equation for continuous wave sources and delta function input pulses to examine the spatial and temporal spreading of beam wave pulses. Through numerical approximations to the obtained solutions, we find that focusing effects of the diffuse intensity are negligible. Finally, we compare these results to those of collimated beam waves and pulsed plane waves. Through these comparisons, we determine that the spatial spreading of focused beams is similar to that of collimated beams, and the temporal spreading of the focused beam wave pulse is similar to that of plane wave pulses.

The aerosol found in the lowest kilometer over the world's ocean is quite different than that found over land. It includes several unique components of marine origin in addition to a background component, which can be similar to that found over land. Large marine aerosol can have significant interaction with infrared propagation in this region and are thus very important for naval applications. This paper will discuss some of the author's research in this area with special emphasis on the aerosol of interest to the Navy. The topics will include the aerosol found from shipboard level to altitudes above the marine inversion, aerosol found in the boundary layers between the wave tops and shipboard level and the effect of surf produced in the coastal regions. This paper will also describe some aspects of recent series of experiments sponsored by the Office of Naval Research called EOPACE (Electro Optical Propagation in A Coastal Environment). This program has concentrated on looking at the history of the sea salt aerosol produced by the breaking of waves in a surf zone as it interacts with the micrometeorology in the ocean atmospheric surface layer.

On a basis of a multiple-forward-scatter propagation model the atmospheric aerosol contributions to laser beam widening for a horizontal propagation path is estimated and compared with beam widening caused by turbulence. It is shown that the beam widening caused by atmospheric aerosols is significant, often even more significant than that caused by turbulence.

Interest in the use of optical communications over terrestrial links has greatly increased during the last several years. In many applications, the path is horizontal so the index of refraction structure parameter can be taken as constant. In addition, optical communication channels offer a number of advantages over conventional RF channels. However, due to the short wavelength, the reliability of an optical link can be seriously degraded over that of an RF system by atmospheric scintillation. In particular, scintillation can cause severe fading of the channel. In our analysis here we assume that the refractive index structure parameter C_n² is constant and use our recently developed gamma-gamma model and the well known lognormal model to consider the fading statistic associated with a spherical wave model for simplicity. The results are similar to a Gaussian-beam wave with perfect pointing. Our analysis show that compared to the gamma-gamma model, the lognormal model predicts optimistic values of probability of fade, underestimate the number of fades per second and consequently does not measure the mean fade time correctly.