Recent advances in the field of tissue engineering have led to the development of complex three-dimensional tissue constructs. It has become clear, however, that the traditional tools used for studying standard cell cultures are not always adequate for diagnostically studying thick, highly-scattering cultured tissues. Furthermore, many techniques used for studying three-dimensional constructs are invasive or require exogenous fluorophores, which damage the tissue and prevent time-course studies of tissue development. An integrated optical coherence tomography (OCT) and multi-photon microscope (MPM) has been constructed for visualizing 3-D engineered tissues. OCT was used for imaging structure and cell organization, while MPM was used for assessing functional properties of cells. We demonstrate technical developments involved in the construction of this instrument and its use in the non-destructive investigation of cell movement and tissue organization in engineered tissues. Cells labeled with GFP and exogenous fluorescent probes have also been imaged with OCT and confocal microscopy. Studies indicate that an integrated microscope has the potential to be an enabling diagnostic tool for future studies in the growth and organization of engineering tissues and in cell-cell and cell-matrix interactions.

We demonstrated a capability of biomechanical characterization by photoacoustic measurement for the purpose of non-invasive functional evaluation of articular cartilage. In this study, the scheme of photoacoustic measurement was improved. For in vivo application, the measurement scheme was changed from a transmittance mode to a reflectance mode in which an optical fiber was coaxially arranged with a piezoelectric transducer. In order to verify the applicability of this measurement for diagnosis of cartilage degeneration, photoacoustic measurements in a reflectance mode were performed using various degenerated cartilages. As a model of degenerated cartilage, cartilage-bone plugs were punched out from a porcine knee joint and treated with trypsin (1 mg/ml). Stress waves were induced by 250-355 nm, 7-ns light pulses delivered through an optical silica fiber from an OPO and were detected by a piezoelectric transducer. The change in relaxation time, which was correlated with the viscosity-elasticity ratio, had a positive correlation with time of trypsin treatment. Our results revealed the applicability of photoacoustic measurement to in vivo preoperative diagnosis of cartilage degeneration.

Shape quantification of tissue and biomaterials can be central to many studies and applications in bioengineering and biomechanics. Often, shape is mapped with photogrammetry or projected light techniques that provide XYZ point cloud data, and shape is quantified using derived flexure and curvature calculations based on the point cloud data. Accordingly, the accuracy of the calculated curvature depends on the properties of the point cloud data set. In this study, we present a curvature variability prediction (CVP) software model that predicts the distribution, i.e., the standard deviation, of curvature measurements associated with surface topography point cloud data properties. The CVP model point cloud data input variables include XYZ noise, sampling density, and map extent. The CVP model outputs the curvature variability statistic in order to assess performance in the curvature domain. Representative point cloud data properties are obtained from an automated biological specimen video topographer, the BioSpecVT (ver. 1.02) (Vision Metrics, Inc.,). The BioSpecVT uses a calibrated, structured light pattern to support automated computer vision feature extraction software for precisely converting video images of biological specimens, within seconds, into three dimensional point cloud data. In representative sample point cloud data obtained with the BioSpecVT, sampling density is about 11 pts/mm² for an XYZ mapping volume encompassing about 16 mm x 13.5 mm x 18.5 mm, average XY per point variability is about ±2 μm, and Z axis variability is about ±40 μm (50% level) with a Gaussian distribution. A theoretical study with the CVP model shows that for derived point cloud data properties, curvature mapping accuracy increases, i.e. measurement variability decreases, when curvature increases from about 30 m^-1 to 137 m^-1. This computed result is consistent with the Z axis noise becoming less significant as the measured depth increases across an approximately fixed XY region.

In this paper, a unique non-contact, minimum invasive technique for the assessment of mechanical properties of single cardiac myocyte is presented. The assessment process includes following major steps: (1) attach a micro magnetic bead to the cell to be measured, (2) measure the contractile performance of the cell under the different magnetic field loading, (3) calculate mechanical loading force, and (4) derive the contractile force from the measured contraction data under different magnetic field loading.

Accelerated proliferation of smooth muscle cells (SMC) is known to play an integral role in atherosclerotic lesion formation. Thus, there has been significant interest in defining both positive and negative regulators of SMC growth. We have applied a novel optical technique referred to as four-dimensional light scattering fingerprinting (4D-ELF) that enables non-invasive assessment of living cells. 4D-ELF can serve for highly sensitive detection of slight alterations in cellular and subcellular microstructure. Using 4D-ELF, we characterized the proliferation of SMC grown on two different substrates: laminin and fibronectin. Fibronectin-grown SMC have been previously shown to be more proliferative. Our results indicate that light scattering can be used to monitor the changes in the intracellular structure caused by the cell-substrate interaction and differentiate between more and less proliferative SMCs. Thus, light scattering fingerprinting may potentially provide a quick, inexpensive, and accurate means to noninvasively characterize the proliferation of living cells as well as cell-biomaterial interaction.

Photodynamic Therapy (PDT) is the best-known biomedical application of photochemical approaches to therapeutics. Although regulatory approvals for this modality have been obtained only over the last decade, the concept and indeed clinical studies of PDT are over a century old. During the first part of the last quarter century the focus of PDT applications had been on the treatment of cancer. However in recent years this has been broadened to a variety of non-cancer and diagnostic applications. This may be considered a return to the “roots” of PDT where one of the initial observations that form the basis of this approach was the inactivation of paramecium when exposed to acridine orange and light and the fluorescence of tumors when treated with porphyrins and light. Currently the most successful application of PDT is non-oncologic in the treatment of age-related macular degeneration (AMD) with Visudyne; so far this is the only first line use of PDT. At the present time, other diseases are being explored as targets for PDT in laboratories worldwide with a variety of strategies and PDT may now be considered a platform technology where photochemistry may be directed to specific anatomical sites and molecular targets with potentially a large number of applications.

Cancer is a leading cause of death among modern people, largely due to metastatic disease. The ideal cancer treatment should destroy both the primary tumor and distant metastases with minimal toxicity to normal tissue. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species that eventually cause vascular shutdown and tumor cell apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens are likely to emerge in the future to even further enhance immunity. Green fluorescent protein is used as an optical reporter to non-invasively image the progression of mouse tumors, and in addition, may act as a foreign (jellyfish) antigen. We asked whether the response of tumor bearing mice to PDT differed when a non-immunogenic tumor cell line was transfected with GFP? We injected RIF-1 or RIF1-EGFP cells in the leg of C3H/HeN mice and both the cells and tumors grew equally well. We used two PDT protocols (benzoporphyrin derivative (BPD) with 15-minute interval or Photofrin with 24-hour interval). The results showed significant differences between the responses of RIF1 or RIF1-EGFP tumors after BPD or Photofrin PDT and complete cures and mouse survival when RIF-1 EGFP tumors were treated with BPD. This increased tumor response may be due to antibody-mediated cytotoxicity and the presence of an artificial tumor antigen (GFP) that can produce a CD8 T-cell response against the whole tumor. The presence of antibodies against EGFP in mouse serum correlates with the hypothesis.

Since Photodynamic therapy(PDT) is able to increase the antitumor immunity, in our laboratory we examine the antitumor effect of combination of PDT,with photoactivated Aluminium disulfonated Phthalocianine(ALS₂Pc),adoptive immunotherapy, with immune lymphocytes, and chemotherapy on aggressive murine tumor. Mice bearing L1210 tumor were treated at day +4 with PDT ( 5mg/Kg of AlS₂Pc and 100mW/cm² x 10’ of exposure of laser light 24hrs. later),at day +6 with Adriamycin(ADR 2mg/Kg) and at day + 7 with immune lymphocytes(IL),collected from L1210 bearing mice pretreated with PDT(2x10⁷ cells).The results show that the combination ADR + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug and the adotive immunotherapy treatment are slightly effective. The same positive results were obtained with the combination of PDT,Cisplatin(CDDP 2mg/Kg) and IL,while the CDDP treatment alone and the Il treatment alone are slightly effective. In conclusion these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.

The tumor response to photodynamic therapy (PDT) involves a complex interplay between direct cytotoxicity to the tumor cells and secondary damage as a result of the effects of PDT on the vasculature and stimulation of the host inflammatory response. Pre-clinical and clinical studies have suggested that the combination of direct and indirect effects of PDT culminate in an activation of host anti-tumor immune responses. We have begun to examine the direct effects of PDT on tumor immunogenicity and have made the novel discovery that PDT treatment of tumor cells in vitro enhances tumor cell immunogenicity. We have further demonstrated that the increase in tumor cell immunogenicity by PDT can be correlated with the ability of PDT-generated tumor cell lysates to stimulate dendritic cell maturation and activation. The mechanisms by which PDT is able to enhance tumor cell immunogenicity and stimulate dendritic cell maturation and activation is unclear, however our finding suggest that alterations in tumor immunogenicity correlate with enhanced release of dendritic cell stimulating factors such as heat shock proteins.

Glycated chitosan (GC), a novel immunoadjuvant, has been used in combination with selective photothermal interaction in treatment of metastatic tumors. It has shown to be able to induce anti-tumor immunity and to enhance treatment efficacy. To further study the effects of glycated chitosan, photodynamic therapy (PDT) was used as the mechanism of direct tumor killing. Specifically, Photofrin-based PDT was used to treat EMT6 mammary tumors in mice and mTHPC-based PDT was used to treat Line 1 lung tumors in mice. In both cases, GC was administered immediately after the PDT treatment around the treated tumors. With EMT6 tumors, the use of GC improved the PDT-mediated tumor cure rate from 37.5% to 62.5% with 0.1 ml of 0.5% GC solution and to 75% with 0.1 ml of 1.5% GC solution. With the Line 1 tumors, the non-curative PDT treatment was converted into a 37.5% cure-rate by using a post-PDT peritumoral injection of 0.09 ml of 1.67% GC solution. In comparison, the treatments with GC alone or GC plus PDT light (no photosensitizer) produced no tumor regression and had no influence on the tumor growth rate, when compared to non-treated control tumors. GC was also used for the treatment of B16 melanoma in mice, using a combination of in situ application of GC and an irradiation of an 805-nm laser. The survival rates of the mice bearing melanoma tumors increased significantly when the laser and GC were applied, particularly when GC was applied 24 hours prior to the laser irradiation. These results strongly suggested that glycated chitosan played a significant role in the treatment of tumors.

Treatment of solid tumors by photodynamic therapy (PDT) induces a host reaction, coordinated through a network of inflammatory and immune responses, that plays an important role in the therapy outcome. It is suggested that this host response is initiated by altered self-associated endogenous danger signals massively released from PDT-treated tumors. Toll-like receptors, localized predominantly in the membrane of immune cells, are the major sensors of the recognition arm of the innate immune system. The engagement of these receptors by PDT-generated danger signals prompts the activation of the networks of innate immunity signaling pathways leading to the downstream activation of nuclear transcription factors responsible for the transcription of inflammatory/immune response-associated genes. The contribution of PDT-induced host response to the therapeutic outcome depends on the balance between the tissue-destructive action of inflammatory/immune effectors and the impact of concomitantly mobilized negative regulatory mechanisms evolved for controlling the intensity and duration of inflammatory and immune responses.

Photodynamic therapy (PDT) mechanism with high-intensity pulsed laser excitation has not been well understood. We think complete understanding of this unknown effect in PDT leads perfect treated depth control at various lesions. To realize the depth controlled PDT for atheromatous plaque therapy with a fibrous cap intact and surrounding damage free, we studied PDT against murine macrophage-like cells in vitro with the second-generation chlorin photosensitizer manufactured by Photochemical Co. Ltd. (Okayama Japan). The relation between the excitation conditions (pulse energy density and repetition rate) and PDT photocytotoxicity was examined in vitro. The XeCl excimer laser pumped dye laser (wavelength: 669±3 nm, pulse duration: 7ns in FWHM) was used with the pulse energy density from 1.2 to 9.5 mJ/cm², and the pulse repetition rate from 5 to 80 Hz. Under higher pulse energy density condition, no significant PDT photocytotoxicity was obtained. We examined the photobleaching of the protein containing photosensitizer medium solution, which is considered to correlates with the generation of singlet oxygen. Under higher pulse energy condition, the photobleaching efficiency decrease was observed and the measured PDT effect decrease in terms of laser pulse energy density could be explained by the photobleaching. We measured the oxygen partial pressure in photosensitizer medium solution immediately after the laser exposure. The decrease of oxygen partial pressure, i.e., the amount of the oxygen consumption during the laser exposure was observed 46 mmHg under the excitation condition of the pulse energy density of 9.5 mJ/cm², the total fluence of 5 J/cm², the repetition rate of 80Hz, and correlated with the bleaching efficiency 87% under the same condition. We calculated cell death distribution in depth direction based on measured photocytotoxicity under various pulse energy densities. The possibility of depth controlled PDT for safety atheromatous plaque therapy was suggested by the PDT effect alteration depending on pulse energy density.

The precise and selective destruction of tumor tissue through hyperthermia, when combined with immunoadjuvant applications, has shown promise in the treatment of cancer. The combination of an 805-nm laser and indocyanine green (ICG) has been shown to be highly effective in laboratory experiments in satisfying both the precision and selectivity requirements of such a procedure. A systematic study was conducted to further understand the interaction of the laser and the dye, using both Monte Carlo simulation and direct temperature measurement in gel phantom and chicken tissue. A phantom system, with a spherical, dye-enhanced target in the center, was constructed to simulate a tumor buried in deep tissue. The temperature increase in the target tissue under the irradiation of the 805-nm laser was significantly higher than the surrounding tissue. The same tumor-tissue configuration was also used in the theoretical simulation. The theoretical and experimental results for the temperature distributions and the treatment parameters can be used to provide the optimal thermal effect in cancer treatment.

Many research works have explored the use of the low power laser as a tool for the control of inflammatory processes. The anti-inflammatory effect of low power optical radiation and its ability to induce analgesia has been reported for different experimental conditions. Many published works are very qualitative in nature. In this work the action of low power laser radiation on acute inflammatory process is evaluated. The time evolution of rat paw edema and pain induced by carrageenan was experimentally monitored. A 632.8 nm He-Ne laser was used for the treatment. The laser treatment, at a dosage of 2,5 J/cm², was applied at the first, second and third hour after the induction of the inflammation. A hydroplethysmometer was used for the evaluation of the inflammation. The measurement of pain sensitivity was performed according to the method described by Randall and Selito, (1957). The laser treatment was capable of inhibiting the carrageenan-induced hyperalgesia by 49% (p<0,001) at the second hour after the induction, as compared to the non-treated group. At the fourth hour (peak of the carrageenan action on hyperalgesia) and at the sixth hour, the achieved inhibition was 49% (p<0,001) and 61% (p<0,001), respectively. In the treated groups, the edema evolution was inhibited by 38% (p<0,01), at the second hour after induction, as compared to the non-treated groups. At the fourth hour (peak of the carrageenan action on leakage) and at sixth hour the achieved inhibition was 35% (p<0,01) and 30% (p<0,05) respectively.

We present a new interference pattern based scheme for the cell diagnostics. In this approach the biological cell is illuminated by a spherical light source. The interference pattern that is created by the unscattered incident light and the light that is scattered by a cell is used for cell diagnostics. A three-dimensional computational code (AETHER) for solving the full set of Maxwell’s equations with the Finite-Difference Time-Domain (FDTD) method has been used to numerically determine the interference light intensity pattern between the unscattered incident light and the scattered light. Features of the interference patterns that are obtained for different cellular parameters and structures are discussed. We have numerically shown that the interference intensity pattern can replace the purely scattered light patterns currently used in cell diagnostics.

It is well known that the exact and analytical theoretical solution of any physical tasks can be a powerful instrument for the analysis. But it is well known too, that in the general light transport and scattering theories, which are always used in different laser medical applications, there are only few exact and analytical approaches exist to solve the important model tasks. It can be shown that the conventional mathematic theory of the Markov processes can also provide some exact and analytical solutions for a number of practically important cases. As an example the analytical solution of 1-D pure scattering task using the Markov processes formalism is presented. Some consequences of that for the general scattering theory and for the noninvasive medical diagnostic problems are discussed as well. For instance, this solution can predict an enhanced value of the experimental estimated transport scattering coefficient if the thin sample of biotissue is used. For the laser Doppler medical flowmetry in the case of a strong scattering and a low level power of laser our result can predict the appearing of additional spectra into the tissue’s output signals which can be wrong interpreted like a conventional Doppler spectrum.

The purpose of this study is to investigate the feasibility of using microneedles in comparison to Er:YAG skin surface laser ablation as a means to modify the epidermis of in-vitro hamster skin to facilitate delivery of topically applied hyper-osmotics such as glycerol into the skin to achieve optical skin clearing. This allows to temporarily reduce scattering of light in otherwise turbid tissues with potential applications pertaining to non-invasive optical imaging techniques such as optical coherence tomography (OCT) or therapeutic applications like laser blood vessel coagulation to treat port wine stains in skin. A portable, battery powered Er:YAG laser (Lasette) manufactured by Cell Robotics Inc. was used to produce holes in the stratum corneum and epidermis using individual 400 μs pulses causing localized ablation. Following each laser pulse the tissue was mechanically translated by 1 mm before another pulse was delivered. As an alternative method to the use of an expensive laser source requiring some kind of light scanning mechanism to treat larger skin areas efficiently, microneedles were investigated. They do not require an energy supply, are also pain-free and can be manufactured into arrays allowing treatment of larger skin areas. A single application forms micron scale holes in the stratum corneum through which topically applied skin clearing agents such as glycerol can penetrate into the tissue. In this feasibility study individual microneedles were used to manually induce holes in the skin each spaced approximately 1 mm apart from the other. Upon such epidermal modification by either technique, glycerol was then applied to the tissue surface and amplitude OCT measurements monitored changes of the optical properties of the tissue over time. Due to the geometry of the microneedle used in this study the cross sectional area of each hole in the epidermis was about 68% smaller than the comparable ablation site caused by an individual laser pulse. Results indicate enhanced skin clearing rates due to the induced holes in the stratum corneum in both cases by a factor of 5 to 8. Due to the larger area of laser ablation in comparison to the holes caused by microneedles, overall skin clearing rates are higher with the laser. However, localized data analysis near holes produced by either technique yields comparable results which show an increase in the clearing rate of up to 10 to 13 times over intact skin without any holes.

A fast scanning fiber-based system of Mueller-matrix optical coherence tomography was built to characterize the polarization properties of biological tissues with high spatial resolution. A polarization modulator with its fast-axis oriented at 45° in the source arm of the Michelson interferometer, driven by a sinusoidal wave, was used to continuously modulate the incident polarization states of both the sample and the reference arms. Two detection channels were used to detect the horizontal and vertical polarization components of the interference signals, which were used to calculate the roundtrip Jones matrix of the sample. The roundtrip polarization parameters of the sample were calculated from the measured Jones matrix. The system was successfully tested for both standard optical polarization elements and various types of biological samples.

Most optical tomography work within highly scattering media has employed coherence domain and time domain methodologies, both detecting the shortest path photons over the dominant randomly scattered background. Angular domain imaging instead uses micromachined collimators to observe only those photons within a small angle of the aligned laser light source, which simulations show are the shortest path photons, while rejecting heavily scattered light. These angular filters consist of micromachined silicon collimator channels 51 micron wide by 10 mm or 20 mm long on 102 micron spacing giving acceptance angles of 0.29 to 0.15 degrees on a CCD detector. Phantom test objects were observed in mediums ranging from 1 to 5 cm thick at scattered to ballistic ratios of 500,000:1 to 10,000,000:1 depending on the illumination pattern. Object detection was retained at the same scattering levels for either 1 cm or 5cm thick mediums, demonstrating little dependence on medium thickness. Detection was also independent of the object size: phantoms ranging from thin structures of 100 micron wide lines and spaces to 4 mm spheres were detected at approximately the same scattering ratios. Minimum size resolution depends on CCD pixel size, not the collimator characteristics. Furthermore, detection was a function of the scattering ratio produced after the phantom's position, not of the whole medium’s scattering ratio. This means objects nearer the detector are much more observable. Longer collimators significantly increase the scattered light rejection. Monte-Carlo simulations with angular tracking demonstrate the object size independence and are undertaken to verify the other behaviors.

We review the basic theory for the radiative transport equation governing light propagation in biological tissues. The Green's function is the fundamental solution to the transport equation from which all other solutions can be computed. We compute the Green's function as an analytical expansion in plane wave modes. We calculate these plane wave modes numerically using the discrete ordinate method. We use the Green's function to compute the point spread function in a half space composed of a uniform scattering and absorbing medium.

We designed the method for prevention of restenosis after balloon angioplasty using laser-induced bubble-collapse acoustic wave. This study was performed to evaluate the effect on smooth muscle cells (SMCs) by Ho:YAG laser (λ=2.10μm)-induced acoustic wave, in vitro and in vivo. The laser energy was delivered by a silica glass fiber into water. Sound pressure was measured with a hydrophone changing the laser energy. The laser-induced acoustic wave was loaded to SMCs in vitro. This acoustic effect on SMCs was measured by MTT assay. The acoustic wave loaded SMCs were controllably injured with the laser energy and laser shots. The balloon denudated rabbit aorta was used to evaluate in vivo effect. The laser-induced acoustic wave loaded aorta was extracted at 42 days after the laser irradiation, and was examined by Hematoxylin-Eosin staining. We found that the laser irradiation of 20 pulses with 60mJ/pulse prevented SMCs proliferation. We think the mechanism of this effect might be same as brachytherapy. We demonstrated the applicability of Ho:YAG laser-induced acoustic wave against vascular restenosis after balloon angioplasty.

Laser induced breakdown has the lowest energy threshold in the femtosecond domain, and is responsible for production of threshold ocular lesions. It has been proposed that multiphoton absorption may also contribute to ultrashort-pulse tissue damage, based on the observation that 33 fs, 810 nm pulse laser exposures caused more DNA breakage in cultured, primary RPE cells, compared to CW laser exposures delivering the same average power. Subsequent studies, demonstrating two-photon excitation of fluorescence in isolated RPE melanosomes, appeared to support the role of multiphoton absorption, but mainly at suprathreshold irradiance. Additional experiments have not found a consistent difference in the DNA strand breakage produced by ultrashort and CW threshold exposures. DNA damage appears to be dependent on the amount of melanin pigmentation in the cells, rather than the pulsewidth of the laser; current studies have found that, at threshold, CW and ultrashort pulse laser exposures produce almost identical amounts of DNA breakage. A theoretical analysis suggest that the number of photons delivered to the RPE melanosome during a single 33-fsec pulse at the ED₅₀ irradiance is insufficient to produce multiphoton excitation. This result appears to exclude the melanosome as a locus for two- or three-photon excitation; however, a structure with a larger effective absorption cross-section than the melanosome may interact with the laser pulses. One possibility is that the nuclear chromatin acts as a unit absorber of photons resulting in DNA damage, but this does not explain the near equivalence of ultrashort and CW exposures in the comet assay model. This equivalence indicated that multiphoton absorption is not a major contributor to the ultrashort pulse laser damage threshold in the near infrared.

To develop the noninvasive transdermal drug delivery system, pulsed lasers (argon-fluoride excimer laser (ArF laser) and erbium:yittrium aluminum garnet laser (Er:YAG laser)) were used to partially ablate the stratum corneum (SC), the upper layer of the skin. Because of the barrier function of the SC to drug permeation, the number of drugs especially macromolecules used in transdermal drug delivery system without skin irritation has been limited. Ultrastructural changes on the SC surface of ablated Yucatan micropig skin in vitro were observed with Environmental Scanning Electron Microscope. The result indicated that the structural changes varied according to each laser sources and irradiation conditions (laser fluences and numbers of pulses). Many granular structures of about 2 μm in diameter were observed in the ablated sites on ArF laser with lower fluence exposure (30 mJ/cm², 200 pulses), and plane structures in the sites with higher fluence exposure (80 mJ/cm², 80 pulses). In contrast, the ablation of Er:YAG laser created some pores of about 20 μm across on the surface of the SC. Under the irradiation condition of partial ablation, the skin permeability of macromolecule compound was enhanced. This partial SC ablation by pulsed laser could be possible candidate of the noninvasive transdermal drug delivery system with good physiological conditions of skin.

Using a Ti:Sapphire laser operating at 800nm and a repetition rate of 1 kHz, we investigated the damage induced to fresh cadaveric porcine liver after laser irradiation for pulse widths of 120-fs, 8ps, and 7-ns. The laser was held constant at a focal spot diameter of 100μm yielding a maximum fluence of 9J/cm². Then, using polarization optics, the energy per pulse was controlled to well below ablation threshold fluences. The tissue samples were translated under the laser via 0.1μm resolution encoded X-Y-Z motorized stages. After irradiation and fixation, we evaluated the tissues using brightfield light microscopy of Hematoxylin and Eosin stained 4 μm thick cross sections, scanning electron microscopy, and transmission electron microscopy. The tissue samples were examined for both removal rates of material, thermal damage to surrounding tissue, and cell disruption for equivalent fluence levels across the temporal range. We found an increase in removal rate along with a decrease in thermal damage as the pulse widths approached the femtosecond regime for a constant fluence. With femtosecond pulses, ablation still occurred below fluences of 2J/cm². However, for nanosecond pulses, ablation no longer occurred, showing a decrease in ablation threshold as the pulse width decreases. Because of the reduced thermal effects compared to nanosecond pulses, ultrafast lasers may offer a solution to more precise tissue removal with less damage to surrounding cells.

In this work we employ a novel probe-beam technique to access the plume dynamics in the laser ablation process. A helium-neon laser in the scattered-beam mode was used to probe the plume. The sample used was a chicken myocardium tissue. The scattered probe light was collected by a 600 mm optical fiber and sent to a ¼ m spectrometer. After finding an adequate acquisition time window, the scattered He-Ne laser light was analyzed for several delay times. The plume luminescence was also collected. While no defined spectral lines could be observed for a 100 μs acquisition time window, a large number of emission lines could be analyzed for the 5 ms gate, the optimum gate window. Our results also show that the ejected material starts to be registered by the spectrometer at about 40 μs after the ablation pulse has been fired. The measurement of the relative atomic composition by means of the laser ablation and the analysis of the ablation plume dynamics were performed. The results pointed out the presence of many elements including sodium, hydrogen and others. Understanding the mechanisms involved in tissue ablation allows reduced damage and better results depending on the application goals.

Optical spectroscopy stands as a powerful tool to gather information on the physical phenomena involved in the laser ablation process. This work reports on the time-resolved measurement of the ablation plume generated by a Q-switched Nd:YAG laser. Chicken heart tissue was used as sample. A helium-neon laser, aligned perpendicularly to the Nd:YAG laser beam and parallel to the ablated surface, was used as a probe beam. The probe light is collected by an optical fiber and sent to a spectrophotometer for analysis. A CCD detector was connected to the spectrometer. The technique reveals the differences in the plume behavior. Our results show that the plume is formed with some delay time with respect to the high energy laser pulse. The data show the constitution of the plume for different moments in time allowing the identification of the sodium emission lines in the chicken myocardium. For times lower than 40 μs just light components are ejected from the tissue while the heavier components of the plume takes much more time to be ejected and later to dissipate from the path of the probe beam.

A gas discharge strontium vapor laser has been shown to operate with up to 90% of its light emitted at 6.45 μm. We have investigated the use of this laser as a potential stand-alone, tabletop alternative to the FEL for ablation of soft tissue. This custom-made laser currently delivers up to 2.4 watts of average power at 13 kHz pulse repetition rate (range 5-20 kHz). Despite a poor spatial beam profile the laser has been shown to ablate both water and soft tissue. However, current pulse energies (< 185 μJ) are insufficient for single pulse ablation even when focused to the smallest possible spot size (130 μm). Instead, the high pulse repetition rate causes the ablation to occur in a quasi CW manner. The dynamics of ablation studied by pump-probe (Schlieren) imaging and macroscopic white light imaging showed micro-explosions but at a rate well below the pulse repetition frequency. Histological analysis of ablation craters in bovine muscle exhibited significant collateral thermal damage, consistent with the high pulse frequency, thermal superposition and heat diffusion. Efforts to increase the pulse energy in order to achieve the threshold for pulse-to-pulse ablation are ongoing and will be discussed.

Pulsed mid-infrared (6.45 μm) radiation has been shown to cut soft tissue with minimal collateral damage (<40 mm); however, the mechanism of ablation has not been elucidated to date. The goal of this research was to examine the role of the unique pulse structure of the Vanderbilt Mark-III FEL and its role in the efficient ablation of soft tissue with minimal collateral damage. The pulse structure consists of a 2.865 GHz train of one picosecond micropulses within a 4-5 μs macropulse envelope operated between 2 and 30 Hz. The effect of the picosecond micropulses was examined by running the native FEL pulse structure through a pulse stretcher in order to increase the micropulse length from 1 picosecond up to 100 picoseconds. This allowed us to determine whether or not the picosecond train of micropulses played any role in the ablation process. The pulse stretcher was varied between 1, 30, 60, and 100 picoseconds. The ablation threshold was determined for water and mouse dermis for each micropulse length using PROBIT analysis of 100 individual observations of the macropulse. The results of the analysis showed no statistical difference between 1 and 100 picoseconds. The ablation efficiency was also measured on 90% w/w gelatin and mouse dermis for the different micropulse lengths. Multiple ablation craters were made by varying the number of pulses delivered between 5 and 500. The ablated crater depth was measured using OCT. No significant difference was observed between 1 and 60 picoseconds; however, the 100 picosecond micropulse did show a reduction in the efficiency of ablation. We have shown that the effect of micropulse duration of the FEL on the ablation process is negligible between 1 and 100 picoseconds. Further analysis is needed beyond 100 picoseconds.

Theoretical work has been previously reported in which the full thermo-mechanical response of an absorber to an incident laser pulse has been calculated. For a laser pulse of any energy or duration, the temperature rise, explosive bubble formation, and shock wave generation in the surrounding medium can be predicted. This work allows the assessment of danger to biological or opto-electronic systems from laser pulses. The work also allows the thermo-mechanical properties of micro and nano particle absorbers, such as the thermal expansion coefficient and bulk modulus, to be calculated from measurements of the pressure waves generated in the medium. Previous results assumed a temporal profile for the laser pulse that was a square wave with infinitely fast rise and fall times. We report on new calculations that use a more realistic gaussian-like temporal profile for the laser pulse. We compare how the resulting thermo-mechanical responses are altered compared to the idealized temporal square wave laser pulse.

Ultrashort laser pulses are widely used now for biomedical diagnostics in order to reveal various tissue abnormities, such as tumors. In particular, the use of these pulses is preferable to investigate relatively thin objects (e.g., layers of human skin). In this case, femtosecond rather than longer pulses should be used due to more pronounced broadening of their temporal profiles for forward detected radiation. However ultrashort pulses are characterized by continuous spectra covering a range of wavelengths. As the optical properties of the probed medium depend on the wavelength, this may cause problems in deconvoluting the signal. In this paper, an attempt is made to investigate this problem. A two-layer model of the object mimicking the upper layers of skin (epidermis and dermis) are considered. Laser pulses of 3.9 to 62.5-fs duration are simulated to impinge onto a plane two-layered medium. The layers differ by optical parameters. The laser beam radius is 0.1 mm. Central wavelengths of spectra of the incident pulses lie in UV, blue, or red spectral regions. Absorption and scattering coefficients of the medium correspond to the real skin; the thicknesses of the successive layers are 0.1 and 0.2 mm respectively. It is shown that the δ-function spectrum approximation may be applied to laser pulses, which central wavelengths lie within the red spectrum region for all tested pulse durations. If the central wavelengths lie within the blue or UV regions, the spectrum width should be taken into account for pulses shorter than 60 fs.

Human retinal pigment epithelial (RPE) cells (hTERT-RPE1) were used to detect photo-oxidation products generated from chronic NIR (810 nm) laser exposure. Exposure of a discrete area within cell monolayers provided a means of distinguishing fluorescence above background levels. Oxidative stress was detected using the fluorescent dye H₂DCF-DA and its analog CM-H₂DCF-DA. Fluorescence was detected in cells exposed to mode-locked (76 MHz, ~160 femtoseconds) but not CW laser exposure. Detection of photo-oxidation from the mode-locked laser was dependent upon radiant exposure, but only if irradiance was greater than a threshold value. The CM-H₂DCF-DA dye proved a more sensitive indicator of oxidation than H₂DCF-DA, and the radiant exposure threshold for detection was dependent upon dye concentration. No oxidation was detected from CW exposures (using the most sensitive fluorescent dye conditions) when using 3 times the irradiance, and 10 times the radiant exposure needed to detect fluorescence from mode-locked exposure.

There are several data sources for collecting laser incidents. All reviewed sources collect information differently for varying purposes. An effort was undertaken to combine laser exposure reporting data into a single database so that trends in laser incidents could be identified. A review of available datasets revealed significant disparities in laser exposure reporting. As a result, utilizing the existing database to predict personnel at increased risk for laser exposure and injury is challenging if not impossible. For example, many of the data sources do not contain information about physical examinations, diagnosis, or medical follow-up, which are important for studying laser injury outcomes. This study proposes using the Delphi Technique to identify relevant fields that should be collected for a laser incident database based on the experiences of three groups of United States Air Force (USAF) professionals: (1) Engineers (Bioenvironmental Engineers), (2) Health Physicists, and (3) Physicians (Ophthalmologists and Flight Surgeons). In broad terms, these three professional groups coordinate laser incident analyses and investigations. Knowing what information is most important for studying laser incidents is the first step in establishing an effective database that will assist in identifying occupations that are at high-risk for laser injury. Robust data sets obtained for analysis by these healthcare professionals can be an effective tool for laser injury prevention and management.

We performed measurements to validate damage threshold trends in minimum visible lesion (MVL) studies as a function of spot size for nanosecond laser pulses. At threshold levels, nanosecond pulses produce microcavitation bubbles that expand and collapse around individual melanosomes. This microcavitation process damages the membranes of retinal pigment epithelium (RPE) cells. A spot size study on retinal explants found cell damage fluence (energy/area) thresholds were independent of spot size when microcavitation caused the damage, contradicting past in vivo retinal spot size experiments. The explant study (ex vivo) used a top-hat beam profile, whereas the in vivo studies used Gaussian beams. The difference in spot size trends for damage in vivo versus ex vivo may be attributed to the optics of the eye but this has not been validated. In this study, we exposed artificially pigmented human RPE cells (hTERT-RPE1)-in vitro-to 7 ns pulsed irradiation from a Ti:Sa TSA-02 regenerative amplifier (1055 nm) with beam diameters of 44, 86, and 273 μm (Gaussian beam profiles). We detected the microcavitation event with strobe illumination and time-resolved imaging. We used the fluorescent indicator dye calcein-AM, with excitation by an Argon laser (488 nm), to assess cell damage. Our current results follow trends found in the in vivo studies.

As the laser technology advances and the availability of high power femtosecond pulsed laser systems increase, the urgency to have damage thresholds and ED50 data on these new laser systems becomes more and more prominent. In this study, we have used >50 mJ, 30-50 femtosecond laser pulses at ~810 nm that produced self-focusing filaments in the atmosphere. Then, these high-powered (1-3 terawatt) filaments were placed on a grid pattern on a piece of chamois. The effects and the damage caused by the filaments were investigated. The results were compared to the damage threshold data. Ultimately, our purpose is to extend this study to porcine skin and to measure Minimum Visible Lesion (MVL) thresholds and to determine the ED50 for exposures at above mentioned laser pulses.

The dependence of retinal damage thresholds on laser spot size, for annular retinal beam profiles, was measured in vivo for 3 μs, 590 nm pulses from a flashlamp-pumped dye laser. Minimum Visible Lesion (MVL)ED₅₀ thresholds in rhesus were measured for annular retinal beam profiles covering 5, 10, and 20 mrad of visual field; which correspond to outer beam diameters of roughly 70, 160, and 300 μm, respectively, on the primate retina. Annular beam profiles at the retinal plane were achieved using a telescopic imaging system, with the focal properties of the eye represented as an equivalent thin lens, and all annular beam profiles had a 37% central obscuration. As a check on experimental data, theoretical MVL-ED₅₀ thresholds for annular beam exposures were calculated using the Thompson-Gerstman granular model of laser-induced thermal damage to the retina. Threshold calculations were performed for the three experimental beam diameters and for an intermediate case with an outer beam diameter of 230 μm. Results indicate that the threshold vs. spot size trends, for annular beams, are similar to the trends for top hat beams determined in a previous study; i.e., the threshold dose varies with the retinal image area for larger image sizes. The model correctly predicts the threshold vs. spot size trends seen in the biological data, for both annular and top hat retinal beam profiles.

This study examined the role of oxidative stress and the effect of a single dose treatment with N-Acetylcysteine (NAC) on the temporal development of acute laser-induced retinal injury. We used the snake eye/Scanning Laser Ophthalmoscope (SLO) model, an in vivo, non-invasive ocular imaging technique, which has the ability to image cellular retinal detail and allows for studying morphological changes of retinal injury over time. For this study 12 corn-snakes (Elaphe g. guttata) received 5 laser exposures per eye, followed by either a single dose of the antioxidant NAC (150mg/kg, IP in sterile saline) or placebo. Laser exposures were made with a Nd: VO₄ DPSS, 532nm laser, coaxially aligned to the SLO. Shuttered pulses were 20msec x 50 mW; 1mJ each. Retinal images were taken using a Rodenstock cSLO and were digitally recorded at 1, 6, 24-hrs, and at 3-wks post-exposure. Lesions were assessed by two raters blind to the conditions of the study yielding measures of damaged area and counts of missing or damaged photoreceptors. Treated eyes showed a significant beneficial effect overall, and these results suggest that oxidative stress plays a role in laser-induced retinal injury. The use of NAC or a similar antioxidant shows promise as a therapeutic tool.

Our recent research has shown that skin becomes temporarily transparent when a hyper-osmotic agent such as glycerol is introduced into the tissue. Local dehydration and index matching reduce light scattering which increases the penetration depth of collimated light. We have shown that when glycerol is applied to in vivo hamster skin, the resulting transparency is sufficient to allow visualization of blood vessels, and there is a temporary reduction in local blood flow. The reduced blood flow combined with greater light delivery significantly reduces the laser fluence rate [W/cm²] required to coagulate dermal blood vessels.

The possibility to induce selective hyperthermia in a target tissue or organ is of great interest for the treatment of cancer and other diseases. An emerging application of thermotherapy is for choroidal neovascularization, a complication of age-related macular degeneration. The therapy is currently limited because the temperature required for optimal tissue response is unknown. We report here an investigation of near infrared laser-induced heating in an ocular phantom. Magnetic resonance thermography (MRT) was used as a non-invasive method to determine the temperature distribution inside the phantom during exposure to a continuous wave diode laser at 806 nm wavelength with 1 watt maximum output. The laser beam had a quasi-gaussian profile, with a radius of 0.8-2.4 mm at target. High quality temperature images were obtained from temperature-dependent phase shifts in the proton resonance frequency with a resolution of 1deg C or better, using a 2T magnet. A phantom with a layer of bovine RPE melanin of 1.5 mm thickness was used to determine the spatial resolution of the MRT measurements. Three dimensional temperature maps were also constructed showing a spatial resolution of 0.25 mm in all direction. The heat distribution depended on the laser parameters, as well as the orientation of the melanin layer with respect to the incident laser beam. The temperature profiles determined by MRT closely followed predictions of a heat diffusion model, based on the optical properties of infrared light in melanin. These results support the use of MRT to optimize laser-induced hyperthermia in a small organ such as the eye.

We have measured the Minimum Visible Lesion (MVL) thresholds for porcine skin and determined the ED50 for exposures at 1314 nm and 0.35 ms laser pulses. An in-vivo pigmented animal model, Yucatan mini-pig (Sus scrofa domestica), was used in this study. We also have measured the thermal response using a high-speed Infrared camera for single pulse temperature recordings for Gaussian beams of 1 mm diameter. Several 2-D measurements of temperature as a function of time were made with an IR array detector thermal camera using a sampling rate of 100 frames per second. In Vitro samples of the same pig skin were used for measurements of the optical properties (absorption coefficient, μ_a, and reduced scattering coefficient μ_s) as a function of wavelength around 1315 nm wavelength. A measured surface temperature distribution for one IR laser pulse of 0.37J at a spot size of 1.2 mm diameter gave approximately a 43° C rise at a hot spot. Temperature distributions as a function of time and space will be presented and compared with the measured thresholds.

A new source-term thermal model was used to determine the skin temperature rise using porcine skin parameters for various wavelengths, pulse durations, and laser spot sizes and is compared to the Takata thermal model. Expanding on this preliminary source-term model using a Gaussian profile to describe the spatial extent of laser pulse interaction in skin, we report on the coupling of temporal consideration to the model. Computer simulation of the new source-term model and the Takata thermal model are presented to highlight the theoretical extent of thermal damage. Laser exposures of 1.54 μm, 0.60 ms in duration and using spot sizes of 0.7 mm and 1.0 mm were applied to the porcine skin. The damage thresholds were determined at 1 hour and 24 hours post-exposures using probit analysis. The ED₅₀ for these skin exposures at 24 hours post-exposure were 20 J/cm²and 8.1 J/cm²respectively. These damage thresholds are compared with our model predictions and another thermal model with the damage integral predicting damage levels. They are also compared with previously published skin thresholds and with the ANSI Standard’s MPE for 1540 nm lasers at 0.60 ms.

To properly assess the retinal hazards from several lasers using multiple wavelengths, the retinal effects of 10-second laser irradiation from 532 and 860 nm were determined in non-human primates for several different power combinations of these wavelengths. A total of 12 eyes were exposed using four different ratios of power levels to determine the contribution to the damage levels from each wavelength. The data are compared to the calculations resulting from use of the currently accepted method of predicting hazards from simultaneous laser. The ANSI-Z136 - 2000 standard was used to calculate the combined maximum permissible exposure (MPE) and for comparison with the measured visible lesion thresholds, i.e., ED₅₀s.

Two-dimensional electrophoresis and histomorphometry were used to determine if equivalent protein changes occurred within native rabbit corneas and engineered corneal tissue models following in vitro exposure to single pulse, 1540 nm laser light operating at a pulse width of 0.8 milliseconds. Frozen sections of exposed tissues were processed to detect laser-induced protein changes. Isoelectric points, molecular weights and relative densities were used to characterize corneal proteins of interest that were then identified using MALDI-MS peptide fragment analysis. Increasing radiant exposures of corneal tissues were associated with progressively more severe necrosis of the epithelium and stroma in both the native and engineered tissues.

Five male Yorkshire pigs were exposed on their flank to 4 microsecond pulses of laser light from a Deuterium Fluoride 3.8 micron Laser at varying energies. A preliminary ED₅₀ threshold for various skin reactions was determined for this laser exposure combination. The animal’s skin was assessed for injury immediately, 1 hour, 24 hours and 72 hours post exposure. In general, energies below 3.2 J/cm² leave no lasting skin reaction. As energy increased above the threshold, erythema or skin reddening was easily visualized. High-energy pulses appear to produce a “rug burn” erythema without evidence of punctate hemorrage (bleeding) or coagulation. Laser exposure sites on the pigs were also biopsied to obtain histopathological results. These findings suggest that the principal effect of this type of in-vivo laser exposure is removal of the epithelium, while not damaging the papillary dermis or structures beneath the Basement Membrane Zone (BMZ).

The purpose of this review is to compile information on the optical and healing properties of the cornea when exposed to infrared lasers. Our long-term goal is to optimize the treatment parameters for corneal injuries after exposure to infrared laser systems. The majority of the information currently available in the literature focuses on corneal healing after therapeutic vision correction surgery with LASIK or PRK. Only a limited amount of information is available on corneal healing after injury with an infrared laser system. In this review we will speculate on infrared photon energy absorption in corneal injury and healing to include the role of the tear layer. The aim of this review is to gain a better understanding of infrared energy absorption in the cornea and how it might impact healing.

The inflammatory process can be considered as a tissue protective response to an aggressive stimulus. That process leads to an increase in vascular permeability and, consequently, edema formation. In this study it is shown that the electrical capacitance can be used as a tool for the monitoring of the time evolution of an edema in biological tissues and that the method can sense the modulating effect of low power laser therapy. The electrical capacitance was measured during the edema settling up in rats after induction of acute inflammation by carrageenan injections, associated or not with low power laser therapy. A LCR meter model LCR-815B from HP, was used to measure the electrical capacitance between two electrodes positioned onto the rat skin, in the edematous site. Measurements were taken every 15 min. All rats were anesthetized to overcome electrical capacitance variations. Rats were divided into three groups: i) only anesthetic was injected (0.3 ml of Zoletil 50); ii) anesthetic and 1 ml of carrageenin at 2%; iii) same as group (ii) plus treatment with 2.5 J/cm² from a GaAlAs laser (650 nm). A maximum on the capacitance variation was observed when the anesthetic and the carrageenin were injected. Lower values were obtained for the laser treated group, which corroborated with the anti-inflammatory effect of the laser therapy. The electric capacitance accompanied the settling up and down of the edemas for all animals.

The near-infrared (NIR) laser radiation due to its high penetration depth is widely used in phototherapy. In application to skin appendages a high selectivity of laser treatment is needed to prevent light action on surrounding tissues. Indocyanine Green (ICG) dye may provide a high selectivity of treatment due to effective ICG uploading by a target and its narrow band of considerable absorption just at the wavelength of the NIR diode laser. The goal of this study is to demonstrate the efficacy of the NIR diode laser phototherapy in combination with topical application of ICG suggested for soft and thermal treatment of acne vulgaris. 28 volunteers with facile or back-located acne were enrolled. Skin sites of subjects were stained by ICG and irradiated by NIR laser-diode light (803 or 809 nm). Untreated, only stained and only light irradiated skin areas served as controls. For soft acne treatment, the low-intensity (803 nm, 10 - 50 mW/cm², 5-10 min) or the medium-intensity (809 nm, 150 - 190 mW/cm², 15 min) protocols were used. The single and multiple (up to 8-9) treatments were provided. The individual acne lesions were photothermally treated at 18 W/cm² (803 nm, 0.5 sec) without skin surface cooling or at 200 W/cm² (809 nm, 0.5 sec) with cooling. The results of the observations during 1-2 months after the completion of the treatment have shown that only in the case of the multiple-wise treatment a combined action of ICG and NIR irradiation reduces inflammation and improves skin state during a month without any side effects. At high power densities (up to 200 W/cm²) ICG stained acne inflammatory elements were destructed for light exposures of 0.5 sec. Based on the concept that hair follicle, especially sebaceous gland, can be intensively and selectively stained by ICG due to dye diffusion through pilosebaceous canal and its fast uptake by living microorganisms, by vital keratinocytes of epithelium of the canal and sebaceous duct, and by rapidly proliferating sebocytes, new technologies of soft and thermal acne lesions treatment that could be used in clinical treatment of acne were proposed.

We report on a novel mechanism of tissue modification. We demonstrate that if the train of ultrashort laser pulses has sufficient average power, multiphoton absorption can result in a significant heating of a localized area of a collagen-rich tissue leading to its photothermal modification. We propose a simple model describing the effect of local heating and derive a simple way of estimating the contribution of this mechanism for different parameters of incident radiation. Microscopic confocal imaging studies complement the spectroscopic studies to ensure the minimal destruction of surrounding areas of a tissue.

Photothermal (PT) technique was applied to optimizing selective photothermolysis of cancer cells and bacteria into which nanoparticles have been incorporated (selective “nanophotothermolysis”). This technique involved first irradiating nanoparticles-penetrated cells with nanosecond pump-laser pulses in the visible spectral ranges. Laser-induced local thermal effects around the nanoparticles in the cancer cells or bacteria were then detected via time-resolved monitoring of temperature-dependent variations of the refractive index. This procedure was accomplished with imaging of a second probe-laser pulse. Analysis of the distinctive temporal shape of the PT response revealed linear and nonlinear phenomena around nanoparticles, such as alteration of local temperature, and bubble-formation-caused cell death accompanied by laser-induced melting and disintegration of particles. The damage threshold was obtained for live cancer cells in vitro depended on the size (range: 2-250 nm) and number of particles, laser energy, and number of pulses. Local heat-based induction of apoptosis and necrosis was controlled in parallel with conventional kits (e.g. trypan blue, Annexin V-propidium iodide) and optical and electron microscopy. The PT technique potentially allowed for the detection of nanoparticles that had been delivered into live cells by direct microinjection, natural diffusion, and selective targeting with antibodies.

A physical model describing the propagation of low frequency surface waves in relation to the viscoelastic behavior of porcine skin is presented along with a series of empirical studies testing the performance of the model. The model assumes that the skin behaves as a semi-infinite, locally isotropic, viscoelastic half-space. While the assumption of a semi-infinite body is violated, this violation does not appear to have a significant impact on the performance of the model based upon the empirical studies. One Hertz surface waves in the skin propagate primarily as Rayleigh waves with a wavelength and velocity of approximately 3 m and 3.0 m/s, respectively. The amplitude of the acoustic wave as measured by tracking the acoustic stress wave - induced shift in a backscattered laser speckle pattern, decreased exponentially with lateral distance from the acoustic source. Using this model of surface wave propagation, the mechanical loss factor or tan δ of the skin was measured to be on the order of 0.14±0.07. The results presented herein are consistent with earlier works on the propagation of low frequency acoustic waves in biological tissues and should serve as a theoretical and empirical basis for using the wave characteristics of propagating surface waves in combination with the mechanical behavior of the tissue for biomechanical studies and for potential diagnostic applications.

Purpose: The aim of the present study was to characterize permissible exposure limits (MPE) for safety analysis, with an emphasis on the immediate retinal damage following SHG of Nd:YAG Q-Switched laser radiation and to test its correlation to physical parameters. Methods: Pigmented rabbits (n=14) were exposed to single pulses of Nd:YAG laser radiation (532nm, pulse duration:8-12ns) in various energies ranging from 10 to 150 μJ. Exposures were conducted in retina tissue, very close to the optic nerve, with a total of 20 exposures per retina. Retinas were viewed during the first 15 min following exposure, using an on-line digital video camera. Thereafter, animals were sacrificed for histological evaluation. A quantitative analysis of the clinical findings, based on a severity score scale and a morphometric analysis of the extent of the lesions, was used to test the relationship with the laser energy. In addition, hemorrhage thresholds were computed using Probit Analysis. Results: Retinal damage, at various levels of severity, was observed immediately after exposure to energies above 26 μJ, characterized by edema and sub-retinal hemorrhages. The appearance and severity of the lesions varied among animals, between fellow eyes and even within the same retina. The ED50 for immediate pre-retinal hemorrhage was determined as 83μJ and the lesions’ diameter ranged from 141-640μ. A significant correlation (R=0.80, P<0.0001) was found between the extent of the lesions and energy levels. The diameter of the lesions showed a linear (P<0.008) increase with the laser energy. The histological observations indicated elevation of retinal layers and extensive damage in the outer segment of the photoreceptors and in the pigmented epithelial cells layer. Conclusions: A linear, laser-retinal tissue interaction was found immediately following exposure to single pulses of Nd:YAG laser radiation. It is suggested that unlike argon laser, which produces a thermal burn to the eye, Nd:YAG laser damage is a result of a combination of photo-mechanical and thermal mechanism.