This PDF file contains the front matter associated with SPIE Proceedings Volume 7904, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Although femtosecond laser cell surgery is widely used for fundamental research in cell biology, the mechanisms in the so-called low-density plasma regime are largely unknown. To date, it is still unclear on which time scales free electron and free radical-induced chemical effects take place leading to intracellular ablation. In this paper, we present our experimental study on the influence of laser parameters and staining on the ablation threshold. We found that the ablation effect resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. In addition, fluorescence staining of subcellular structures significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade.

Laser microbeam has enabled highly precise non-contact delivery of exogenous materials into targeted cells, which has been a highly challenging task while using traditional methods without compromising cell viability. We report distinct spatial localization of impermeable substances into mammalian cells and goldfish retinal cells in explants subsequent to ultrafast laser microbeam assisted injection, realized by focusing a near infrared tunable Ti: sapphire laser beam. Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time constant for injection. Using optimized laser intensity, exposure and spatial irradiation pattern, desired spatial transfection patterns in goldfish retina explants were achieved as confirmed by expression of injected plasmids encoded for light-activable channelrhodopsin-2 (ChR2) ion channel tagged with fluorescent protein. Laser assisted delivery of exogenous material into specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the functioning of neuronal circuitry of normal and degenerated retina.

Endovenous Laser Ablation (EVLA) has become a popular minimally invasive alternative to stripping in the treatment of saphenous vein reflux. Several wavelengths have been proposed; of which 810, 940 and 980- nm are the most commonly used. However, the most appropriate wavelength is still the subject of debate. Thermal shrinkage of collagenous tissue during EVLA plays a significant role in the early and late results of the treatment. The aim of this study is to compare the efficacy of 980 and 1940-nm laser wavelengths in the treatment of varicose veins. In this study, 980 and 1940-nm lasers at different power settings (8/10W for 980-nm, 2/3W for 1940-nm) were used to irradiate stripped human veins. The most prominent contraction and narrowing in outer and inner diameter were observed with the 1940-nm at 2W, following 980-nm at 8W, 1940-nm at 3W and finally 980-nm at 10W. The minimum carbonization was observed with the 1940-nm at 2W. As a conclusion, 1940-nm Tm-fiber laser which has a significant effect in the management of varicose veins due to more selective energy absorption in water and consequently in the vein is a promising method in the management of varicose veins.

We explore the potential of intravascular or endoscopic optical coherence tomography (OCT) to extract relevant mechanical properties of a tissue deformed by an inflating balloon. Tubular OCT phantoms with different mechanical properties are fabricated. The phantoms are deformed by an inflating balloon, and the deformation is monitored with OCT. A quantitative description of the phantom deformation is obtained by segmenting the OCT images. Two strategies to extract the mechanical properties from this quantitative data are presented: by comparing to a finite-element simulation and by performing a mechanical analysis.

For a systemically administered drug to act, it first needs to cross the vascular wall. This step represents a bottleneck for drug development, especially in the brain or retina, where tight junctions between endothelial cells form physiological barriers. Here, we demonstrate that femtosecond pulsed laser irradiation focused on the blood vessel wall induces transient permeabilization of plasma. Nonlinear absorption of the pulsed laser enabled the noninvasive modulation of vascular permeability with high spatial selectivity in three dimensions. By combining this method with systemic injection, we could locally deliver molecular probes in various tissues, such as brain cortex, meninges, ear, striated muscle, and bone. We suggest this method as a novel delivery tool for molecular probes or drugs.

Re-orientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The cell gets detached after irradiation for few seconds, followed by vertical orientation. The detached cell was transported along axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by Gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. The cell could be brought back to the substrate by reducing the laser beam power using a polarizer or blocking the laser beam. Viability of the detached and manipulated cell was found not to be compromised as confirmed by PI fluorescence exclusion assay. The re-oriented cell was allowed to re-attach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for non-contact modification of cellular orientations that will enable study of inter-cellular interactions and design of engineered tissue.

Terahertz (10¹² Hz) pulsed imaging is a totally non-destructive and non-ionising imaging modality and thus potential applications in medicine are being investigated. In this paper we present results using our hand-held terahertz probe that has been designed for in vivo use. In particular, we use the terahertz probe to perform reflection geometry in vivo measurements of human skin. The hand-held terahertz probe gives more flexibility than a typical flat-bed imaging system, but it also results in noisier data and requires existing processing methods to be improved. We describe the requirements and limitations of system geometry, data acquisition rate, image resolution and penetration depth and explain how various factors are dependent on each other. We show how some of the physical limitations can be overcome using novel data processing methods.

Metal meshes work as band-pass filters in the terahertz (THz) region, with their transmission spectra acutely affected by the refractive index of the material inside and above the metal mesh openings. We used a metal mesh for high-sensitivity observations by focusing on the "dip", that is, a sudden change in transmittance that only appeared when the THz wave was obliquely incident onto the metal mesh. Here we report a measurement of stratum corneum to inspect the feasibility of applying the metal mesh sensor to observations of human skin.

Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm^-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

Finite-difference time-domain (FDTD) methods are widely used to model the propagation of electromagnetic radiation in biological tissues. High-performance central processing units (CPUs) can execute FDTD simulations for complex problems using 3-D geometries and heterogeneous tissue material properties. However, when FDTD simulations are employed at terahertz (THz) frequencies excessively long processing times are required to account for finer resolution voxels and larger computational modeling domains. In this study, we developed and tested the performance of 2-D and 3-D FDTD thermal propagation code executed on a graphics processing unit (GPU) device, which was coded using an extension of the C language referred to as CUDA. In order to examine the speedup provided by GPUs, we compared the performance (speed, accuracy) for simulations executed on a GPU (Tesla C2050), a high-performance CPU (Intel Xeon 5504), and supercomputer. Simulations were conducted to model the propagation and thermal deposition of THz radiation in biological materials for several in vitro and in vivo THz exposure scenarios. For both the 2-D and 3-D in vitro simulations, we found that the GPU performed 100 times faster than runs executed on a CPU, and maintained comparable accuracy to that provided by the supercomputer. For the in vivo tissue damage studies, we found that the GPU executed simulations 87x times faster than the CPU. Interestingly, for all exposure duration tested, the CPU, GPU, and supercomputer provided comparable predictions for tissue damage thresholds (ED₅₀). Overall, these results suggest that GPUs can provide performance comparable to a supercomputer and at speeds significantly faster than those possible with a CPU. Therefore, GPUs are an affordable tool for conducting accurate and fast simulations for computationally intensive modeling problems.

A two-dimensional, time-dependent bioheat model is applied to evaluate changes in temperature and water content in tissues subjected to laser irradiation. Our approach takes account of liquid-to-vapor phase changes and a simple diffusive flow of water within the biotissue. An energy balance equation considers blood perfusion, metabolic heat generation, laser absorption, and water evaporation. The model also accounts for the water dependence of tissue properties (both thermal and optical), and variations in blood perfusion rates based on local tissue injury. Our calculations show that water diffusion would reduce the local temperature increases and hot spots in comparison to simple models that ignore the role of water in the overall thermal and mass transport. Also, the reduced suppression of perfusion rates due to tissue heating and damage with water diffusion affect the necrotic depth. Two-dimensional results for the dynamic temperature, water content, and damage distributions will be presented for skin simulations. It is argued that reduction in temperature gradients due to water diffusion would mitigate local refractive index variations, and hence influence the phenomenon of thermal lensing. Finally, simple quantitative evaluations of pressure increases within the tissue due to laser absorption are presented.

Laser radiation has many applications in biomedical field, such as wound healing, tissue repairing, heating and ablation processes. Intravenous low power laser radiation is used clinically for skin and vascular disorders. Laser radiation improves microcirculation and modulates the rheological properties of blood. FTIR (Fourier Transform Infra Red Spectra) is used to see the structural changes in erythrocyte membrane. In the present work He Ne laser (λ= 632nm, power=2mW) is used to irradiate human Red blood cells. Red blood cells are separated from human whole blood using centrifugation method (time=10 min., temperature=15°C and RPM=3000) and then exposed to HeNe laser radiation. Laser exposure time is varied from 10 min. to 40min for Red blood cells. Absorption spectrum, FTIR and fluorescence spectra of RBC are compared before and after HeNe laser irradiation. The absorption spectrum of RBC after exposure to HeNe laser shows a significant decrease in absorbance. The FTIR spectrum of non irradiated RBC clearly show the peaks due to O-H (free group), C=O (amide I group), N=O (nitro group), C-O (anhydride group) and C-H (aromatic group). Laser radiation changes in transmittance in FTIR spectra related to C=O group and percentage of transmittance increases for O-H, C=C, N=O, C-O and C-H group.

Current laser safety standards for multiple pulse lasers are based primarily on modeling and the results of single pulse studies. Previous thermal effects studies have focused on histological and visible endpoints, with only a few studies examining the actual temperatures achieved. The goal of this research was to probe the actual vertical temperature profile produced by 2.01 micron laser pulses in the cornea. In this study the corneal temperature rise from multiple 2.01 micron Tm:YAG laser pulses was investigated using ex-vivo rabbit eyes. A thermal-measurement data set for a different number of pulses was collected and compared. An infrared thermal camera employing microbolometer detectors captured surface temperature rises resulting from laser pulses. Single 10 ms pulses as well as two, three, and four pulse sequences were utilized while the total energy delivered was held constant. A comparison of the data to temperatures required for denaturing proteins and the current laser safety guidelines will be presented.

Laser exposure duration dictates whether tissues subjected to short visible wavelengths ( ≤ 514 nm) are damaged by thermal (e.g. 0.1 s) or non-thermal ( ≥ 100 s) mechanisms. Somewhere between these extremes, an abrupt transition between the two damage mechanisms has been found for both in vitro and animal retinal models (J. Biomed. Opt. 15, 030512, 2010). Non-thermal (photochemical) damage is characterized by an inverse relationship between damage threshold irradiance and exposure duration (irradiance reciprocity). We have found that exposures of 40 - 60 s in an in vitro retinal model require radiant exposures well above the expected requirement for nonthermal damage, introducing the concept that damage was forced to be thermal in mechanism. Here we quantify and compare photo-oxidative processes at ambient temperatures between 35 - 50 °C.

Familial Adenomatous Polyposis (FAP) is an autosomal dominant disease characterized by the development of multiple colonic polyps at younger age with a near 100% lifetime risk of colorectal cancer in later years. The determination of FAP is made after extensive clinical evaluation and genetic testing of at risk individuals. Genetic testing is expensive and in some cases deleterious mutations are not found in all patients with a clinical diagnosis of FAP. As such, the early identification of affected individuals could substantially eliminate associated morbidity and mortality. We investigated a novel spectro-polarimetric imaging system to capture images of the oral mucosa at different wavelengths in an attempt to distinguish patients with FAP from controls. Total diffused oral mucosal reflectance (OMR) and oral mucosal vascular density (OMVD) were calculated from spectral data collected from 33 patients with gene positive FAP, 5 patients who tested negative for FAP, and 45 controls. A statistically significant difference in OMVD (p < 0.001) was observed between individuals with FAP and controls. Analysis of OMR showed no significant difference between the two subject groups.

Light propagation in fibrous biological tissue is quite different from that in isotropic medium. Several studies showed that the measured reduced scattering coefficient in fibrous tissue strongly depends on the measurement direction. In this study, we investigated the possibility of retrieving optical properties in anisotropic tissue using time-domain measurements. A Monte Carlo model was used to simulate light propagation in a fibrous tissue consisting of aligned cylinders and spherical shape scatters. An isotropic diffuse model was then used to determine the optical properties from simulated time-resolved reflectance. When the measurement position was parallel to the fiber direction, the derived reduced scattering coefficient had good agreement with the true background scattering coefficient values with a less than 10% error. In contrast, when measuring in a direction perpendicular to the cylinders, the derived reduced scattering coefficient was close to the summation of the reduced scattering coefficients of both cylinders and background only in samples with small cylinder diameters. If the fiber size in the medium is known, the reduced scattering coefficient associated with the cylinders can be derived by using a correction coefficient.

Goals: Improving cancer diagnosis is one of the important challenges at this time. The precise differentiation between benign and malignant tissue is in the oncology and oncologic surgery of the utmost significance. A new diagnostic system, that facilitates the decision which tissue has to be removed, would be appreciated. In previous studies many attempts were made to use tissue fluorescence for cancer recognition. However, no clear correlation was found between tissue type and fluorescence parameters like time and wavelength dependent fluorescence intensity I(t, λ). The present study is focused on cooperative behaviour of cells in benign or malignant prostates tissue reflecting differences in their metabolism. Material and Methods: 50 prostate specimens were obtained directly after radical prostatectomy and from each specimen 6 punch biopsies were taken. Time-resolved fluorescence spectra were recorded for 4 different measurement points for each biopsy. The pathologist evaluated each measurement point separately. An algorithm was developed to determine a relevant parameter of the time dependent fluorescence data (fractal dimension D_F ). The results of the finding and the D_F -value were correlated for each point and then analysed with statistical methods. Results: A total of 1200 measurements points were analysed. The optimal algorithm and conditions for discrimination between malignant and non-malignant tissue areas were found. The correct classification could be stated in 93.4% of analysed points. The ROC-curve (AUC = 0.94) confirms the chosen statistical method as well as it informs about the specificity (0.94) and sensitivity (0.90). Conclusion: The new method seems to offer a very helpful diagnostic tool for pathologists as well as for surgery.

Determination of tissue optical parameters is fundamental for application of light in either diagnostics or therapeutical procedures. However, in samples of biological tissue in vitro, the optical properties are modified by cellular death or cellular agglomeration that can not be avoided. This phenomena change the propagation of light within the biological sample. Optical properties of human blood tissue were investigated in vitro at 633 nm using an optical setup that includes a double integrating sphere system. We measure the diffuse transmittance and diffuse reflectance of the blood sample and compare these physical properties with those obtained by Monte Carlo Multi-Layered (MCML). The extraction of the optical parameters: absorption coefficient μ_a, scattering coefficient _μs and anisotropic factor g from the measurements were carried out using a Genetic Algorithm, in which the search procedure is based in the evolution of a population due to selection of the best individual, evaluated by a function that compares the diffuse transmittance and diffuse reflectance of those individuals with the experimental ones. The algorithm converges rapidly to the best individual, extracting the optical parameters of the sample. We compare our results with those obtained by using other retrieve procedures. We found that the scattering coefficient and the anisotropic factor change dramatically due to the formation of clusters.

Low level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably go from the photobiostimulation/photobioinibition at cellular level to the molecular level. The detailed mechanism underlying this effect is still obscure. This work is dedicated to quantify some relevant aspects of LLLT related to molecular and cellular variations. This goal was attached by exposing malignant breast cells (MCF7) to spatially filtered light of a He-Ne laser (633 nm) with 28.8 mJ/cm² of fluency. The cell viability was evaluated by microscopic observation using Trypan Blue viability test. The vibrational spectra of each experimental group (micro- FTIR technique) were used to identify the relevant biochemical alterations occurred due the process. The red light had influence over RNA, phosphate and serine/threonine/tyrosine bands. Light effects on cell number or viability were not detected. However, the irradiation had direct influence on metabolic activity of cells.

We studied a pre-charring optical behavior of blood at a laser catheter-tip during a red laser irradiation (663 nm, CW) with around 50 W/cm² in blood to prevent charring at the laser catheter-tip. The laser irradiated red-blood-cell shape changes were microscopically observed. A round formation, aggregation, and hemolysis were found until blood charring (ex vivo). A time-history of diffuse-reflected light power and transmitted light power from a thin blood layer which was irradiated by the red laser were measured with microscope optics to investigate the charring process. The diffusereflected light power decreased following a gentle peak before the charring. This decrease indicated the pre-charring behavior which might be induced by scattering and absorption changes due to red-blood-cell degenerations described above. Using the laser catheter located in porcine heart, we successfully detected the pre-charring behavior by a backscattering light power (in vivo). We demonstrated charring prevention availability with the laser power control (ex vivo). We think that the backscattering light power measurement and laser power control via the laser catheter might be useful to detect pre-charring behavior, and to prevent the charring for therapeutic laser irradiation in blood under catheterization such as arrhythmia treatment with photodynamic therapy.

Angular Domain Imaging (ADI) has been previously demonstrated to generate projection images of attenuating targets embedded within a turbid medium. The imaging system employs a silicon micro-tunnel array positioned between the sample and the detection system to reject scattered photons that have deviated from the initial propagation direction and to select for ballistic and quasi-ballistic photons that have retained their forward trajectory. Two dimensional tomographic images can be reconstructed from ADI projections collected at a multitude of angles. The objective of this work was to extend the system to three dimensions by collecting several tomographic images and stacking the reconstructed slices to generate a three dimensional volume representative of the imaging target. A diode laser (808nm, CW) with a beam expander was used to illuminate the sample cuvette. An Angular Filter Array (AFA) of 80 μm × 80 μm square-shaped tunnels 2 cm in length was used to select for image forming quasi-ballistic photons. Images were detected with a linear CCD. Our approach was to use a SCARA robot to rotate and translate the sample to collect sufficient projections to reconstruct a three dimensional volume. A custom designed 3D target consisting of 4 truncated cones was imaged and reconstructed with filtered backprojection and iterative methods. A 0.5 mm graphite rod was used to collect the forward model, while a truncated pseudoinverse was used to approximate the backward model for the iterative algorithm.

Conventional fluorescence imaging often does not have a mechanism to remove the scattering effect in biological tissue. We use Angular Domain Imaging (ADI) to improve the detection of smaller structures in fluorescence layer over that can be provided by existing systems. ADI is a high resolution, ballistic imaging method that utilizes the angular spectrum of photons to filter multiple-scattered photons and accepts only photons with small angular deviation from their original trajectory. Advantages of the ADI technique are that it is insensitive to wavelength and the sources are not required to be high quality, coherent, or pulse, as with OCT or time domain. Our target is to perform fluorescence ADI at shallow tissue such as skin (≈ 1mm) with a buried collagen layer. To experimentally model shallow tissue with phantoms, a thin layer of scattering medium with similar scattering characteristic (μs = 200cm^-1, g = 0.85) is placed on top fluorescence plastic (415nm excitation, ≈ 555-585nm emission) which is patterned by strips of non-emitting structures (200-400μm). Positioning multiple collimated arrays with acceptance angles of 5.71° on top of the scattering medium, test structures (200μm wide) can be detected at shallow scattering medium thickness (1mm). Monte Carlo simulation confirms that fluorescence ADI can image structures at shallow tissue depth by using collimator array with modest filtration angles. Results show micromachined collimator arrays provide both high spatial resolution and angular filtration on scattered photons.

Light-tissue interaction is common in clinical treatments and medical researches, therefore investigation of light path in irradiated tissues is of high importance. In this research, simulations and experimental measurements of the reflected light intensity from one-layered lattices and phantoms are presented. Our results suggest that random walk simulations fit well the photon migration model and enable the extraction of the lattice absorption parameter. The experimental results present a partial fitting to the random walk model: while phantoms presenting different absorption coefficients are distinguished by different reemitted light profiles, the model does not apply an adequate description for the phantom absorption coefficient extraction. This calls for further investigation.

Photothermal therapy offers a solution for the destruction of cancer cells without significant collateral damage to otherwise healthy cells. Several attempts are underway in using carbon nanoparticles (CNPs) and nanotubes due to their excellent absorption properties in the near-infrared spectrum of biological window. However, minimizing the required number of injected nanoparticles, to ensure minimal cytotoxicity, is a major challenge. We report on the introduction of magnetic carbon nanoparticles (MCNPs) onto cancer cells, localizing them in a desired region by applying an external magnetic field and irradiating them with a near-infrared laser beam. The MCNPs were prepared in Benzene, using an electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements showed the size distribution of these MCNPs to be in the range of 5-10 nm. For photothermal irradiation, a tunable continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely guide photothermal therapy.

In the present work, we demonstrate a potential use of gold nanorods as a contrast agent for selective photothermal therapy of human acute leukemia cells (HL-60) using a near-infrared laser. Gold Nanorods (GNR) are synthesized and conjugated to CD33, a 67 kDa glycoprotein found on the surface of myeloid cells that belongs to the sialoadhesin family of proteins. After pegylation, or conjugation with CD33 antibody, GNR were non-toxic for acute and chronic leukemia cells. We used a Quanta System q-switched titanium sapphire laser emitting at a center wavelength of 755 nm. Each sample was illuminated with 1 laser shot at either high or low fluence. Both laser modes were used in 3 independent cell probes. HL-60 cells were treated for 45 min with GNR conjugated with mAb CD33, or with GNR-Pegylated particles. After laser application, the cells were resuspended and analyzed to cell viability with Trypan blue exclusion assay. GNR-CD33 conjugates significantly increase the percentage of cell death as compared with a control group after laser illumination: a 3 fold increase is observed.

ABSTRACT Damage thresholds (ED₅₀) for skin using Yucatan mini-pig (Sus scrofa domestica) have been determined at the operational wavelength of 1319 nm with beam diameters of 0.61 cm and 0.96 cm. Exposure durations of 0.25, 1.0, 2.5 and 10 seconds were used to determine trends in damage threshold with respect to exposure time and beam diameter at this moderately-high penetrating wavelength. A relatively narrow range of total radiant exposure from 37.4 J/cm² to 62.3 J/cm² average was observed for threshold damage with laser parameters encompassing a factor of two in beam area and a factor of forty in exposure duration.

We report on an investigation aimed to increase the efficiency of photodynamic therapy (PDT) through the influence of localized surface plasmon resonances (LSPR's) in metal nanoparticles. PDT is based on photosensitizers that generate singlet oxygen at the tumour site upon exposure to visible light. Although PDT is a well-established treatment for skin cancer, a major drawback is the low quantum yield for singlet-oxygen production. This motivates the development of novel methods that enhance singlet oxygen generation during treatment. In this context, we study the photodynamic effect on cultured human skin cells in the presence or absence of gold nanoparticles with well established LSPR and field-enhancement properties. The cultured skin cells were exposed to protoporphyrin IX and gold nanoparticles and subsequently illuminated with red light. We investigated the differences in cell viability by tuning different parameters, such as incubation time and light dose. In order to find optimal parameters for specific targeting of tumour cells, we compared normal human epidermal keratinocytes with a human squamous skin cancer cell line. The study indicates significantly enhanced cell death in the presence of nanoparticles and important differences in treatment efficiency between normal and tumour cells. These results are thus promising and clearly motivate further development of nanoparticle enhanced clinical PDT treatment.

Balb/c wild type mice were used to perform in vivo experiments of laser-induced thermal damage to the retina. A Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope with a spectral domain optical coherence tomographer was used to obtain fundus and cross-sectional images of laser induced injury in the retina. Sub-threshold, threshold, and supra-threshold lesions were observed using optical coherence tomography (OCT), infrared reflectance, red-free reflectance, fluorescence angiography, and autofluorescence imaging modalities at different time points post-exposure. Lesions observed using all imaging modalities, except autofluorescence, were not visible immediately after exposure but did resolve within an hour and grew in size over a 24 hour period. There was a decrease in fundus autofluorescence at exposure sites immediately following exposure that developed into hyper-fluorescence 24-48 hours later. OCT images revealed threshold damage that was localized to the RPE but extended into the neural retina over a 24 hour period. Volumetric representations of the mouse retina were created to visualize the extent of damage within the retina over a 24 hour period. Multimodal imaging provides complementary information regarding damage mechanisms that may be used to quantify the extent of the damage as well as the effectiveness of treatments without need for histology.

Even though catheterization or electric stimulation are used for treatment of neurogenic bladder, invasiveness and inconvenience of these approaches prompt us to develop a new possible therapeutic method to control urination by using optical stimulation. The optical method using femtosecond pulsed laser (FSPL) has advantages of focused and subsurface stimulation. Irradiation of FSPL induced a rapid increase of intracellular calcium level followed by contraction of primary cultured human bladder smooth muscle cells. Short exposure of bladder detrusor ex-vivo to FSPL also induced a controlled contraction of detrusor. Collectively, we propose that FSPL can be considered as a potential therapeutic approach for intractable neurogenic bladder.

We were unable to reproduce published inactivation results, or show any interaction, between 90 femtosecond (fs) pulses of 850 nm or 425 nm laser radiation and buffer/water, DNA, protein, M13 bacteriophage or E. coli. Using agarose electrophoresis and polyacrylamide gel electrophoresis, we examined purified plasmid DNA (pUC19), bovine serum albumin, and DNA and coat proteins extracted from M13 following exposures to irradiances of up to 120 MW/cm². We measured M13 viability using an assay for plaque-forming ability in soft agar after exposure to the same irradiances used for the protein and DNA experiments. Exposures of up 1 GW/cm² at 850 nm had no effect on the viability of E. coli as measured by a colony forming assay in soft agar. Peroxynitrite, known to be toxic, to cause single strand breaks in DNA, and fragment proteins in vitro gave positive results in all assays.

Computational molecular docking simulations (Dock and AutoDock) may provide a wealth of structural information related to the bound configuration of protein-ligand complexes, but they require verification to ensure their results correctly predict the bound complex. Resonance Raman spectroscopy data has been collected to correlate normal mode vibrations observed in the bound configurations to computationally generated structures in order to determine the best match between the in silico model and experiment. This methodology was used to determine the bound structures at an atomistic level of β-lactoglobulin (BLG) and meso-tetrakis (p-sulfonatophenyl) porphyrin (TSPP) in aqueous solutions at pH 7 and 9. Comparisons of Raman spectra of TSPP before and after binding to BLG yield line shifts that are generated by the noncovalent binding of the ligand to the protein. Previous studies have shown that the Tanford transition in BLG, which occurs above pH 7.9, destabilizes the protein, allowing it to undergo a laser-induced structural change when bound to TSPP and illuminated by at least 0.3 J of laser energy. By examining the structures at pHs above and below the transition, we hope to reveal the mechanism of action that initiates the laser-induced changes in the protein. Future studies will use the computed bound configuration as an initial condition for molecular dynamics simulations of the laser-protein-complex interaction to predict the final state of the protein after irradiation.

Fluorescent imaging of cells and tissues cultured within a rotating wall vessel bioreactor offers quantitative assessment of the 3-dimensional aggregation of cells into tissue constructs. We present the design of a rotating wall vessel system optimized for real-time fluorescent analysis. The modulation transfer function of our system is found to be superior to the commercially-available vessel used in previous fluorescence imaging studies. We demonstrate dynamic fluorescent imaging of DAPI-stained porcine pancreatic islets.

Tissue scaffolds are an integral part of the tissue engineering process, assisting in the culturing of cells in three dimensions. It is important to understand both the properties of the scaffold and the growth of cells within the scaffold. This paper describes a system to characterise scaffolds using acoustic techniques alone and the development of an ultrasound modulated optical tomography system to study the growth of cells within the scaffolds. Our interest is in characterising the properties of gel-based and polymer foam-based scaffolds. Results from a purely acoustic system have been used to investigate the properties of foam scaffolds manufactured from synthetic polyesters poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) via a supercritical fluid process. As these are porous materials, they are particularly challenging acoustically as the pores scatter sound significantly. However, it is demonstrated that acoustic signals are detectable through a 6mm thick scaffold. Although acoustics alone can be used to characterize many properties of the scaffolds, useful information can also be obtained from optical techniques e.g. monitoring the growth of cells within the scaffold via optical absorption or fluorescence techniques. Light scattering is of course a significant problem for relatively thick engineered tissue (~5mm). The acoustic approach has been extended to include laser illumination and detection of the ultrasound modulated optical pulse. Images of optically-absorbing materials embedded in gel-based tissue phantoms will be presented demonstrating that a lateral resolution of 250μm and an axial resolution of ~90μm can be achieved in scattering samples.

With the increasing complexity of the chemical composition of pharmaceuticals, cosmetics and everyday substances, the awareness of potential health issues and long term damages for humanoid organs is shifting into focus. Artificial in vitro testing systems play an important role in providing reliable test conditions and replacing precarious animal testing. Especially artificial skin equivalents ASEs are used for a broad spectrum of studies like penetration, irritation and corrosion of substances. One major challenge in tissue engineering is the qualification of each individual ASE as in vitro testing system. Due to biological fluctuations, the stratum corneum hornified layer of some ASEs may not fully develop or other defects might occur. For monitoring these effects we developed an fully automated Optical Coherence Tomography device. Here, we present different methods to characterize and evaluate the quality of the ASEs based on image and data processing of OCT B-scans. By analysing the surface structure, defects, like cuts or tears, are detectable. A further indicator for the quality of the ASE is the morphology of the tissue. This allows to determine if the skin model has reached the final growth state. We found, that OCT is a well suited technology for automatically characterizing artificial skin equivalents and validating the application as testing system.

Aligned, electrospun fibers have been used in a wide variety of applications from filters to scaffolds for tissue engineering. In this study we demonstrate a quick and accurate method to quantify fiber alignment using the Radon Transform. To test the accuracy of this method, we generated mock images fibers with varying degrees of fiber alignment. Images were filtered to detect edges and analyzed with the Radon Transform from 1 to 180 degrees at 1 degree intervals. The absolute values of each column were summed and used to create a normalized probability distribution function. The probability distribution function was quantified using both the full width half- maximum (FWHM) and calculating the entropy of the function. These results were compared to an analysis method using the fast Fourier transform. The FWHM for the Radon transform was consistent and statistically different at all fiber orientations for different degrees of fiber variation. Both the entropy analysis for the Radon transform and the FWHM for the fast Fourier transform did not show statistical difference. The FWHM method for the radon transform was performed on electrospun fibers and showed statistical difference between two groups known to be statistically different by manual analysis.

In 1999 we have introduced a new approach for treatment of spine diseases based on the mechanical effect of nondestructive laser radiation on the nucleus pulposus of the intervertebral disc. Laser reconstruction of spine discs (LRD) involves puncture of the disc and non-destructive laser irradiation of the nucleus pulposus to activate reparative processes in the disc tissues. In vivo animal study has shown that LRD allows activate the growth of hyaline type cartilage in laser affected zone. The paper considers physical processes and mechanisms of laser regeneration, presents results of investigations aimed to optimize laser settings and to develop feedback control system for laser reparation in cartilages of spine and joints. The results of laser reconstruction of intervertebral discs for 510 patients have shown substantial relief of back pain for 90% of patients. Laser technology has been experimentally tested for reparation of traumatic and degenerative diseases in joint cartilage of 20 minipigs. It is shown that laser regeneration of cartilage allows feeling large (more than 5 mm) defects which usually never repair on one's own. Optical techniques have been used to promote safety and efficacy of the laser procedures.

Red blood cells (RBC) possess unique viscoelastic characteristics which allow them to pass through capillaries narrower than their size. Measurement of viscoelastic property of cells (e.g. RBC) in low-force regime is of high significance as it represents conditions of membrane fluctuation in response to physiological conditions. Estimation of visco-elastic properties of RBC requires measurement of extent of deformation in RBC subjected to known force. Optical tweezers, being gentle and absolutely sterile, are emerging as the tool of choice for application of localized force on cells. However, stretching of RBC in very low force regime has not been quantified. Further, though deformations in transverse directions have been measured, vertical deformations due to stretching of cells cannot be quantified by classical microscopic images. Here, we report realization of offaxis digital holographic microscopy (DHM) for highly sensitive axial changes in RBC shape due to stretching by optical tweezers without attaching microscopic beads. The RBC was stretched in axial direction with nanometer precision by change of divergence of the trapping beam. The obtained deformation patterns were compared with the axial position of the tweezers focus. Since the pathophysiology of progression of diseases like malaria and cancer is reflected in the biophysical (both mechanical and material) properties of the cells, it is possible to identify the changes by simultaneous measurement of refractive index and elasticity using this approach.

We firstly investigate the mechanic and acoustic properties of human teeth by using the laser generation of surface acoustic wave (SAW) technique. The materials investigated included normal and decayed teeth, which have the same grain size and different thickness, are used as the samples. The tissue responds to the laser-induced stress by thermoelastic expansion. We can obtain the shape of acoustic pulse and the phase velocity was determined for the teeth system and extract information on the teeth thickness, density, and transverse sound velocity that could be used as diagnostic parameters.

Vascular endothelial growth factor (VEGF) is known for its role in neovascularization and cellular signaling pathways of sub-threshold retinal lesions. The objective of this study was to elucidate potential protection mechanisms to laser-induced heat stress utilizing an in vitro retinal model. The cell line was characterized to determine the relative abundance of VEGF-C protein. Cells, preconditioned via water bath and controls, were then exposed to 2 μm laser radiation to assess whether increases in protein production following preconditioning could confer any protection. There was no significant increase in threshold damage irradiance (ED₅₀) in the preconditioned cells versus control.

Uneven distribution of skin color is one of the biggest concerns about facial skin appearance. Recently several techniques to analyze skin color have been introduced by separating skin color information into chromophore components, such as melanin and hemoglobin. However, there are not many reports on quantitative analysis of unevenness of skin color by considering type of chromophore, clusters of different sizes and concentration of the each chromophore. We propose a new image analysis and simulation method based on chromophore analysis and spatial frequency analysis. This method is mainly composed of three techniques: independent component analysis (ICA) to extract hemoglobin and melanin chromophores from a single skin color image, an image pyramid technique which decomposes each chromophore into multi-resolution images, which can be used for identifying different sizes of clusters or spatial frequencies, and analysis of the histogram obtained from each multi-resolution image to extract unevenness parameters. As the application of the method, we also introduce an image processing technique to change unevenness of melanin component. As the result, the method showed high capabilities to analyze unevenness of each skin chromophore: 1) Vague unevenness on skin could be discriminated from noticeable pigmentation such as freckles or acne. 2) By analyzing the unevenness parameters obtained from each multi-resolution image for Japanese ladies, agerelated changes were observed in the parameters of middle spatial frequency. 3) An image processing system modulating the parameters was proposed to change unevenness of skin images along the axis of the obtained age-related change in real time.

Monte Carlo (MC) simulation was implemented in a three dimensional tooth model to simulate the light propagation in the tooth for antibiotic photodynamic therapy and other laser therapy. The goal of this research is to estimate the light energy deposition in the target region of tooth with given light source information, tooth optical properties and tooth structure. Two use cases were presented to demonstrate the practical application of this model. One case was comparing the isotropic point source and narrow beam dosage distribution and the other case was comparing different incident points for the same light source. This model will help the doctor for PDT design in the tooth.

Angular Domain Spectroscopic Imaging employs an array of micro-channels to perform angular filtering of light that traverses a turbid sample to reject moderately to highly scattered light. In this work, we experimentally characterized an ADSI system by measuring transmission spectra and the first and second derivatives obtained from absorbing and scattering targets. The derivative analysis was used to estimate the concentration of indocyanine green mixed in a scattering liquid. The experimental results provided support for ADSI as a potential method for quantitative spectroscopic imaging of ex vivo tissue samples.

In this research, we present the first multi-GPU FDTD implementation of Maxwell's equations in dispersive media that uses the Open-MP API to synchronize the operation of GPUs and their corresponding CPUs. By taking advantage of the CUDA programming model, we present a unique implementation of the FDTD scheme that exploits the memory hierarchy of GPUs, including the global, texture, and shared memory. This enables us to tackle problems that are otherwise computationally prohibitive. Practical results will be presented along with a measure of speedup factors achieved when using multiple GPU processors.

The aim of this work was to establish measurement conditions under which endogenous skin fluorescence ("auto-fluorescence") is relatively invariant, so that changes in exogenous agents can be accurately determined. Fluorescence emission was measured on the volar forearm of 36 subjects, chosen to be equally representative of all 6 Fitzpatrick skin types. All subjects were exposed to approximately 40 minutes of optical excitation at 450 and 500 nm with 4 irradiances between 0.3 and 9 mW/cm². Both non-optically-induced (e.g. tissue settling and fluctuation) and optically-induced variations were observed in the measured fluorescence and mechanisms explaining these effects are proposed. The optically-induced auto-fluorescence decay was independent of skin type when excited at 450 nm, but significantly dependent on skin type when excited at 500 nm. Further, the extent of decay over time was linearly related to irradiance at 500 nm, but at 450 nm was non-linear, with the extent of decay rolling off between 2 and 9 mW/cm². In order to maintain the auto-fluorescence signal within 95% of its original value over a 30 minute period, the excitation at 450 nm would need to be limited to 1.5 mW/cm², while excitation at 500 nm should be limited to 5 mW/cm².

The detailed mechanism of blast-induced traumatic brain injury (bTBI) has not been revealed yet. Thus, reliable laboratory animal models for bTBI are needed to investigate the possible diagnosis and treatment for bTBI. In this study, we used laser-induced shock wave (LISW) to induce TBI in rats and investigated the histopathological similarities to actual bTBI. After craniotomy, the rat brain was exposed to a single shot of LISW with a diameter of 3 mm at various laser fluences. At 24 h after LISW exposure, perfusion fixation was performed and the extracted brain was sectioned; the sections were stained with hematoxylin-eosin. Evans blue (EB) staining was also used to evaluate disruption of the blood brain barrier. At certain laser fluence levels, neural cell injury and hemorrhagic lesions were observed in the cortex and subcortical region. However, injury was limited in the tissue region that interacted with the LISW. The severity of injury increased with increasing laser fluence and hence peak pressure of the LISW. Fluorescence originating from EB was diffusively observed in the injuries at high fluence levels. Due to the grade and spatial controllability of injuries and the histological observations similar to those in actual bTBI, brain injuries caused by LISWs would be useful models to study bTBI.

To estimate the error of scattering coefficient spectrum determined by using double-integrating sphere system and inverse Monte Carlo method, optical properties of tissue phantom were measured. The tissue phantom was composed of hemoglobin, intralipid and gelatin. The thickness of samples (0.1-1.0 mm) and hemoglobin concentration (0.5-4.0 mg/ml) were changed and the effects of optical properties spectra were investigated. As the results, when the value of μa was large, μ's spectrum was not consistent with scattering theory. The higher hemoglobin concentration of samples was the lager the errors of μ's spectra were. The thinner the sample was, the smaller the errors were. However μa spectrum was not accurate when the sample was thin. It was predicted that when the sample thickness was 0.1 mm μ's spectrum was accurate. And when the sample thickness was 1.0 mm, μa spectrum was accurate.

When imaging through small aquatic creatures, scattered photons produce problems in image quality and resolution. Angular Domain Imaging (ADI) reduces scattered photons and improves the image quality and resolution. ADI is an imaging technique which utilizes the angular spectrum of photons to filter multiple-scattered photons and accept only photons with small angular deviation from their original trajectory. Advantages of the ADI technique are that it is insensitive to wavelength and the sources are not required to be high optical quality, coherent, or pulsed, as with OCT or time domain. Our target is to image a small species called Branchiostoma lanceolatum, a lancet that is 5-8cm long and 5mm thick, by using ADI to remove the scattering in order to image internal structures. A laser illuminates the lancelet in a water-filled container and a spatiofrequency filter removes the scattered photons before the imager. Experimentally, a coherent Nd:Yag second harmonic (533nm) laser creates images but also optical interference occuring within the internal structures of the lancelet. Conversely, an incoherent broad-band white light source eliminates the structural interference effect; however, the wavelength variation of the scattering coefficient combined with the limitation of the image sensor's dynamic range limit the ability to distinguish the internal structures in many areas. Thus, an IR diode laser (780nm) is used to lower the scattering coefficient as compared to conventional visible light source and to diminish the interference effects due to its shorter coherence length.

The function of a protein is correlated to its structure. Thus, the ability to control the structure by unfolding the protein becomes of considerable interest. One novel method of unfolding a protein involves using a light activated ligand bound to the protein which then triggers a photochemical reaction. Many porphyrins are natural products and are cell-friendly within certain limits of concentration. Many of them have been used for Photodynamic Therapy (PDT) of cancer. The specific porphyrin used in this study is meso-tetrakis (sulfonatophenyl) porphyrin (TSPP). In this study we investigated the binding of TSPP to Tubulin and the effects of irradiating the porphyrin/protein complex in an attempt to induce unfolding of Tubulin. Tubulin is an important protein which forms microtubules and has been the target of many anticancer therapies. A combination of various spectroscopic methods can be used to gain insight into the structural changes induced by the photosensitizer on the protein and provide a blueprint of the molecular interactions. Absorption and fluorescence spectroscopy yields information on how the electronic transition energy levels may be changing. Resonance Raman spectroscopy (RRS) provides structural information based on the changes of the vibrational modes of the ligand and circular dichroism (CD) probes the secondary structure of the protein. Thus by taking spectroscopic measurements of the protein/porphyrin complex before and after irradiation we can obtain structural information of the effects induced by light absorption. The results indicate that there is indeed significant unfolding of Tubulin as a result of irradiating the bound Porphyrin.