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A novel immunoadjuvant, glycated chitosan, has been used in combinations with a near-infrared laser and a laser- absorbing dye to treat metastatic tumors in rats. The laser-dye combination provides selective photothermal tumor destruction. The addition of the in situ immunoadjuvant works in tandem with the photothermal interaction to induce a host antitumor immunity. Our previous experiments have shown the efficacy of this novel modality against a metastatic breast cancer in rat model, using the three components. The current study is to investigate the roles of different components, namely, the laser, the dye and the immunoadjuvant. Firs, the selective photothermal laser- tissue interactions are studied in vivo using rat leg muscles and rat tumors. Our results showed that with appropriate combination of laser parameter and dye does, an optimal selective photothermal tissue interaction could be achieved. The immune response is crucial in control of tumor metastasis and the immunoadjuvant has played pivotal role in the induction of the immunity in our experiment. Therefore, the role of immunoadjuvants in the laser cancer treatment is also investigated in the current study. Specifically, three different concentrations of glycated chitosan solutions - 0.5%, 1% and 2% - were used. In comparison, the 1% solution provided the best treatment outcome. Two additional immunoadjuvants, incomplete Freund's adjuvant and complete Freund's adjuvant were also used in the same laser-dye-adjuvant treatment protocol. The functions of different adjuvants are compared.
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The immune system is implicated in the mechanism of tumor destruction following photodynamic therapy (PDT). Several investigators have shown that immune stimulation can augment PDT. In this study, a single intratumoral injection of ENHANZYNTM adjuvant was administered to tumor-bearing mice immediately following verteporfin PDT in a therapeutic modality referred to as Photodynamic Vaccination (PDV). After optimal PDT, little difference in the rate of tumor re-growth or time to tumor reappearance was seen upon addition of the adjuvant. This may be as expected as this treatment regimen results in effective long-term tumor cure in mice. The effect of adjuvant and sub-optimal PDT was less clear as both groups treated with either a high or low does of adjuvant showed tumor re-growth earlier than those animals treated with PDT alone. However, tumors of mice receiving sub-optimal PDT followed by high dose immune adjuvant did not show the rapid, uncontrolled growth seen in other groups and, in the majority of cases, tumor volume decreased steadily with time. This resulted in a superior period of survival despite the animals being tumor-bearing. Interestingly, the data obtained in this study clearly demonstrates the ability of PDT to protect against re- challenge with a second round of tumor implantation. This was seen in all groups and stresses the importance of the immune response in PDT tumor control. Addition of the high immune adjuvant does to sub-optimal PDT appeared to be the most effective treatment group in this respect, giving complete protection against tumor re-implantation.
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The treatment of solid cancerous lesions by photodynamic therapy (PDT) elicits an acute host reaction primarily manifested as a strong, rapidly developing inflammatory response. It is becoming increasingly clear that the destructive impact of the inflammatory process is directly responsible for the so-called indirect damage in PDT-treated tumors. The loss of vascular homeostasis followed by massive damage to vascular and perivascular regions in PDT- treated tumors and the ensuing tumor antigen-specific immunity, are direct consequences of critical initiating events including the action of complement, activation of poly(ADP-ribose)polymerase (PARP) and ischemia/reperfusion insult, and the associated cascades of tissue-destructive responses. Hence, the effectiveness of PDT as an anti- cancer modality is largely owed to the fact that it instigates a comprehensive engagement of powerful innate host defense mechanisms.
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Several groups, including our own, have reported that PDT enhances the host anti-tumor immune response and it is known that the enhanced immune response plays a role in the overall tumor response to PDT. The mechanism behind this enhancement is unknown, however it has been shown that the initiation of an inflammatory response and the infiltration of neutrophils into the tumor bed is critical to the tumor response. We have shown that PDT induces the expression of chemokines that play a critical role in neutrophil infiltration. Recent studies in our laboratory have shown that in addition to affecting the inflammatory and chemokine/cytokine response, PDT also alters the immunogenicity of the tumor, either through changes in antigen structure or enhancement of presentation of tumor associated antigens by host antigen presenting cells to tumor specific T cells. These recent studies and the underlying mechanisms will be discussed.
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This article is an attempt to analyze the concept, distinguishing features and possible application of photo- pharmaceutical therapy (PPT). Besides photopheresis, PUVA, and photodynamic therapy, PPT also embraces a broad spectrum of various combinations of light and drugs. PPT techniques can be classified according to the role of light in drug therapy into several groups: 1) Light activation of drugs before, during or after their administration, 2) light activation of cells of biotissue to potentiate the pharmaceutical effect of drugs, 3) light assisted drug delivery, 4) optical sensing of drug action at cellular and subcellular levels, and 5) selective photochemistry of drugs during their manufacturing. PPT seeks to describe the mechanisms of light-drug interaction, to time and sequence light-drug action, and to verify their synergetic effect. This article yields the results of developing new PPT modifications created in collaboration with some Russian scientific institutes and medical centers. The developed modifications are as follows: 1) drug pre-administration photoactivation, 2) antibody-photoconformation photoimmunotherapy, 3) photophonophoresis with a blend of photosensitizers and antibiotics, 4) photoelectrophoresis, 5) drug effect enhancement due to laser-induced blood circulation activation, 6) photoimmunization with alpha- fetoprotein, 7) photo-pharmaceutical dosimetry, and 8) a rapid drug toxicity photoassay.
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An indwelling balloon applicator developed for postoperative intracavity brachytherapy was evaluated for photodynamic therapy. Measurements of light distributions in a brain phantom show that the applicator can be used to deliver sufficiently uniform light doses during PDT. The light distribution is uniform to within 5% when the balloon is filled with a scattering medium. Based on simple assumptions, it is shown that the applicator can be used to deliver a threshold optical dose to brain tissue at depths of 1.4 cm in less than 90 minutes. A mathematical model of the thermal distribution around the applicator suggests that tissue temperatures will be below the hyperthermic threshold at the input powers required for treatments to depths of 1.4 cm in the resection cavity. The delivery of threshold light doses to depths exceeding 1.4 cm is likely to result in hyperthermic effects to tissues near the applicator surface.
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The FDA has approved PDT using Photofrin for certain esophageal and lung cancers, specifying an approved prescription of administered drug (mg/kg body weight) and administered light (J/linear cm of cylindrical fiber). This paper describes our development of a multi-optical fiber catheter for endoscopic use which documents the drug accumulated in the target tissues and the light penetration into the target tissues. The catheter uses reflectance to specify the light penetration depth and uses reflectance- corrected fluorescence to document drug accumulation. The goal is to document the variation in drug and light received by patients who are administered the FDA-approved prescription.
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The aim of the present work was to study the response of hemoglobin oxygen saturation and relative blood volume in human skin in vivo to laser irradiation. The hemoglobin oxygen saturation and relative hemoglobin concentration in skin were evaluated from diffuse reflectance spectra in visible wavelength range. The skin spot at human hand was irradiated with laser beam and hemoglobin oxygen saturation and relative hemoglobin concentration were sampled every two seconds from the center of the irradiated spot. It was evidently observed that hemoglobin oxygen saturation is increased after starting irradiation. During occlusion the oxygen consumption rate was higher in the presence of laser irradiation. However, these effects were observed only at sufficiently high laser fluence rates. The most probable reason is that it is due to thermal effects.
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This paper considers the fraction PDT-induced oxidizing radicals that react with a specific oxidizable target within a cell rather than with all possible oxidizable sites. There are many oxidizable sites within the cell, each with a different efficiency of oxidation (Y_ox_j) and a different in vivo concentration (C_iv_j). One measures the efficiency of oxidation of a single ith chemical species in vitro (Y_it_i), then measures the oxidation of the same species in vivo (Y_iv_i). The concentration of this ith species in vivo must be measured (C_iv_i). A convenient test chemical species is chosen, such as a photobleachable fluorophore. Then the in vivo yield is approximately: Y_iv_i = (C_iv_i*Y_it_i)/sum_all_j(C_iv_j*Y_iv_j) (eq.1). Rearranging to solve for the total oxidation: Sum_all_j(C_iv_j*Y_iv_j) = (C_iv_i*Y_it_i)/Y_iv_I (Eq.2) Once the sum_all_j() in Eq.2 is specified, one can measure the in vitro oxidation efficiency and the in vivo concentration of any ith species and use Eq.1 to predict the fraction of PDT_generated singlet oxygen that will attack that ith species in vivo. Of course, the above is only a first approximation toward a complex problem but is a beginning. This paper illustrates the experimental specification of the Y_ox_j for NADPh oxidation in a cuvette using the photosensitizer Photofrin.
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Mueller matrices provide a complete characterization of the optical polarization properties of biological tissue. A polarization-sensitive optical coherence tomography (OCT) system was built and used to investigate the optical polarization properties of biological tissues and other turbid media. The apparent degree of polarization (DOP) of the backscattered light was measured with both liquid and solid scattering samples. The DOP maintains the value of unity within the detectable depth for the solid sample while the DOP decreases with the optical depth for the liquid sample. Two-dimensional depth-resolved images of the full Mueller matrices of biological tissues were measured with this system. These polarization measurements revealed some tissue structures that are not perceptible with standard OCT.
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In recent years, several investigators have begun to explore polarized light imagery as a potential diagnostic tool. For example, polarimetric images have shown promise in identifying a variety of dermatological conditions. Because tissues tend to depolarize a large fraction (~85%) of incident light, the Mueller calculus lends itself well to these applications. A particular property of the Mueller matrix, the Depolarization Index, has demonstrated promise in discriminating between cancerous and benign moles. In this paper, we discuss the depolarizing aspects of tissues, however we primarily attempt to analyze the small fraction of light that has retained a polarization state. Analyzing the residual polarizing properties of a sample is challenging, and it requires a polar decomposition of the measured Mueller matrix into the basic properties of diattenuation, retardance, and depolarization. The diattenuation and retardance images contain information about the complex refractive index of the tissue, including any spatial variations in the index. We present measurements of the diattenuation and retardance of laser light reflected from skin as a function if incident angle and scattered angle.
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Using radiative transfer, we investigate linear and circular polarized light normally impinging a plane-parallel medium containing a random distribution of identically sized latex spheres in water. The focus of this study is to understand fundamental properties of polarized light scattering. In particular, we analyze backscattered and transmitted flux responses computed form direct numerical simulations. Form these numerical computations, we observe that circular polarized light depolarizes at a slower rate than linear polarized light. In addition, circular polarized light shows a more noticeable dependence on the size of the scatterers than linear polarized light. Furthermore, the helicity flip observed in circular polarized backscattered light is a fundamental phenomenon manifested by low order scattering.
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Monte Carlo method was used to simulate time resolved polarization imaging in turbid media. Mie theory was used to calculate the Meuller matrix of a single scattering event. In the simulation, the Stokes vector of each incident photon package was traced. The summation of the Stokes vectors of the traced photon packages gave the total output Stokes vector. The time integrated Mueller matrix of transmittance and reflectance light of a turbid media were calculated. The transmittance Mueller matrix and reflectance Mueller matrix have very different patterns. The time resolved 2D images of degree of polarization (DOP) for transmitted light and reflected light were calculated. The patterns showed different features for linearly polarized incident light and for circularly polarized light. The DOP patterns were also related to the scattering properties of the sample. The time resolved 2D DOP of the internal optical flux was also calculated. The DOP evolution was demonstrated vividly by the simulation results. The different patterns for linearly/circularly polarized light were compared. Linearly polarized light survived longer in turbid media with a small particle size. Circularly polarized light survived longer in turbid media with a larger particle size.
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Propagation of polarized light through liver, muscle and skin was studied using the Mueller Matrix formalism. Collimated HeNe laser light was passed through a set of polarization elements to create one of four possible polarization states (horizontal (H), vertical (V) and 45- degree (P) orientations of linearly polarized light, and right circularly (R) polarized light). The beam passed through thin sections of tissue of varying thickness (0.2- 0.9 mm thick). The unscattered, collimated, transmitted light passed through a second set of polarization elements to analyze for transmission of each of the 4 possible polarization states (H,V,P,R). Transmitted intensities for 16 possible combinations of source and detector polarization yielded a data matrix that was converted into a Mueller matrix describing the propagation properties of the tissue. The results were roughly consistent with all three tissue types behaving as ideal retarders whose birefringent values, dn = (Delta) *wavelength/(2*(pi) *thickness), were in the range of 1x10-3 to 5x10-3 which is consistent with the literature. The order of the strength of birefringence was liver < muscle < skin. Although the above birefringence values may apply to muscle, the structure of liver and skin are not necessarily consistent with the ideal retarder model and further work is needed.
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We have shown in previous work that the threshold for laser- induced breakdown is higher than the threshold for ophthalmoscopically visible retinal damage, but they approach each other as pulse duration decreased form several nanoseconds to 100 femtoseconds. We discuss the most recent data collected for sub-50 fs laser induced breakdown thresholds and retinal damage thresholds. With these short pulse durations, the chromatic dispersion effect on the pulse should be considered to gain a full understanding of the mechanisms for damage. We discuss the most likely damage mechanisms operative in this pulse width regime and discuss relevance to laser safety.
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We have measured the optical density of various laser eye protection samples as a function of increasing irradiance. We show that the protective quality of some eyewear degrades as irradiance increases. In previous studies this problem has been demonstrated in samples irradiated by nanosecond pulses, but the current study shows that the modern laser eye protection seems to be robust except for the irradiance possible with ultrashort laser pulse exposure. We discuss the most likely saturation mechanisms in this pulse duration regime and discuss relevance to laser safety.
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Retinal pigment epithelial (RPE) cells of the vertebrate eye contain melanin packaged in structures called melanosomes. Previously, we reported that the bovine melanosome has a laser photodisruption threshold of 153.6 mJ/cm2 at 25°C and 532 nm, and an internal absorption coefficient of 2237 cm-1. Those values used an estimate of melanosome density inferred from studies of skin laser effects. We now revisit that calculation using a density value obtained from density centrifugation analysis of bovine and baboon RPE melanosomes. Stepped-density gradients (60% to 80%) of the nonionic medium, Nycodenz, were formed, and samples of melanosomes were spun on the gradients in a swinging-bucket rotor at 10,000 rpm for 60 m. Bovine melanosomes formed two populations, one at the interface between 70% and 75%, and the other between 75% and 80%, corresponding to the densities of ~1.38 and ~1.41 gm/cm3, respectively. Baboon melanosomes migrated to within the same density region. Using a density value of 1.41 gm/cm3, and assuming a water content of 52% for hydrated melanosomes, the internal absorption coefficient was calculated as 2339 cm-1. Although this calculation uses an objective density measurement, the water content remains an estimate, and the actual value in situ may differ.
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Purpose: The direct comparison of in-vivo OCT images with fixed tissues sections assumes the fixation of tissue has no effect on the size and configuration of final pathology images such as light micrographs. Fixation artifact has been a concern in numerous studies of the pathology of retinal laser lesions. We tested this hypothesis. Methods: The Humphrey OCT model 2000 with a custom mirror and lens assembly was used to scan tissue phantoms and both fresh and fixed ex-vivum tissue samples. The optical configuration was determined by optimization of the contrast and signal strength on tissue phantoms. Fresh porcine retinas were scanned using this optimal configuration, then fixed using either glutaraldehyde or formalin. OCT images were taken of the tissue at various stages during the fixation process. Additionally, we examined fixed retinal tissue containing retinal laser lesions as a part of our study of ultrashort-pulsed laser effects on the macacca mulatta retina. Histologic sections were prepared and evaluated. Results: In this presentation, we describe our optical setup and image optimization process and assess the effects of glutaraldehyde and formalin processing on OCT image quality. The OCT images of glutaraldehyde-fixed laser lesions are compared with similar images of laser lesions in-vivo. Fixation artifacts appeared on OCT at 2 to 24 hours. Opacification of the lumen of large vessels was seen at two hours with both glutaraldehyde and formalin, while fixation induced retinal detachment appeared at 24 hours. Overall, there was a grater delineation of the laser lesions by OCT at 24 hours when compared to at 1 or 2 hours of fixation. Conclusions: Fixations induced changes in OCT scans of retinal tissue are present as early as 2 hours after immersion in fixative. Although both glutaraldehyde and formalin fixation preserve much of the tissue structure, these method of fixation have s significant effect on OCT imaging of both normal retinal tissue and laser lesions.
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We have developed a detailed theoretical model that allows for the prediction of the shockwave strength and bubble size that are expected to result in the retina as a result of a laser pulse of any pulse duration or energy. The results of the calculations for the shockwave and bubble size depend on the absorption coefficient and unknown thermo-mechanical properties of the absorbing melanosomes. We discuss how the shock strength and bubble size depend on melanosome parameters such as absorption coefficient, thermal coefficient of expansion and bulk modulus. We also describe experiments that could be performed to measure these coefficients in spite of the difficulty presented by the small size of the melanosomes.
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Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.
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We investigated the interaction of a laser-induced cavitation bubble with an elastic tissue model by high-speed photography with up to 5 Mill. frames/sec. The elastic material consisted of a transparent polyacrylamide (PAA) gel whose elastic properties can be controlled by modifying the water content to mimic various biological tissues. The elastic modulus E of the PAA sample was varied between 0.017 and 2 MPa. The dimensionless bubble-boundary distance γ(distance between laser focus and sample boundary, scaled by the maximum bubble radius) was for each value of E varied between γ = 0 and γ = 2.2. In this parameter space, we determined the jetting behavior, jet velocity, jet penetration into the PAA sample and bubble- induced removal of PAA material. The jetting behavior varies between unidirectional jets towards or away from the boundary, and formation of an annular jet which results in bubble splitting and subsequent formation of two very fast axial jets flowing simultaneously towards the boundary and away from it. General principles of the formation of annular and axial jets are discussed which allow to interpret the complex dynamics. The liquid jet directed away form the boundary reaches a maximum velocity between 300 m/s and 600 m/s (depending on E) while the peak velocity of the jet directed towards the boundary ranges between 400 m/s and 960 m/s. The peak velocities near an elastic material are 10 times higher than close to a rigid boundary. The liquid jet penetrates PAA samples with an elastic modulus in the intermediate range 0.12 < E < 0.4 MPa. In this same range of elastic moduli and for small γ-values, PAA material is ejected into the surrounding liquid due to the elastic rebound of the sample surface that was deformed during bubble expansion. The surface of the PAA sample is, furthermore, lifted during bubble collapse when a region of low pressure develops between bubble and sample. For stiffer boundaries, only an axial liquid jet towards the boundary is formed, similar to the bubble dynamics next to a rigid wall. For softer sample, the liquid jet is directed away from the boundary, and material is torn off the PAA sample during bubble collapse, if the bubble is produced close to the boundary. These processes play an important role for the efficiency and side effects of pulsed laser surgery inside the human body.
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The effects of wavelength on infrared (IR) laser ablation with a free electron laser were studied by analyzing the acoustic signals produced during ablation of gelatin and tissue samples. The acoustic signals resulting from surface ablation of the samples were recorded with a piezoelectric microphone and the acoustic energy contained in the signal was calculated for samples of varying mechanical strength. Gelatin samples of different mechanical strengths were made by varying the water concentration in the gels to 70% and 90% wt./vol. The gels were irradiated at wavelengths of λ = 2.94, 2.80, and 6.45 μm with the measured acoustic energy normalized to the incident laser pulse energy. The results showed that while there was a statistically significant difference in the average acoustic energy measured for both concentrations of gelatin at λ = 2.94 and 2.80 μm, there was no difference in the average acoustic energy for the two concentrations of gelatin at λ = 6.45 micrometers . This supports the model of mechanical weakening of the sample by breaking the amide II molecular bonds in proteins, originally proposed by Edwards et al.
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Holmium laser pulses (λ= 2.1 m) are often used for medical laser applications inside the human body, because they can be transmitted through low-OH quartz fibers, and they are relatively well absorbed in water and biological tissues. However, large thermal damage zones were observed after application of free-running holmium laser pulses for arthroscopic surgery. The aim of our study is to reduce thermal damage without introducing additional mechanical damage and without impairing the hemostatic action of the laser radiation. For that purpose we use double pulses from a custom-made acousto-optically Q- switched thulium laser (λ = 2.0 μm) that can emit pulses with energies of up to 150 mJ. The penetration depth of the thulium laser radiation (170 μm) as well as the thermal damage zone are only half as large as that of the holmium laser. The use of Q-switched pulses creates stress confinement conditions leading to a more efficient ablation than with free running pulses. For a given ablation depth, the residual heat deposited in the tissue is therefore smaller than with free running pulses and, hence, also the thermal damage zone. This reduction of thermal damage is possible even though the free-running pulses already fulfil the condition for thermal confinement. The thermal damage zone was only 100 μm for the Q-switched thulium pulses but 200 μm for the free-running pulses. The degree of thermal damage was, in addition, much more severe for the free-running pulses. Q-switched pulses lead to an explosive ablation of the target material. In a liquid environment, this gives rise to the formation of cavitation effects, we release a pre-pulse with small energy (40 mJ) before each ablation pulse of up to 150 mJ. The pre-pulse produces a small cavity that is then filled by the ablation products of the main pulse. The ablation pulse is emitted about 100 μs after the pre-pulse when the bubble is maximally expanded. This way, no additional cavitation effects are induced, and the transformation of laser energy into mechanical energy is minimized. The pre-pulse creates, furthermore, a channel through which the laser light is transmitted, avoiding absorption losses in the liquid between fiber tip and target. The ablation efficiency was 2-3 times better for the Q-switched pulses than for the free-running pulses.
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Laser reshaping of mechanically deformed cartilage specimens accelerates stress relaxation and results in permanent shape change. The mechanism of laser-mediated cartilage reshaping is still unknown, but clearly depends upon the complex molecular interactions between the physio-chemical environment and matrix proteins (collagen, and proteoglycans). It is well known in articular tissues that the mechanical properties of cartilage are sensitive to changes in tissue pH and osmolarity. The objective of this study was to determine the effect of osmolarity on shape change during laser reshaping in morphologic cartilage tissues. Porcine nasal septal cartilage specimens were cut (20 x 5 x 1.5 mm) and immersed in osmotically graded NaCl (0.2NS, 0.8NS, 1.0NS, 1.2 NS and 5 NS) or Phosphate buffered (0.2NS, 0.9NS, 1.0NS, 1.1NS, and 5NS) solutions for 12 hours to establish equilibrium. Then, specimens were bent into semicircular shapes, secured with clamps, and irradiated with an Nd:YAG laser (λ= 1320nm, 5W, 15 secs, 5 mm spot size) along the region of maximum curvature. Resultant bend angle was measured. Shape retention was calculated by comparing resultant curvature with pre-irradiation measurements. Non-irradiated, untreated (negative controls) cartilage retained less than 46% of the original bend. There was no difference in shape retention with respect to varying osmolarity (changed tissue water content) in either group. Resultant bend angles varied from 84 degree(s) to 194 degree(s) corresponding to shape retention varying from 42% to 72% in specimens which were immersed in either NaCl of Phosphate buffered solutions. While laser heating of deformed specimens does result in significant reshaping, the alterations in osmolarity do not seem to effect this process significantly over the range of values evaluated in this study.
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In this study, a computer-controlled instrument was designed and constructed in order to systematically evaluate the effect of laser dosimetry on shape change during laser- mediated cartilage reshaping. Porcine nasal septal cartilage specimens, stripped of periocondrium were cut (25 x 5 x 2 mm) using a custom guillotine microtome and bent into a cylindrical shape (7.38 mm bend radius) using a jig constructed from aluminium tubing and high tension wire. Each specimen was irradiated (Nd:YAG laser λ= 1.32 μm, 6-10 W, 2-16 s pulse duration, 31-51 W/cm2) in five distinct regions using a pattern that maximized cooling between laser exposures. Real-time measurements of tissue optical properties and surface temperatures were recorded during each laser exposure as these features correlate with the onset of stress relaxation and shape change. After irradiation, each specimen was re-hydrated in saline solution (15 minutes), removed from the jig, and photographed with a digital camera. The radius of curvature of the specimen was compared to the radius (7.38 mm) of the reshaping jig. Optimal reshaping (greatest degree of curvature) was observed at 6W, with an irradiation time of 16s, or alternately at 10W, with an irradiation time of 8s.
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Mechanically deformed morphologic cartilage grafts undergo temperature dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, the porcine nasal septal cartilage specimens were evaluated histologically following laser mediated reshaping using H&E. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, 1 = 1.32 mm) while recording simultaneously radiometric surface temperature, internal stress, and backscattered light intensity from a probe laser. Each specimen received one, two, or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in backscattered light intensity that correlate with accelerated stress relaxation. A five minute time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Specimens were then fixed in formalin, serially dehydrated in ethanol, embedded in paraffin, and sectioned with a microtome for histologic examination using light microscopy. Large variation in native tissue histology was observed among individual tissue samples, and vascular were identified in several specimens. Large lacunae with shrunken chondrocytes were identified along with cells with pyknotic nuclei, although these histologic observations did not correlate with the degree of laser exposure. These observations are discussed.
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The elastic modulus of rabbit nasal septal cartilage was measured during Nd:YAG laser (λ= 1.32 μm, 10 seconds, 21.22 W/cm2) radiation. Cyclical force versus displacement curves were generated in cantilevered specimens (9.5 mm x 3.0 mm x 1.0 mm) using a calibrated thin beam load cell and a single axis motorized micropositioner (velocity = 0.3 mm/sec). The laser and a thermopile were positioned above the secured specimen. Samples were irradiated three times with 30 second cooling intervals between each sequential laser exposure. Surface temperature reached a maximum of 65°C. Measurements were recorded before, during, and after each laser irradiation, and then following complete rehydration in normal saline (NS) for 1 hour at 25°C. Following each laser exposure, the sample was sprayed with normal saline delivered via an atomizer to prevent desiccation. Elastic modulus was calculated using a model assuming linear elastic behavior. The modulus in native tissue was 6.08 ± 0.17 Mpa, and this decreased during and after each successive laser exposure (5.41 ± 0.39 Mpa, 4.94 ± 0.46), (5.05 ± 0.104 Mpa, 4.17 ± 0.46 Mpa), (4.23 ± 0.53, 3.71 ± 0.60 Mpa), for the first, second, and third laser exposures, respectively. Following rehydration for one hour in normal saline, the modulus returned to near-baseline values (5.33 ± 0.40 Mpa). The results suggest that molecular changes that occur in the cartilage tissue matrix during laser cartilage reshaping are not accompanied by irreversible changes in the matrix modules.
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In this study, the rheological and phase behavior of porcine nasal cartilage were determined using dynamic mechanical thermal analysis (DMTA), differential scanning calorimetry, and thermogravimetric analysis and the principles of thermal analysis (TA). The results were then incorporated in a finite element analysis used to estimate thermal residual stress and temperature distributions during laser irradiation. The finite element analysis was conducted by using a commercially available code ABAQUS (Hibbitt, Karlsson & Sorensen, Inc, USA) to solve the fully coupled thermo-mechanical equations. Arrhenius kinetics were used to obtain the activation energy associated with the phase transition as determined using DMTA and the results were compared with the energy of the phase transformation calculated using DSC. Laser-induced stress relaxation produced an increase in the von Mises stress within the simulation. The values generated during photo thermal heating were calculated assuming cartilage as an isotropic linear visoelastic material. The advantages and limitations of this approach technique are discussed, in particular with relevance to optimizing this procedure for use in clinical settings.
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Above the ablation threshold the removal of tissue is characterized by a luminous plasma, followed by a plume of non-luminous debris. Both the plasma and the plume are capable of shielding the ablation site, attenuating the beam and decreasing the ablation rate significantly at high numbers of pulses (n) and high fluence. The ablation of several biological tissues by a XeCl excimer laser at 308 nm has been studied. The laser pulse length is 200 ns, around a factor of 10 longer than previously reported studies. In order to study the plume's effect on the ablation rate is has been captured using an Imacon 468 camera capable of 1x108 frames per second. We have calculated the evolutionary speed and the extent of the plasma and ensuing debris with respect to pulse repetition rate (PRR), n and energy for a range of tissues. Probe beam experiments have also been carried out to confirm these results. With this data we can determine the amount of time that the tissue is shielded on the time scale of the incoming pulses and use the results to help explain the ablation rate measurements. A maximum velocity of 2.58x104 ms/s was found for dentine and the tissue was found to be shielded for a maximum of 120 microsecond(s) by the luminous plasma and 10 ms by the non-luminous plume.
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Free running Er:YAG lasers are used for a precise tissue ablation in various clinical application as, for example, laser skin resurfacing. The ablated material is ejected from the tissue surface in the direction of the incident laser beam. We investigated the influence of the shielding by the ablation plume on the energy deposition into the irradiated sample because it influences the ablation dynamics and the amount of ablated material. The shielding was investigated for gelatin with different water content, skin and water. Laser flash photography combined with a dark field Schlieren technique was used to visualize the gaseous and liquid ablation products. The distance traveled by the ablating laser beam through the ablation plume was evaluated from the photographs for various times after the beginning of the laser pulse. The temporal evolution of the transmission through the ablation plume was probed using a second free running Er:YAG laser beam directed parallel to the sample surface. The ablation dynamics shows two phases: Vaporization and material ejection. The photographic observations give evidence for a phase explosion to be the driving mechanism for the material ejection. The photographic observations give evidence for a phase explosion to be the driving mechanism for the material ejection. The transmission is only slightly reduced by the vapor plume, but it decreases by 25-50% when the ejected material passes the probe beam. The laser energy deposited into the sample amounts to only 61% of the incident energy for gelatin samples with 90% water content and 86% for skin samples. The shielding must therefore be considered in modeling the ablation dynamics and determining the dosage for clinical applications.
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We have developed the fast time-response measurement of thermal radiation with 15ns rise time to monitor the corneal surface temperature during ArF excimer laser ablation. In this study, e aim to investigate the influence of the relation between the corneal penetration depth and sampling depth of the measurement system on the measured temperature using 193 nm and 247 nm pulsed lights which have different penetration depths of cornea. When the sampling depth was defined as the penetration depth of cornea at the thermal radiation wavelength, we obtained about 3 micrometers of the sampling depth by pulsed photothermal radiometry (PPTR). In the case of the 247 nm light irradiation, where the corneal absorption coefficient at 247 nm was approximately equal to that for the thermal radiation, we found that the measured temperature rises were same as the estimated temperature rises based on the photothermal process. In contrast, in the case of the 193 nm light irradiation, where the absorption coefficient at 193 nm was larger than that for the thermal radiation, we found that the measured temperature rises were lower than the estimated temperature rises.
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We investigated Q-switched, TEM00 thulium laser induced bubble formation at a silica-water interface in the absence of a speckle pattern. An optical bubble detection unit of high sensitivity was developed to observe on-line the bubble formation onset. Additional fast flash photographs revealed a heterogeneous bubble onset close by the silica surface, at a maximum distance of 8micrometers from the surface. Under control of the optical detection unit, threshold radiant exposures for bubble formation were determined. Strong threshold variation was observed, when replacing a silica surface by another. By performing a series of 45 threshold measurements on different silica surfaces of same 60/40 (scratch and dig) quality, threshold radiant exposures in a range between 26 mJ/mm2 and 95 mJ/mm2 were found, with equal distribution of the 45 threshold values over this range. With help of the gas entrapping crevice model we present a first interpretation of our results. They show, in conclusion, that accurate predictions of the bubble onset solely as a function of irradiation parameters cannot be made.
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We investigated in vitro the mechanism of myocardium tissue ablation with nanosecond pulsed laser at the visible and near-infrared wavelengths. In experiments, porcine myocardium tissue was used as sample. It was found that the ablation rate at 1064 nm was larger than that at 532 nm in spite of lower absorption coefficient at 1064 nm than that at 532 nm for the tissue. During ablation the laser-induced optical emission intensity was measured and it was correlated with the ablation depth. Ablated tissue samples were fixed and stained, and histological analysis was performed with an optical microscope and a polarization microscope. For the 1064-nm laser-ablated tissues thermal damage was very limited, although damage that was presumably caused by mechanical effect was observed. The optical emission intensity during the 1064-nm laser ablation was higher than that during the 532-nm laser ablation at the same laser intensity. And for the 1064-nm laser ablation the ablation threshold was nearly equivalent to the optical emission. Based on these experimental results, we concluded that with the 1064-nm laser light, the tissue removal was achieved through a photodisruption process. Application of 1064-nm, nanosecond pulsed laser photodisruption to transmyocardial laser revascularization (TMLR) was discussed.
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A difficulty with using an OPO laser to reshape the cornea by ablation is the tendency for ablation to stop prematurely. We report on using an OPO laser (2.94 um wavelength, 5 ns pulse duration, 7 mJ pulse energy, approximately 0.5-mm 1/e2-radius Gaussian beam) to ablate a 20% acrylamide gel as a model for the cornea. Experiments demonstrated that ablation proceeds at an average rate of ablation of 3-4 μm/pulse then stops at about 1 mm depth. A computer model was developed to simulate the ablation and desiccation processes. Using a range of operating parameters, the model could achieve ablation rates of 2.8-3.5 μm/pulse and cessation of ablation after 0.25-2.1 mm. A key factor is the absorption coefficient of desiccated gel which was measured experimentally to be about 1700 cm-1. In conclusion, desiccation from residual heat after an ablative pulse creates a dried layer that attenuates subsequent pulses. If the threshold energy density required for ablation is too high, then too much residual energy remains after each pulse and the consequent dried layer halts the ablation process.
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In laser ablation of tissues, tomography of the tissue surface is necessary for measurement of the crater depth and observation of damage of the surrounding tissue. We demonstrate here OCT images of craters made by UV laser ablation of different tissues. The maximum depth of a crater is found among several OCT images, and then the ablation rate is determined. The conventional OCT of the spatial resolution of 15 μm was used in our experiment, but OCT of the resolution of the order of 1 μm is required because the ablation rate is usually a few microns per pulse. Such a high-resolution OCT is also demonstrated in this paper, where the light source is a halogen lamp. Combination of laser ablation and OCT will lead to in situ tomographic observation of tissue surface during laser ablation, which should allow us to develop new laser surgeries.
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We theoretically investigated variable-function (cutting/coagulating) characteristics of the continuous wave 3 μm, 2 μm cascade Ho3+:ZBLAN fiber laser using 3D heat-conduction calculation with finite element method. We have modified a commercial-available simulator in order to calculate heat conduction and thermal ablation process in soft tissue. In this calculation we considered specific heat rise due to the thermal denaturation of protein and volume shrinkage caused by temperature elevation. Beam profile, beam traveling speed, output power, and absorption coefficient were employed to describe the laser beam. The configuration of cutting groove and temperature distribution were calculated by varying the power ratio of the two wavelengths. Coagulation layer was defined as the region that was over 60°C for 1 second because we found that birefringence loss in porcine myocardium observed by a polarizing microscope occurred on this temperature history. When we increased the power ratio of 2 micrometers radiation to the total power of 0.9 W from 0% to 100% at the traveling speed of 0.5 mm/s, the incision depth decreased form 1.45 mm to 0.25 mm, while the coagulation layer thickness increased from 0.17 mm to 0.70 mm. We experimentally performed laser cutting on the same condition by our calculation using extracted porcine myocardium and compared this experimental results with the calculated results. We demonstrated that the incision depth and coagulation layer thickness estimated by our calculation indicated good agreement with the experimental results within 20% differences regarding the function variability by 3 μm/2 μm light mixing.
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We have examined the temporal characteristics of the optical properties of blood undergoing laser-induced photocoagulation during long pulse (10 ms) 532-nm irradiation. The time-domain optical properties were probed at 532 nm, 594 nm, 633 nm and 1064 nm using a newly developed pump-probe technique in a double integrating sphere apparatus. During the 10 ms illumination period, blood evolves from liquid to a liquid blood-coagulum mixture to a system at the liquid/vapor transition in an essentially adiabatic manner. As with previous studies, a sharp rise in the 532 nm signal remitted from the sample can be linked to the onset of coagulation and a concomitant increase in scattering caused by microscopic coagulum particles. We also observe a subsequent decay in this remittance and, at sufficiently high radiant exposures, acoustic and visual transients indicating the onset of microvaporization. Probing the sample at the other wavelengths, we show that the optical properties of the system display highly complex behavior in multiple time frames. We believe that this rich behavior results from the interplay of: i) a time/temperature-dependent red-shift in the absorption spectrum of the oxy-hemoglobin chromophore, ii) coagulation dynamics occurring on at least two distinct time and length scales, and, iii) the creation of at least one new chemical species possessing a different absorption spectrum to that of oxy-hemoglobin. The thermal properties of the system were measured in a time- and spatially-resolved manner using a newly developed technique, and modeled using finite-element analysis incorporating the effects of time-dependent changes in the absorption coefficients of the blood, and phase changes representing coagulation and the liquid/vapor transition. Cross-correlating the optical and thermal studies, we show that the temporal properties of the 532 nm and 633 nm remittance signals can potentially be used to develop a sensitive real-time probe of the onset of coagulation, which in turn will lead to accurate dosimetry during clinical procedures. We also show that the three features of the coagulation highlighted above have profound implications for the design of lasers for vascular therapeutic applications.
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Our work addresses laser tissue interaction for skin exposures from 1318 nm laser pulses. Single pulse data from 0.5 milli-second exposures, along with the mechanisms of photon energy absorption in tissue are investigated. We offer preliminary ED50 data and its implication within the realm of laser tissue interaction for discussion. A comparison will be made between the skin reaction of the Yucatan mini-pig (highly pigmented model) and Yorkshire pig (lightly pigmented model). This study represents the first systematic histological investigation of skin reaction to 1318 nm laser pulses.
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Completion of recent studies within our group indicates a breed-based difference in dermal response to 1540 nm 0.8 millisecond laser pulses. Laser exposure to Yucatan Mini- Pigs (highly pigmented skin) and Yorkshire pigs (lightly pigmented skin) demonstrate statistical differences between the ED50's of the two breeds. Laser delivery is accomplished using an Er:Glass system producing 1540 nm of light at millisecond exposure times and in the range of 5 to 95 J/cm2. Dermal lesion development was evaluated for acute, 1 hour, and 24-hour post exposure presentation. Our data contradicts the theory that water absorption is the sole mechanism of dermal tissue damage observed from 1540 nm laser exposures, as skin chromophores appear to play a role in lesion development.
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The concept of complex laser treatment of localized cancer is recently suggested with the focus on optimization, increasing efficiency and selectivity of Interstitial Laser Therapy (ILT) with interactive imaging and temperature feedback. This treatment is based upon a combination of ILT, photoacoustic (PA) and photodynamic therapy (PDT) with microwave radiometric remote control of the temperature in the treated zone. The features of this concept for primary breast and head and neck cancer are: 1) the application of microwave thermometry for non-invasive real-time overheating control during ILP; 2) direct intralesional injection of a photosensitizer and dye enhance through a tiny needle, followed by PA and ultrasonic impregnation and partly cancer cells damage; 3) combination ILT and PDT therapies; 4) post- operative PDT of the tumor by positioning LED arrays around breast; 5) using RODEO MRI for control of location of the tumor, needle and fiber and to monitor tissue changes during complex laser treatment. This paper focuses more on development of microwave radiometry temperature control. The previous experiments are presented concerning the study of remote microwave radiometric sensor for diagnostic purpose including the results of the clinical trials that have been conducted among over 1000 patients.
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In the past the effects of laser irradiation upon tissue have been assessed based on structural and macroscopic characteristics such as temperature, pressure and tissue mass removal. However, the effects of laser irradiation on a cellular level are not well understood and it is postulated that cellular injury caused by laser treatment may affect the efficacy of the laser procedure. In this research we have used an alternative method of detecting injury by targeting the heat shock protein (Hsp70). A stable cell line was generated containing the luciferase reporter gene attached to the heat shock protein (Hsp70). After thermal injury luciferase is produced in tandem with the heat shock protein to emit bioluminescence at 563 nm. The luminescence was quantified with a photon counting ICCD camera system. The heat shock to bring about Hsp70 transcription was created by immersing the cells in a water bath or by irradiating the cells with a Holmium:YAG pulsed laser (λ= 2.1 μm, τ p = 250 μs). For the laser experiments, radiant exposures varied from 5 to 30 mJ/mm2 and the number of pulses varied at 15, 25 and 35. The peak expression of luciferase was found to be 3 to 4 hours post heat shock for lower exposures but increase to between 6 and 9 hours if higher radiant energies are used. An experiment was also done to assess to what extent the cellular response to heat followed the Arrhenius rate process.
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One of the major problems of cancer chemotherapy is slow diffusion of anti-cancer drugs in the interstitium and their poor penetration from blood through tumor capillary wall and cancer cell membrane. To enhance delivery of the drugs in cancer cells we proposed to use interaction of exogenous microparticles with laser or ultrasonic radiation. This interaction results in cavitation near the particles upon certain irradiation conditions. Our previous pilot studies demonstrated feasibility of enhanced delivery of model and real anti-cancer conditions. Our previous pilot studies demonstrated feasibility of enhanced delivery of model and real anti-cancer drugs in tissues in vitro and in vivo if laser pulsed or ultrasonic radiation is applied. In this work we performed studies in tissue phantoms in order to find optimal parameters that can be used for safe and efficient delivery of anti-cancer drugs in tumors. Water solutions and gelatin were used as tissue phantoms with well-controlled parameters. Cavitation in the phantoms was studied by using optical dn ultrasound techniques. Results of our studies indicate that efficient cavitation-driven drug delivery can be achieved with no or minimal damage to normal tissues.
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Recently we proposed to use laser- and ultrasound-induced cavitation to enhance delivery of anti-cancer agents from blood into tumor cells through tumor capillary wall, interstitium, and cancer cell membrane. Cavitation threshold can be lowered by using microparticles (with certain optical and acoustic properties) which can accumulate in tumors after injection in blood. Lower cavitation threshold allows for local and pronounced cavitation in tumors and, therefore, may provide safe and efficient delivery of anti-cancer drugs in cancer cells without damage to normal tissues by laser or ultrasound radiation. In this paper, we studied enhanced penetration of model macromolecular (rhodamine-dextran) and real anti- cancer (5-FU) drugs and efficacy of cancer therapy with the use of this technique in nude mice bearing human colon tumors KM20. Our studies showed enhanced penetration of the drugs in irradiated tumors and significant improvement of cancer therapy when radiation was applied in combination with polystyrene particle and 5-FU injections. Complete tumor regression of irradiated tumors was obtained when optimum conditions were used. Our results suggest that this technique can potentially be used for efficient and safe cancer chemotherapy.
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Fiber optic probes used to deliver and collect light for biomedical fluorescence spectroscopy applications have varied widely in design. Critical design parameters include fiber diameter, tissue fiber-tip spacing, and illumination- collection fiber separation distance. While device design has been shown to influence spectral distributions, previous studies have not thoroughly addressed how probe geometry affects the spatial origin of detected fluorescence or how probe design might be customized for specific tissue sites or applications. We have developed a Monte Carlo model of laser-induced fluorescence and investigated the effect of design parameters on light propagation using gastrointestinal tissue optical properties. The distribution of emission locations for detected fluorescence were calculated. Initial results indicated that average fluorescence emission depth and total signal intensity are highly dependent on fiber size and tissue-fiber spacing. The implications of these results for optimization of probes used in the detection of neoplasia are discussed.
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Heat alters the bulk physical properties of cartilage tissue, including the optical scattering and absorption coefficients. The purpose of this investigation was to measure wavelength dependent scattering of light using three different probe lasers (λ= 488 nm, 670 nm, 808 nm) during Nd:YAG laser (λ= 1.32 micrometers , 50 Hz pulse repetition rate) heating of porcine septal cartilage. An integrating sphere was used to collect diffusely backscattered light from these probe lasers and three lock- in amplifiers were used to discriminate between the different signals. Peak signal intensity of the backscattered light was observed at different temperature depending on the probe laser wavelength and specimen thickness. The observed changes are unlikely due to axial thermal gradients created during Nd:YAG laser heating and do not correlate with the fluence distribution of the three probe laser wavelengths evaluated. The observed wavelength dependent differences suggest that tissue matrix alterations during heating are due to macromolecular conformation changes that occur on the scale of the wavelength of the probe laser light. As changes in the bulk properties of cartilage can be inferred by using simple non-contact techniques such as light scattering, the characterization of the wavelength dependence of these phenomena will become increasingly important.
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The results of a study of deep (several centimeters) light penetration into biological tissue are presented in order to estimate its significance to potentially photosensitive structures and processes including the fetal eyes. In order to accomplish this goal, samples of various tissues (fat, muscle, and uterus) from surgical patients and autopsies were examined with a double integrating sphere arrangement to determine their optical properties. The results were implemented in a Monte Carlo modeling program. Next, optical fiber probes were inserted into the uterus and abdominal wall of patients undergoing laparoscopic procedures. The fibers were couples to a photomultiplier tube with intervening filters allowing measurements of light penetration at various wavelengths. To determine the feasibility of stimulation in utero, a xenon lamp and waveguide were used to transilluminate the abdomen of several labor patients. Light in the range of 630 to 670 nm where the eye sensitivity and penetration depth are well matched, will likely provide the best chance of visual stimulation. Fetal heart rate, fetal movement, and fetal magnetoencephalography (SQUID) and electroencephalography (EEG) were observed in different studies to determine if stimulation has occurred. Since internal organs and the fetus are completely dark adapted, the amount of light required to simulate in our opinion could be on the order of 10(superscript -8 Watts.
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Optical Technologies to Solve Problems in Tissue Engineering
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Re-narrowing or restenosis of a human coronary artery occurs within six months in one third of balloon angioplasty procedures. Accurate and repeatable quantitative analysis of vessel shape is important to characterize the progression and type of restenosis, and to evaluate effects new therapies might have. A combination of complicated geometry and image variability, and the need for high resolution and large image size makes visual/manual analysis slow, difficult, and prone to error. The image processing and analysis described here was developed to automate feature extraction of the lumen, internal elastic lamina, neointima, external elastic lamina, and tunica adventitia and to enable an objective, quantitative definition of blood vessel geometry. The quantitative geometrical analysis enables the measurement of several features including perimeter, area, and other metrics of vessel damage. Automation of feature extraction creates a high throughput capability that enables analysis of serial sections for more accurate measurement of restenosis dimensions. Measurement results are input into a relational database where they can be statistically analyzed compared across studies. As part of the integrated process, results are also imprinted on the images themselves to facilitate auditing of the results. The analysis is fast, repeatable and accurate while allowing the pathologist to control the measurement process.
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Optical coherence tomography (OCT) has developed as a promising medical diagnostic imaging technology for non- invasive in situ cross-sectional imaging of biological tissues. We present this technique to image bone tissue and to monitor the redox state of mitochondria enzyme Cytochrome oxidase (CytOx) in bone for applications in tissue engineering. Superluminescent diode (SLD) with its peak emission wavelength (λ = 820nm) on the absorption band of oxidized form of CytOx was used in the experiments. The results demonstrate that the OCT system is capable of imaging the calvaria of newborn rats tomographically with a resolution at 9 microns, which could only be previously obtained by the conventional excisional biopsy. The thickness of periosteum of various calvarias from different ages of rats can be accurately determined by the system. The backscattered power-versus-depth profile form the liquid phantoms (naphthol green B with intralipid) and tissue specimens (periosteum of calvaria from newborn rats) are used to quantify the absorption changes of the sample. Absorption coefficients of naphthol green B could be quantified accurately by the linear relationship between attenuation coefficients from the slopes of the reflected signals and naphthol green B concentration. The results also show that the attenuation coefficient decreases in periosteums as CytOx being reduced by sodium dithionite, demonstrating the feasibility of this method to monitor the redox state of tissues studied.
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The optical bi-directional reflectance distribution functions (BRDR) and bi-directional scattering distribution functions (BSDF) of human incisors were measured form -180° to 180° using a scatterometer at 632.8 nm, 1.064 μm, and 3.39 μm. Results from these measurements show that multiple scattering events dominate the optical characteristics of the tooth at the measurement wavelengths in the visible and near-IR. Results form the 3.39 μm wavelength indicate that very little scatter or absorption occurs. This allowed us to obtain an estimated absorption coefficient of about 0.7 cm-1, which is much smaller than previously reported for the visible and near-IR wavelengths.
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Speckle-based strain measurements in biological tissues can be useful for many applications. In using the speckle strain gauge the goal is to observe and track speckles that are translating in both time and space as a result of an applied load. Usually, speckle images are processed with the FFT or the Radon transform. Here we have attempted to apply the novel technique of the Directional Continuous Wavelet Transform (DCWT) for image processing. This method yields two kinds of image decompositions 1) in terms of dilation scale and rotation angle (scale-angle representation) or 2) in terms of time and rotation angle (time-angle representation). In our study these properties of DCWT have been used to track the dynamics of speckle motions.
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A tissue engineering problem that we anticipate will become increasingly of interest is how to grow protein layers and filaments in preferred orientations. For example, the polymerization of monomers into an oriented structure which may exert influence on adherent cells. In this paper, we report on an optical solution using polarized light measurements to probe the structure and orientation of fibers. In particular in this initial study, we measure the fast-axis orientation and retardance of micro-domains in thin sections of liver, muscle, and skin tissues using a polarizing microscope. The size of microdomains of iso- retardance is in the range 10-100 μm, which suggests that optical measurements with laser beams that are on the order of 1-mm in diameter or with imaging cameras with pixels sizes on the order of 100 s of μm will average over several microdomains and consequently complicate interpretation of measurements.
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Two complementary techniques for the imaging of tissue subunits are discussed. A computer guided light microscopic imaging technique is described first, which confocally resolves thick serial sections axially. The lateral area of interest is increased by scanning a mosaic of images in each plane. Subsequently, all images are fused digitally to form a highly resolved volume exhibiting the fine structure of complete respiratory units of lung. A different technique described is based on microtomography. This method allows to image volumes up to 3x3x3 cm at a resolution of up to 7 microns. Due to the lack of strong density differences, a contrast enhancement procedure is introduced which makes this technique applicable for the imaging of lung tissue. Imaging, visualization and analysis described here are parts of an ongoing project to model structure and to simulate function of tissue subunits and complete organs.
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To create a model of development of atherosclerosis and other major human diseases the structural and functional peculiarities of bradytrophic tissues were studied in series of investigations. Sclera, cornea, tendon, cartilage and some other tissues belong to bradytrophic tissues, which nutrition is the result of the diffusion of extravasal liquid. In this fragment of research isotropic properties of cartilage tissue were established before optical enlightenment, induced by tissue immersion, and after it. The sample of sarcoma (muscular tissue's tumor) is also isotropic. By its optical properties and enlightenment dynamics sarcoma is very like cartilage tissue. The enlightenment rate in the indicated tissue significantly higher than in sclera.
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Background: Laser induced protein denaturation is of fundamental interest for understanding the mechanisms of laser tissue interaction. Conjugates of nanoabsorbers coupled to proteins are presented as a model system for investigating ultrafast protein denaturation. Irradiation of the conjugates using repetitive picosecond laser pulses, which are only absorbed by the nanoabsorbers, could result in effects with a spatial confinement of less than 100 nm. Materials and Methods: Experiments were done with bovine intestinal alkaline phosphates (aP) coupled to 15 nm colloidal gold. This complex was irradiated at 527 nm wavelength and 35 ps pulse width with a varying number of pulses ranging form one up to 104. The radiant exposure per pulse was varied form 2 mJ/cm2 to 50 mJ/cm2. Denaturation was detected as a loss of protein function with the help of the fluorescence substrate 4MUP. Results and discussion: Irradiation did result in a steady decrease of the aP activity with increasing radiant exposures and increasing number of pulses. A maximal inactivation of 80% was reached with 104 pulses and 50 mJ/cm2 per pulse. The temperature in the particles and the surrounding water was calculated using Mie's formulas for the absorption of the nanometer gold particles and ana analytical solution of the equations for heat diffusion. With 50 mJ/cm2, the particles are heated above the melting point of gold. Since the temperature calculations strongly depend on changes in the state of matter of the particles and water, a very sophisticated thermal model is necessary to calculate exact temperatures. It is difficult to identify one of the possible mechanisms, thermal denaturation, photochemical denaturation or formation of micro bubbles from the dependance of the inactivation on pulse energy and number of applied pulses. Therefore, experiments are needed to further elucidate the damage mechanisms. In conclusion, denaturing proteins irreversibly via nanoabsorbers using picosecond laser pulses is possible. The confinement of the heat to the nanoabsorbers when irradiating with picosecond pulses suggests that the denaturation of proteins could be possible with nanometer precision in cells with this approach. However, the mechanism of protein inactivation, which is part of present investigations, is crucial for the precision of such nanoeffects.
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Internal stress σ(t), diffuse transmitted light intensity I(t) from a He-Ne probe laser (λ= 632.8 nm), and radiometric surface temperature Ts(t) were measured during the photothermal heating of porcine septal cartilage using a pulsed Nd:YAG laser (λ= 1.32 μm). Rectangular specimens, 1-4 mm thick, were secured to a tensile force testing rig and laser irradiated. Force measurements during heating showed significant variation in the rate of deformation, which were found to be strong dependent on tissue orientation; revealing the anisotropic nature of its thermo-mechanical properties. These finding suggest that the collagen and proeoglycan networks lie in a preferential orientation within the extracellular matrix, which must be addressed before this procedure can be used on a wider basis.
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In vivo, laser radiation has been shown to stimulate cartilage repair and proliferation, which is of clinical relevance as light can be delivered using minimally invasive techniques. However, dosimetry and temperature dependence of this phenomenon have neither been determined nor have these findings been conclusively demonstrated ex vivo. In this study, we detected the presence of proliferating chondrocytes in intact laser irradiated rabbit septal cartilage using a novel whole mount Bromodeoxyuridine (BrdU) assay, and determined the dependence of this phenomenon on laser dosimetry. Cartilage specimens were irradiated with light from an Nd:YAG laser (λ= 1.32 μm, 3-16 sec, 10-45 W/cm2) and placed in tissue culture with BrdU for 7-9 days. BrdU (a thymidine analogue) is incorporated into DNA during replication. Specimens were then fixed and treated with an enzyme-linked double antibody system providing a color change to indicate the presence of BrdU in dividing cells. The samples were analyzed in whole mount and with conventional histology. Proliferation was clearly identified for laser exposures greater than 6 seconds at (25 W/cm2), and was observed only on the periphery of the laser spot. This study clearly demonstrates that laser heating of ex vivo cartilage tissue results in chondrocyte proliferation. Inasmuch as this phenomenon was observed in tissue culture, the non-specific cellular and humoral responses present an intact organism were eliminated. Cell division likely results form either changes in the fine structure of the tissue matrix or direct stimulation of chondrocyte metabolism.
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Thermodynamic induced changes in birefringence of nasal septal cartilage following Nd:YAG laser irradiation were investigated using a polarization-sensitive optical coherence tomography (PSOCT) system. Birefringence in cartilage is due to the asymmetrical collagen fibril structure and may change if the underlying structure is disrupted due to local heat generation by absorption of laser radiation. A PSOCT instrument and an infrared imaging radiometer were used to record, respectively, depth-resolved images of the Stokes parameters of light backscattered from ex vivo porcine nasal septal cartilage and radiometric temperature following laser irradiation. PSOCT images of cartilage were recorded before (control), during, and after laser irradiation. From the measured Stokes parameters (I,Q,U, and V), an estimate of the relative phase retardation between two orthogonal polarizations was computed to determine birefringence in cartilage. Stokes parameter images of light backscattered from cartilage show significant changes due to laser irradiation. From our experiments we differentiate dehydration and thermal denaturation effects and observe the birefringence changes only in the dehydration effect. Therefore, a dynamic measurement of birefringence changes in cartilage using PSOCT as a feedback control methodology to monitor thermal denaturation is problematic in non-ablative surgical procedures such as laser assisted cartilage reshaping.
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