Combination therapy has been commonly used in chemotherapy, taking advantage of different effects of different chemotherapeutic agents. The treatment effects are often synergistic. The same approach has been investigated in laser phototherapy. Specifically, different combinations of laser photothermal interaction, laser photochemical interaction, immunotherapy and chemotherapy have been used in the treatment of tumors. These novel approaches showed promise in cancer treatment, particularly against metastatic tumors. The recent development in this area is discussed in this paper. Furthermore, a specific combination of photodynamic therapy (PDT) with a novel immunoadjuvant, glycated chitosan (GC), has shown to be effective in the treatment mammary tumors and lung tumors in mice. In the treatment of EMT6 tumor-bearing mice, the Photofrin-based PDT and GC has significantly increased the survival rates from 37.5% with PDT alone to 62.5% when a 0.1-ml 0.5% GC was peritumoral injected immediately after PDT treatment. The survival rate was further increased to 75.0% when GC of higher concentration was used. In comparison, the individual components of the PDT-GC treatment showed either no effect or very limited effects. In the treatment of a poorly immunogenic tumor model, Line 1 lung tumors in mice, the combination of PDT and GC resulted in a 37.5% survival rate, while no survival mice were observed with PDT alone.

Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of photosensitizers with harmless visible light producing reactive oxygen species. The major biological effects of PDT are apoptosis of tumor cells, destruction of the blood supply and activation of the immune system. The objective of this study is to compare in an animal model of metastatic cancer, PDT alone and PDT combined with low-dose cyclophosphamide (CY). Since the tumor we used is highly metastatic, it is necessary to generate anti-tumor immunity using PDT to both cure the primary tumor and prevent death from metastasis. This immunity may be potentiated by low dose CY. In our model we used J774 cells (a Balb/c reticulum cell sarcoma line with the characteristics of macrophages) and the following PDT regimen: benzoporphyrin derivative monoacid ring A (BPD, 2mg/kg injected IV followed after 15 min by 150 J/cm² of 690-nm light). CY (50 mg/kg i.p.) was injected 48 hours before light delivery. BPD-PDT led to complete regression of the primary tumor in more than half the mice but no permanent cures were obtained. BPD-PDT in combination with CY led to 60% permanent cures. CY alone gave no permanent cures but did provide a survival advantage. To probe permanent immunity cured animals were rechallenged with the same tumor cell line and the tumors were rejected in 71% of mice cured with BPD-PDT plus CY. We conclude that BPD-PDT in combination with CY gives best overall results and that this is attributable to immunological response activation in addition to PDT-mediated destruction of the tumor.

Photodynamic therapy (PDT) has become an established clinical modality for the treatment of a variety of cancers and other lesions. Rapid induction of a massive damage in targeted cancerous tissue by PDT triggers a strong host response whose primary purpose is to contain the perturbed local homeostasis, remove the dead tissue and promote tissue healing at the affected site. Recent characterization of the participation of the complement system in the PDT-induced host response has prompted the hypothesis that further amplification of PDT-elicited complement activation can potentiate the antitumor effect of PDT yielding superior therapeutic gains. After discussing the nature of complement activity directed against autologous tissue and the evidence of complement system engagement following PDT treatment, this report describes various approaches for successfully potentiating the PDT-induced complement activation illustrating the perspectives of this strategy for the clinical management of cancer.

Indocyanine Green (ICG) is clinically used as a fluorescent dye for imaging purposes. Its rapid circulation kinetics and minimal toxicity has prompted investigation into ICG's utility as a photosentitizer for therapeutic applications. Traditionally, optically mediated tumor therapy has focused on photodynamic therapy, which employs a photochemical mechanism resulting from the absorption of low intensity CW laser light by localized photosensitizers such as Photofrin II, Benzoporphyrin Derivative (BPD), ICG. Treatment of cutaneous vascular malformations such as port-wine stains, on the other hand, is based on a photothermal mechanism resulting from the absorption of high intensity pulsed laser light by hemoglobin. In this study, we compared the effectiveness of combining photochemical and photothermal mechanisms during application of ICG in conjunction with laser irradiation with the intention that the combined approach may lead to a reduction in the threshold dose of pulsed laser light required to treat hypervascular malformations. The blood vessels in rabbit ears were used as an in vivo model for targeted vasculature. Irradiation of the ears with IR light (λ=785 nm, Δτ = 3 min, I_o = 120 mW) was used to elicit photochemical damage, while photothermal damage was brought about using pulses from a ruby laser (λ=694 nm, τ = 3 ms) with different fluences. For the combined modality, photochemical damage was induced first and followed by photothermal irradiation. This modality was compared with photothermal irradiation alone. The effectiveness of each irradiation scheme was assessed using histopathological analysis. We present preliminary data that suggests that pretreatment with photodynamic therapy before photothermal coagulation results in more severe vascular damage with lower photothermal fluence levels. The results of this study provide the foundation work for further exploration of the therapeutic potentials of photochemical and photothermal effects during application of ICG in conjunction with laser irradiation.

The use of simple models to understand basic features of the Photodynamic Therapy may contribute to the solid establishment of its dosimetry enhancing its degree of clinical susccess . In this work we have used normal rat liver as a prototype for the determination of threshold dose as a function of photosensitize concentration in the tissue. For this purpose we have investigated the depth of necrosis induced by photodynamic therapy when varying doses of light and photosensitizer (Photogema) concentrations are employed. All experiments were done with a light intensity of 250 mW/cm². We have determined that the depth of necrosis depends on the photosensitizer concentration injected in the animal. Stablishing the correlation of the light intensity at the point where lies the separation line between necrosed and health tissue, we measure the threshold dose.

Despite significant progress in tissue engineering over the last decade, the development of real-time, non-destructive tools for monitoring the development of engineered tissues remains a great challenge. To date, the evaluation of cell proliferation and extracellular matrix production in response to various culture conditions depends upon traditional DNA, RNA and protein analysis which requires extraction of cell components from constructs resulting in loss of tissue morphology and integrity. In this study, we report how optical coherence tomography (OCT) can be exploited to monitor cell profiles in real-time and in a non-destructive manner. Scaffolds made from poly(lactic acid) (PLLA) with various porosities were scanned by OCT. A local porosity analysis method has been developed to quantify the porosity change. The hypothesis is whether the local porosity analysis can correlate with the tissue growth within the scaffold following seeding of the cells within it. Bone cells have been grown in the PLLA scaffolds under different culture conditions. The OCT images of these scaffolds have been collected. It has been found that the porosity of the cultured scaffold-cell constructs reduced under different culture conditions compared to blank scaffolds. A decrease in light penetration depth in OCT images has also been observed. There existed a good relationship between the local porosity and tissue growth. It has been demonstrated that the mean local porosity based on OCT images can become a unique method to correlate and monitor tissue growth.

Background and Objective: The application of nanotechnology for laser thermal-based killing of abnormal cells (e.g. cancer cells) targeted with absorbing nanoparticles (e.g. gold solid nanospheres, nanoshells, or rod) is becoming an extensive area of research. We develop an approach to enhance the efficiency of selective nanophotothermolysis of cancer cells through laser-induced synergistic effects around gold nanoparticles aggregated in nanoclusters on cell membrane. Study Design/Materials and Methods: A concept of selective target damages by laser-induced synergistic interaction of optical, thermal, and acoustic fields around clustered nanoparticles is presented with focus on overlapping bubbles from nanoparticles aggregated on cell's membrane. The experimental verification of this concept in vitro was performed by the use a tunable laser pulses (420-570 nm, 8-12 ns, 0.1-300 μJ, laser flux of 0.1-10 J/cm²) for irradiation of MDA-MB-231 breast cancer cells targeted with primary antibodies to which selecttively 40-nm gold nanoparticles were attached by the means of secondary antibodies. The photothermal, electron and atomic force microscopes in combination with viability test (annexin -V-Propidium iodide) were employed to study the nanoparticle's spatial organization, the dynamics of microbubble formations around the particle's clusters, and cells damage. Results: An aggregation of nanoparticles on cell membrane was observed with simultaneous increase bubble formation phenomena, and red-shifted absorption due to plasmon-plasmon resonances into nanoclusters. It led to a significant enhancement, at least two orders of magnitude, of the efficiency of selectively killing cancer cells with nanosecond laser pulses. Conclusion: Described approach allows using relatively small nanoparticles which would be easier delivery to target site with further creation of nanoclusters with larger sizes which provide more profound thermal and related phenomena leading to more efficient laser killing of cancer cells. This nanocluster model might be promising also for treatment or modification different targets (e.g. bacteria, virus, vascular lesions, fat, etc.) as well as teh use different type energy deposition (focused ultrasound, microwave, magnetic field, etc.).

This paper investigates three different techniques to investigate the dynamic change of the attenuation coefficient in in-vitro hamster skin during optical skin clearing using a hyper-osmotic solution of 50% glycerol. The tissue is imaged using an amplitude based OCT system with a wavelength of 1290 nm during the clearing experiment. The tissue sample rested on a mirror surface and OCT images of the tissue and mirror were acquired in 1 minute intervals over a 45 minute period. Intensity profiles were obtained from averaging A-scans at each time interval. The tissue optical properties were determined by curve fitting an exponential function to the acquired intensity profiles, by linear line fit between front- and mirror surface reflection as well as by fitting the photo detector voltage at front- and mirror surface peaks to a modified expression of Beer's Law. In addition, another experimental set-up was used to evaluate focus effects on the back scattered signal from the mirror underneath an in-vitro porcine skin sample which was dehydrated in air to mimic optically cleared tissue. Both, tissue and mirror were mounted onto two independent micro translation stages in order to determine focal effects on the mirror signal. Results show that exponential curve fitting initially yields acceptable data for the attenuation coefficient but later introduces significant error due to the altered light propagation through the tissue due to the applied clearing agent. Attempting to use a linear fit between front- and mirror reflections does not yield physically meaningful data while using the detector voltage read-out in combination with Beer's law yields acceptable results comparable with tissue data published in the literature. Focal effects account for 1.5 to 2.5 dB higher signals than otherwise can be expected from the back reflection of the mirror underneath a scattering tissue sample if the mirror is in the focal plane of the input optics.

The polarization dependence of multiply backscattered light from suspensions of red blood cells diluted with saline and one with additional salt are analyzed. To investigate the polarization dependence, the backscattering Mueller matrices of suspensions are obtained from 36 spatially distributed images of backscattered light intensity with different combinations of incident polarizations and detection analyzers. The spatial dependence of the degree of polarization of backscattering for linearly and circularly polarized incident light are analyzed with the Mueller matrices. To compare the degree of polarization numerically, the relationship between the degree of polarization and the normalized positions on the suspensions by reduced mean scattering free paths are investigated. The results show that the decay of degree of polarization along the distance from the incident point are almost same with or without additional salt when linearly polarized incident light is used, while different properties are observed with circularly polarized incident light.

Cryopreservation is the only method for conserving blood vessels as future allografts with biological immunity controls. Although it affects vessels mechanical structure, no biomechanical integrity simple test is available today. Biological tissues optical properties characterization by spectroscopic methods is of interest due to their types or natures variations. Collected data complementarity contributes to "photodiagnosis" applicative prospects (cancer, vascular...). Pig carotid artery rings were tested after excision and after one month cryopreservation. An uniaxial mechanical testing device was used for ring stretching, and elongation and axial forces measurement. Circumferential large strains and stresses were calculated. Simultaneously, each artery ring optical characteristics was measured using fibered autofluorescence and elastic scattering spectrometers. Mechanical results showed nonlinear strain/stress curves and large deformations in good agreement with other referenced works. Significant differences (p<0.05) between fresh and cryopreserved rings mechanical properties were noticed. Elastic scattering spectra intensity variations were well correlated with artery mechanical properties. The standardized autofluorescence spectra were more clearly correlated with anatomo-histological changes due to cryopreservation, providing rather accurate differentiation between fresh and cryopreserved samples. This study offers a new perspective to detect changes of cryopreserved arterial samples mechanical properties. Coupling mechanical tests (uniaxial traction of arterial rings) and optical spectroscopic measurements (autofluorescence, elastic scattering) is the driving point: it allows correlating mechanical modifications and spectral variations of artery rings before and after cryopreservation. Ultimately, this new approach could help developping a device allowing non-invasive, atraumatic and contactless optical examinations of arterial graft to assess its mechanical state before reimplantation.

Investigation of tissue optics requires accurate values of the bulk optical parameters: the scattering coefficient μ_s, absorption coefficient μ_a, anisotropy factor g and the real refractive index n_r. We employed a coherent reflectance curve method to determine the refractive index of fresh porcine skin dermis at 8 wavelengths between 325 and 1557nm. The surface profiles of fresh dermis samples were measured with a non-contact method of confocal imaging and the roughness parameters were extracted. To determine tissue optical parameters, a single integrating sphere was used to measure the diffused reflectance and transmittance with slab samples of fresh porcine skin dermis. Collimated transmittance was measured with the samples in situ using a spatial filtering setup. Based on these results, a Monte Carlo code capable of handling surface roughness of the tissue sample has been developed and used to inversely determine the bulk values of μ_s, μ_a and g at the 8 wavelengths which are significantly different from the reported values.

The mechanisms of optical clearing in cellular tissue in response to application of a hyperosmotic solute were theoretically considered using the Rayleigh-Gans approximation to scattering. The effect on scattering coefficient due to changes of three input parameters including refractive index ratio, scatterer size, and scatterer volume fraction were investigated. Decreasing refractive index ratio has the greatest effect on decreasing tissue scattering coefficient, and this parameter is likely to undergo a large variation. Decreasing scatterer size can result in a slight increase or decrease in tissue scattering coefficient depending on initial scatterer size, percent decrease in radius, and wavelength. The effect of tissue scattering coefficient due to an increase in the volume fraction of scatterers depends on initial scatterer volume fraction and percent change. Scattering always decreases when initial scatterer volume fraction is greater than .5. Results of simulations illustrate the high sensitivity of changes in tissue scattering due to the initial and final states of these parameters. Improved understanding of the mechanisms of tissue clearing will require accurate experimental measurement of these input parameters.

We present an approximation to light propagation through skin using an improved multilayered diffusion equation. This equation allows for the influence of multiple layers on diffusely reflected light to be considered; which is accomplished by embedding imaginary point sources in the medium that correspond to each layer's transport mean free path. The new approximation is then compared to a previous two-layer solution by Farrell et al. to verify improvement as well as to results from Monte Carlo simulations to verify accuracy.

The microtome has allowed us to observe the uterine extra cervix of the rabbit. This latter is covered with a none keratinized stratified malpighian epithelium made up of five layers. The laser-cervix interaction allows to calculate, by the resolution of the radiative transport equation, the fluence of each layer. The whole fluence is determined by using the optic properties of each layer. These optic properties are obtained by fitting the transmittances measured and calculated by Monte-Carlo method. The obtained results are given below.

This paper presents a study of the use of a single scaleable Monte Carlo simulation to estimate the physiological parameters of skin lesions from data collected in vivo using spectroscopic oblique-incidence reflectometry. Spatio-spectral data from 101 cases are separated into two groups based on their melanocytic conditions. Group-1 consists of (a) cancerous basal cell carcinomas and squamous cell carcinomas and (b) benign actinic keratoses and seborrheic keratoses. Group-2 consists of (a) dysplastic nevi and (b) benign common nevi. Several physiological parameters are estimated, such as the size distribution of the optical scatterers, the relative index of refraction of the scatterers, the total volume concentration of the scatterers, the concentration of the total hemoglobin and the oxygen saturation, and the relative changes related to the values calculated from the neighboring healthy tissues. The most significant features are then combined into one feature. The results show that for both groups the combined feature is significantly different for the benign and cancerous cases than for the dysplastic cases. The ROC area was 0.9 and 0.86 for group-1 and group-2, respectively.

Demonstrated herein is the performance of gradient-based estimators for assessing small motions in speckle patterns. Such estimators are important in a variety of speckle techniques used in non-destructive evaluation. These speckle patterns can be two-dimensional (one spatial dimension and one temporal dimension) as obtained in objective speckle techniques or three-dimensional (two spatial dimensions and one temporal dimension) as typically seen in subjective (imaged) speckle methods. Both theoretical and experimental performance estimates are presented. Through a series of numerical simulations, performance (velocity bias and RMS variation) is characterized in terms of the signal-to-noise ratio. This SNR parameter is a convenient surrogate for the decorrelation of sequential speckle patterns such as those observed in biological tissues. Experimental evaluations are performed on an optically rough metal coupon. We demonstrate that under easily met experimental conditions, estimates of speckle motions on the order of one tenth of a pixel can be obtained reliably.

Quasi-ballistic or "snake" photons carry useable information on the internal structure of scattering mediums such as tissues. By defining quasi-ballistic photons to be those photons that have been scattered but have not exceeded a specified radial distance threshold from their initial trajectory (equivalent to the resolving limit of the quasi-ballistic photons) and by using the Henyey-Greenstein phase function, Monte Carlo modeling has shown that the number of quasi-ballistic photons increases with depth in an isotropic scattering medium until a maximum is reached and then the quantity decreases. The quantity of quasi-ballistic photons at a specified depth can be shown to be governed by two competing processes: the decay of ballistic photons into quasi-ballistic photons and the decay of quasi-ballistic photons into scattered photons. These well-defined behaviors allow one to write a rate equation governing the growth and decay in the quantity of quasi-ballistic photons with depth. It is found that as the anisotropy factor increases with forward scattering and as the resolution limit is widened, the quantity of quasi-ballistic photons begins to exceed the quantity of ballistic photons at a specified depth and the rate of decay of quasi-ballistic photon quantity decreases. The development of a rate equation for the formation of quasi-ballistic photons allows one to analyze how efficient various detection methods are in extracting these quasi-ballistic photons, and it can be seen that there is a compromise between desired resolution and the effective scattering ratio at a detector.

Recently, there has been significant interest in using polarization gating to selectively probe superficial tissue to facilitate the diagnosis of epithelial neoplasia. Thus, understanding the propagation of polarized light in tissue in general and the mechanisms of polarization gating in particular are crucial for biomedical optics applications. However, these investigations have been impeded in part by the lack of realistic tissue models that can replicate both the morphological complexity and the optical properties of biological tissue. Here we report the development of a novel bioengineered connective tissue model to study light transport in tissue. This tissue model was fabricated by combination of scaffolding and crosslinking techniques. It demonstrates great similarity to real connective tissue in its optical properties and microarchitecture. Moreover, the physical and optical properties of the model can be reproducibly controlled. As an example, we demonstrated the application of this tissue model in our investigation of the depth sensitivity of polarization-gating. Specifically, we studied the effects of epithelium and connective tissue on the penetration depth of differential polarization signals. Our results indicate that the penetration depth in both epithelial and connective tissues primarily depends on the optical thickness of the tissue: the polarization gated signal probes the superficial layer of tissue up to the optical depth of ~ 2. The corresponding physical penetration depth depends on the specific tissue type and in the connective tissue is about 6 - 7 times shorter than in the epithelium (~ 40 - 50 microns and ~ 200 - 300 microns, respectively).

Optical tomography within highly scattering media has usually employed coherence domain and time domain imaging, which observe the shortest path photons over the dominant randomly scattered background light. An alternative, Angular Domain Imaging, employs micromachined collimators which detect photons within a small angle of the aligned laser light source. These angular filters consist of micromachined silicon collimator channels 51 micron wide by 10 mm long on 102 micron spacing giving an acceptance angle of 0.29 degrees at a CCD detector. Phantom test objects were observed in turbid mediums ranging from 1 to 5 cm thick at effective scattered to ballistic ratios from 1:1 to greater than 100,000,000:1. Simple line and space test objects detection limits are set by detector pixel size not collimator hole spacing. Restricting the light emission to only the collimating array hole area provides increased detectability by reducing the amount of scattered light background. This is best done using cylindrical spherical cylindrical lens beam expanders/shrinkers to create a wide line of light of small thickness aligned to the collimator array. As object locations within the medium are moved closer to the detector/collimator, image detectability appears to depend on the scattering ratio after the test object rather than the total medium scattering. Hence, objects located closer to the detector than the middle of the medium are observed at a much higher scattering levels than those nearer the light source.

The Mark-III Free Electron Laser (FEL), tuned to 6.45 microns in wavelength has been demonstrated to provide for efficient ablation in ocular, neural, and dermal tissues with minimal collateral damage. To date, the role of the unique pulse structure of the FEL on the ablation mechanism has not been determined. In this study, the native pulse structure of the FEL, a 2.85 gigahertz repetition of picosecond pulses within a five microsecond macropulse envelope, was changed using a pulse stretcher. This device changes the duration of the micropulse from its native one picosecond to 30-200 picoseconds in length, thus reducing the peak intensity of the micropulse down to 1/200th of the original intensity, while the macropulse energy remains unchanged. Two basic metrics were studied: the ablation threshold on water and mouse dermis and the ablation crater depth on gelatin and mouse dermis. These metrics were employed at 6.45 and 6.1 microns in wavelength for 1, 100, and 200 picoseconds in micropulse duration. In addition, bright-field imaging was used to compare the ablation dynamic between 1 ps and 200 ps micropulses on water at 6.1 and 6.45 microns. The effect of changing the micropulse duration was also studied on the ablation of mouse dermis for histological analysis. Craters (500 micron diameter) were created with 25 pulses at three times the ablation threshold as determined for mouse dermis within 8 hours of removal. Three rows of twenty craters were created on each piece of mouse dermis for a given parameter set. The native one picosecond micropulse and 200 picosecond stretched micropulse were compared at 6.1 and 6.45 microns in wavelength. There was no difference seen between the native 1 ps micropulse and the stretched micropulse durations with respect to the ablation threshold, efficiency, dynamics, and thermal damage.

The mechanisms involved in infrared laser tissue ablation are studied using a free electron laser (FELIX) in order to clarify whether the increased ablation efficiency reported in literature for certain infrared wavelengths is due to a wavelength effect or to the specific pulse structure of the lasers that are generally used in these studies. Investigations are presented of ablation of vitreous from pigs’ eyes using several techniques including protein gel electrophoresis and ablation plume visualization. The ablation effects of three different infrared wavelengths are compared: 3 mm, which is currently in clinical surgical use, and the wavelengths associated with the amide I and amide II bands, i.e. 6.2 mm and 6.45mm, respectively. The results suggest a different ablation mechanism to be in operation for each studied wavelength, thus indicating that the generally reported increased ablation efficiency in the 6-6.5 micron range is due to the wavelength rather than the typical free electron laser pulse structure.

The goal of this study was to investigate a Q-switched Er:YAG pumped ZGP crystal Optical Parametric Oscillator (OPO) as a potential alternative source to the Mark-III Free Electron Laser (FEL) for delivering 6.45 micron light for clinical applications. In addition, this research increased the understanding of the role of the unique pulse structure of the FEL with respect to the ablation of soft tissue at 6.45 microns, which has been shown to ablate with very minimal collateral damage (<40 microns). The OPO operates from 6-8 microns in wavelength with a 100 ns pulse. Up to 250 micro-joules per pulse can be obtained with this laser. This provides up to three times the threshold energy for ablation given a diffraction limited spot of ~60 microns in diameter. The ablation threshold was determined using PROBIT analysis of 100 pulses on water at 6.1 and 6.45 microns in wavelength. The ablated crater depth was also measured on 90% w/w gelatin at both wavelengths for craters made with between 5 and 500 pulses. The results obtained with the OPO were then compared with a Mark-III FEL with a similar spotsize (~90 microns) to determine if there were any differences due to the unique pulse structure of the FEL, which consists of a 2.85 GHz train of picosecond pulses within a five microsecond envelope. The results showed no difference with respect to the ablation threshold; while the ablated crater depth was reduced for the FEL pulse for equivalent parameters. In addition, bright-field imaging was performed at three times the ablation threshold for both lasers and will be presented.

We report on our measurements of the Minimum Visible Lesion (MVL) thresholds for porcine skin [Yucatan mini-pig (Sus scrofa domestica)] for laser exposures at 810 nm and sub-50 femtosecond (fs) laser pulses. In this study we measured the ED50 skin thresholds from laser pulses that produced multiple self-focusing filaments while propagating from the laser to the skin. These high-powered (1-2 terawatt) filaments were focused on the flank of mini-pig and three trained readers determined the number of lesions becoming visible at 1-hour and 24-hour post-exposure. The observed damage patterns on the skin surface indicated the number of filaments in the laser pulse and these were photographed for future reference. Histological sections were obtained after both readings and the results will be reported later for sub-surface damage. The threshold using preliminary data at 1-hour was 9 mJ of energy and increased to 25 mJ after 24 hours. This increase in threshold indicated that many of the laser pulses produced only superficial damage (erthemia) that disappeared in 24 hours and that nearly 3 times the pulse energy was required to cause subsurface or cellular damage.

The concept of confinement is that if energy deposition into a system occurs during durations shorter than a confinement time, the response of the system depends only on the total energy deposited and not on the deposition time. For stress confinement, the relevant response is the pressure that is produced. We have shown previously that for laser absorption by a spherical absorber, stress confinement is not valid at the core of the absorber and the tensile stresses continue to grow as the pulse duration shrinks well below any characteristic response time of the system. We have now calculated the pressure response in the cellular medium outside the absorber. We find that for a variety of energies, stress confinement is valid. We find that the characteristic confinement time agrees well with that expected for pressure transmission across the absorber. We show that even though the peak pressure that is produced varies slowly as a function of pulse duration, there is a sudden onset of shock wave production when the pulse duration is shortened below the confinement time. Since damage results from pressure gradients, the sudden onset of shock waves implies a sharp increase in the potential for damage.

Multiphoton Microscopy with a femtosecond pulsed Ti:sapphire laser in the near infrared (NIR) enables the user not only to image cells and tissues with a subcellular resolution but also to perform highly precise nanosurgery. Intratissue compartments, single cells and even cell organelles like mitochondria, membranes or chromosomes can be manipulated and optically knocked out. Working at transient TW/cm² laser intensities, single cells of tumor-sphaeroids were eliminated efficiently inside the sphaeroid without damaging the neighbour cells. Also single organelles of cells inside tissues could be optically knocked out with the nanoscalpel without collateral damage. Tissue structures inside a human tooth have been ablated with sizes below 1 μm. This method may become a useful instrument for nano-manipulating and surgery in several fields of science, including targeted transfection.

Laser-mediated gene transfection is very attractive as a new method for targeted gene therapy because of its high spatial controllability of laser energy. Previously, we demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISW). In this study, we investigated the experimental conditions to increase transfection efficiency in vitro. By applying single-pulse LISW, transfection efficiency was increased with increasing laser fluence. Increase in the number of laser pulses increased transfection efficiency for laser fluences up to 1.3 J/cm², but at higher fluences (>1.7 J/cm²), efficiency showed saturation tendency. Temperature dependence of transfection efficiency was also investigated.

We selectively disrupted the cytoskeletal network of fixed and live bovine capillary endothelial cell using ultrashort laser pulses. We image the microtubules in the cytoskeleton of the cultured cells using green fluorescent protein. The cells are placed on a custom-built inverted fluorescence microscope setup, using a 1.4 NA oil-immersion objective to both image the cell and focus the laser radiation into the cell samples. The laser delivers 100-fs laser pulses centered at 800 nm at a repetition rate of 1 kHz; the typical energy delivered at the sample is 1-5nJ. The fluorescent image of the cell is captured with a CCD-camera at one frame per second. To determine the spatial discrimination of the laser cutting we ablated microtubules and actin fibers in fixed cells. At pulse energies below 2 nJ we obtain an ablation size of 200 nm. This low pulse energy and high spatial discrimination enable the application of this technique to live cells. We severed a single microtubule inside the live cells without affecting the cell's viability. The targeted microtubule snaps and depolymerizes after the cutting. This nanosurgery technique will further the understanding and modeling of stress and compression in the cytoskeletal network of live cells.

Laser photophysical interactions have been used in treatment of cancers. The use of laser energy provides high target selectivity. With photosensitizers and immunoadjuvants, laser treatment can also provide long-term and systemic effects. Photothermal interaction using an 805-nm diode laser was used to treat metastatic melanoma in mice. B16 tumor cells were implanted subcutaneously in mice. When the tumors reached a size of 0.2 to 0.5 cm³, laser energy was applied to the tumors. The temperature increases was measured using temperature probes. In addition, glycated chitosan (GC), an experimental immunoadjuvant, was also used in combination with the laser treatment. Our experimental results showed that the photothermal interaction could reduce the tumor burdens immediately after the treatment. When GC was used, the survival rates were significantly increased. GC was also applied at different time frames in relation to the laser treatment: 24 hours before, at the same time, and 24 hours after. Our results indicated that when GC was applied 24 hours before the laser treatment, the positive responses of the tumor-bearing animals is higher than that of the other groups. It may be due to the fact that the use of GC can facilitate the immunological stimulation and enhance the treatment of laser.

To assess the retinal hazards related to simultaneous exposure from two lasers of separate wavelengths, the retinal effects of 5-second laser irradiation from 532 nm and 647 nm were determined in non-human primates. A total of six eyes were exposed using equal amounts of power to determine the damage levels. The results were combined with those of previous, two-wavelength studies done by our group and compared to damage models developed in our lab. The data were also compared to the calculations resulting from use of the currently accepted method of predicting hazards from simultaneous lasing.

The cells of the retinal pigment epithelium (RPE) are subject to photo-oxidative stress arising from the interaction of incident light with lipofuscin, melanin, and other pigment granules in the RPE cytoplasm. Specific genotypic responses to these stressors are controlled by transcription factors, such as NF-κB (RelA/p50 dimer). The effects of CW laser exposures on NF-κB nuclear translocation have been studied in a line of human-derived RPE cells (hTERT-RPE) that develop melanin pigmentation in culture. The cells were exposed to the CW emission of an Argon-ion laser for 10 m at 0.5 W/cm², a range previously shown to produce oxidation of cellular proteins, DNA, and antioxidants. NF-κB dimer was measured in nuclear extracts by an electrophoretic mobility shift assay. NF-κB nuclear translocation exhibited a modest, early peak at 1 h, and a larger, late peak at 24 h. NF-κB activation could be reduced only by some antioxidants; for example, 20 mM N-acetyl-L-cysteine or 100 μM pyrrolidine dithiocarbamate were ineffective, while 500 μM ascorbic acid was highly effective. These results indicate that interaction of the laser with the RPE melanin granules is a likely source of oxidative reactions, and that the induction of photoxidative stress activates NF-κB, but it remains to be determined if NF-κB is pro- or anti-apoptotic in the RPE cell.

We report pronounced delayed tissue death in photothermal surgery performed with highly concentrated sunlight on the livers of healthy live rats. Pathology reveals that lesion volumes increase by up to a factor of 5 within approximately 24 h after surgery, and then stabilize. Islands of viable cells can persist within damaged tissue, in the immediate vicinity of blood vessels, but also necrose within about 48 h. Delayed cell death is an unambiguously non-thermal process, apparently linked solely to biochemical messengers. The dramatic enlargement of the affected region appears to have been essentially overlooked in laser surgery studies. The ramifications include (a) proper gauging of the required scale of tissue damage during surgery, toward averting excessive destruction of untargeted surrounding tissue; and (b) avoiding false positives from the substantial amount of tissue that appears viable immediately after surgery but will necrose within 24 h. The comparable performance of high-flux solar and concentrated laser light for hyperthermic treatments permits effective surgery and the probing of tissue death dynamics with a solar energy system that is simpler and markedly less expensive than surgical lasers.

Human skin prepared with an optical clearing agent manifests reduced scattering as a result of de-hydration and refractive index matching. This has potentially large effects for laser therapies of several skin lesions such as port wine stain, hair removal and tattoo removal. With most topically applied clearing agents the clearing effect is limited because they penetrate poorly through the intact superficial skin layer (stratum corneum). Agent application modi other than topical are impractical and have limited the success of optical clearing in laser dermatology. In recent reports, however, a mixture of lipofylic and hydrofylic agents was shown to successfully penetrate through the intact stratum corneum layer which has raised new interest in this field. Immediately after application, the optical clearing effect is superficial and, as the agent diffuses through the skin, reduced scattering is manifested in deeper skin layers. For practical purposes as well as to maximize therapeutic success, it is important to quantify the reduced scattering as well as the trans-cutaneous transport dynamics of the agent. We determined the time and tissue depth resolved effects of optically cleared skin by inserting a microscopic reflector array in the skin. Depth dependent light intensity was measured by quantifying the signal of the reflector array with optical coherence tomography. A 1-dimensional mass diffusion model was used to estimate a trans-cutaneous transport diffusion constant for the clearing agent mixture. The results are used in Monte Carlo modeling to determine the optimal time of laser treatment after topical application of the optical clearing agent.

We demonstrated the capability of photoacoustic measurement for viscoelastic characterization. Since tissue viscoelasticity affects the propagation and attenuation of photoacoustic waves generated in the tissue, the relaxation times of the photoacoustic waves give the viscosity-elasticity ratio of the tissue. The relaxation times of photoacoustic waves of articular cartilage tissues engineered under various culture conditions were closely correlated with intrinsic viscosity-elasticity ratios measured by using a conventional viscoelastic analyzer (R > 0.98). In order to apply the photoacoustic measurement method to evaluation of the regeneration of articular cartilage as a method to validate the surgery, the method should enable not only evaluation of engineered tissue during cultivation in vitro but also evaluation after transplantation of engineered tissue in vivo. The aim of this study was to verify the usefulness of the photoacoustic method for repeated measurement of viscoelastic properties in order to evaluate the process of regeneration of a full-thickness defect in rabbit articular cartilage using allografted tissue-engineered cartilage. Photoacoustic waves were induced by 266- and 355-nm, 5-7 ns, light pulses delivered through an optical silica fiber from an Q-switched Nd:YAG laser and were detected by a piezoelectric transducer, which we had designed. About a 40% difference between the viscosity-elasticity ratio of allografted cartilage that of tissue surrounding the defect was shown just after surgery. The difference was significantly reduced at 4 and 12 postoperative weeks. Therefore, since the photoacoustic measurement method enables assessment of the progress of restoration of the viscoelasticity of articular cartilage, its main function, this method would be useful as an evaluation method in regenerative medicine.

We report on the histological results of in-vivo animal follow-up studies on refractive femtosecond laser surgery. Non-invasive flap-free intrastromal ablation as well as flap generation has been performed with MHz nanojoule near infrared femtosecond laser pulses. In particular, the dynamics of corneal wound healing have been studied. Wound-healing effects could be detected up to 90 days post-operation in the case of lasermediated flap generation. The flap-free intrastromal cavity was identified until the 28th day post-treatment. Interestingly, eosinophil granulocytes were observed. The follow-up studies confirmed that the near infrared femtosecond laser at near-nanojoule pulse energy is a highly precise and an attractive tool for intraocular refractive surgery, especially for flap-free intrastromal surgery.

A simple polarization method of mapping of tissue structures that allows for a variation in optical properties of medium in the direction of probing is proposed. This method is based on the use of rotationally invariant polarization parameters of sample. The relation between these parameters and Mueller matrix elements is shown.

Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. In regenerative medicine, it has been reported that phosphorylated proteins are instrumental in the calcification of osteoconnective tissue. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070 cm^-1 (9.4 mm) with Fourier Transform Infrared Spectrometer (FT-IR). This result indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm^-1 with FT-IR method. And we examine laser-dephosphorylation using Free Electron Laser (FEL) as a novel dephosphorylation method. After irradiation of 9.4mm-FEL, infrared absorption peak of phosphate group is reduced. It is suspected that this lowering of the peak of phosphate group is the effect like dephosphorylation. These novel methods can be applied to quality control technology in regenerative medicine.

Collagen gel is a natural biomaterial commonly used in tissue engineering because of its close resemblance to nature, negligible immunogenecity and excellent biocompatibility. However, unprocessed collagen gel is mechanically weak, highly water binding and vulnerable to chemical and enzymatic attacks that limits its use in tissue engineering in particular tissues for weight-bearing purposes. The current project aimed to strengthen and stabilize collagen scaffolds using a photochemical crosslinking technique. Photochemical crosslinking is rapid, efficient, non-thermal and does not involve toxic chemicals, comparing with other crosslinking methods such as glutaraldehyde and gamma irradiation. Collagen scaffolds were fabricated using rat-tail tendon collagen. An argon laser was used to process the collagen gel after equilibrating with a photosensitizing reagent. Scanning electronic microscope was used to characterize the surface and cross-sectional morphology of the membranes. Physico-chemical properties of the collagen scaffolds such as water-binding capacity, mechanical properties and thermostability were studied. Photochemical crosslinking significantly reduced the water-binding capacity, a parameter inversely proportional to the extent of crosslinking, of collagen scaffolds. Photochemical crosslinking also significantly increased the ultimate stress and tangent modulus at 90% of the rupture strain of the collagen scaffolds. Differential scanning calorimetry analysis showed a significantly higher shrinkage temperature and absence of the denaturation peak during the thermoscan comparing with the controls. This means greater thermostability in the photochemically crosslinked collagen scaffolds. This study demonstrates that the photochemical crosslinking technology is able to enhance the physicochemical propterties of collagen scaffolds by strengthening, stabilizing and controlling the swelling ratio of the collagen scaffolds so as to enable their use for tissue engineering.

A cholesterol ester is selective dissociated by MIR-FEL irradiation with wavelength of 5.75 μm correspond to C=O stretching vibration of ester bond. To evaluate the optimum irradiation condition for cholesterol ester decomposition without normal endothelium cell damage, we perform a 5.75 μm-FEL irradiation to a two-layer vessel model which is cholesterol oleate as a model of atherosclerotic lesions and gelatin as a model of endothelial cells. The ester decomposition and gelatin damege depends on total power density of 5.75 μm-FEL provided the two-layer model. Exposure of the FEL with power density exceed 17.8 W/cm² decomposed cholesterol ester thorough gelain layer of 15 μm thickness. If FEL with power density of 25.0 W/cm² is exposed during 10 seconds, the gelatin is evaporized. Therefore, the optimum condition for ester decomposition without gelatin evaporation is between 75 J/cm² from 20 J/cm² in the case of 15 μm gelatin layer thickness. The maximum ester decomposition rate without gelatin vaporization is 71% on FEL of power density of 17.8 W/cm² for 10 seconds.

The goal of this study was to optimize measurement techniques for tissue point spectroscopy during gastrointestinal (GI) endoscopy, as part of a program to enhance and apply autofluorescence/reflectance imaging for early GI cancer detection. The effect of fiberoptic probe pressure on tissue on the measured diffuse reflectance spectra was evaluated, with both fiber-to-fiber probe geometry (standard contact probe) and imaging illumination geometry (wide field illumination and fiber collection) for the wavelength range 440-640 nm, using normal skin in vivo as a model tissue, and by taking continuous spectral measurements while the fiber is approaching the tissue. The most significant finding was a sudden change in the reflectance signal that occurs as the probe comes into contact with the tissue surface.

In selective retina treatment (SRT) spatial confined tissue damage in the absorbing retinal pigment epithelium (RPE) is obtained by applying microsecond laser pulses. The damage in the RPE is caused by transient microbubbles forming around laser heated melanin granules inside the cells. For treatment of RPE related diseases, SRT is thought to share the therapeutic benefits of conventional photocoagulation but without affecting the photoreceptors. A drawback for effective clinical SRT is that the laser-induced lesions are ophthalmoscopically invisible. Therefore, a real-time feedback system for dosimetry is demanded in order to avoid undertreatment or unwanted collateral damage to the adjacent tissue, which is observed at about twofold threshold radiant exposure for RPE damage. We demonstrate that interferometry is capable of detecting microbubbles at threshold irradiation for RPE damage. A porcine ex-vivo RPE model, which was irradiated by a Nd:YLF laser (350ns/1700ns@527nm), was employed. The irradiated area was simultaneously probed by a Michelson interferometer. Cell viability assays were performed after irradiation. Cell damage was compared to the interferometric data. At threshold for cell damage a change in the interferometric transients was observed due to microbubble formation. Thus, interferometry could serve as a non-contact real-time dosimetry control during selective targeting of the RPE in vivo. From the time resolved motion of the bubble interface, which is determined by interferometry, the maximum bubble size is obtained. This gives an estimation for the damage range in the tissue. Knowledge of the damage range as a function of the irradiation parameters is helpful in optimizing SRT.

Many laser therapies involve significant heating of tissue with pulses varying from picoseconds to minutes in duration. In some of the applications heating is a primary goal, while in others it is an undesirable side effect. In both cases, if a hyperthermia is involved, the knowledge about the threshold temperature leading to irreversible cellular damage is critically important. We study the dependence of the threshold temperature on duration of the heat exposure in the range of 0.3 ms to 5 seconds. Thin layer of cells cultured in a Petri dish was exposed to a pulsed CO₂ laser radiation. Laser beam was focused onto sample providing Gaussian intensity distribution in the focal plane with a beam diameter (2w) 2-10 mm. Surface temperature in the central part of the focal spot (1mm in diameter) was measured by thermal infrared (IR) emission from the sample, recorded with a fast IR detector. For pulses shorter than 1 s the temperature profile across the focal spot was found to closely correspond to the radial distribution of the laser beam intensity, thus allowing for accurate determination of temperature at any given distance from the center of the spot. Immediate cellular damage was assessed using vital staining with the live/dead fluorescent assay. Threshold temperatures were found to vary from 65 °C at 5 s of heating to 160 °C at pulses of 0.3 ms in duration. The shorter end of this range was limited by vaporization, which occurs during the laser pulse and results in mechanical damage to cells. Dependence of the maximal temperature on pulse duration could be approximated by Arrhenius law with activation energy being about 1 eV.