This PDF file contains the front matter associated with SPIE Proceedings Volume 7562, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

A massively-parallel molecular delivery system for mammalian cells is demonstrated by pulsed-laser irradiation of a gold-nanoparticles-coated substrate situated below a cell monolayer. This system is capable of high throughput and spatially-targeted delivery into desired areas of a cell culture by designing the laser irradiation pattern. Large area, rapid fabrication of the gold-nanoparticle-coated substrate is achieved by pulsed laser annealing of a continuous gold thin film. Randomly distributed gold nanoparticles and periodic gold nanoparticle arrays were obtained by pulsed laser flood exposure and by polymer mold guided laser annealing respectively. Optical image patterned molecular delivery into adherent cells were demonstrated in HeLa and HEK 293T cells. Delivery efficiencies of fluorescent dye, calcein, were as high as >90% with high cell viability (>90%) in HEK 293T using this device.

A linear superposition method for estimating the plane wave farfield scattering pattern from multiple biological cells computed by the finite difference time domain (FDTD) method is presented. The method allows the FDTD simulation results of scattering from a small number of complex scatterers, such as biological cells, to be used to estimate the farfield pattern from a large group of those same scatterers. This method can be used to reduce the computational cost of FDTD simulations by allowing a single large scattering problem to be broken into smaller problems with more practical computational requirements. It has been found that the method works best in cases where there is little multiple scattering interaction between adjacent cells, so the farfield pattern of multicell geometry can simply be calculated as a phase-adjusted linear superposition of the scattering from individual cells. A strategy is also presented for choosing the minimum number of cells in cases with significant multiple scattering interactions between cells.

It is hoped that the non-invasive optical characterization of physiological features of normal and diseased epithelia can be assessed through the fluorescent emission of such tissues. With a high percentage of cancers arising in the epithelium, the characterization of carcinogenesis in such tissues is imperative. Fluorescent emission from the epithelium, e.g. oral mucosa, has been shown to be sensitive to physiological features, such as cellular morphology, and the amount and types of biochemical agents present in the tissue. Efforts to distinguish the spectral signatures of diseased and healthy states of tissues from fluorescence have been confounded by the distortion of the intrinsic fluorescent signature as a result of wavelength dependent absorption and scattering within the tissue. Theoretical models of light propagation in biological media are required for understanding the distortion of the intrinsic fluorescence arising from compromised tissues. In this work we model the distortion of the intrinsic fluorescence emitted from a tissue with wavelength dependent optical properties, arising from varying blood and water content, using the radiative transport equation. As an example, we demonstrate the ability of blood and water content to distort the signal of a white light source as it is embedded deeper into a tissue.

Angular Domain Imaging (ADI) is a high resolution, ballistic imaging method that utilizes the angular spectrum of photons to filter multiply-scattered photons which have a wide distribution of angles from ballistic and quasi-ballistic photons which exit a scattering medium with a small distribution of angles around their original trajectory. An advantage of the ADI method is that it is suitable with a wide variety of light sources, as it is not sensitive to coherence or wavelength and does not require a pulsed source or a highly collimated beam. We extend the ADI method to transmissive imaging of scattering media using incoherent, collimated sources with a spatial filter comprised of a converging lens (focal distance of 50 to 100 mm) and pinhole aperture (diameter of 100 to 500 μm) giving acceptances angles of 0.06 to 0.6° to produce wide-beam, full-field images of planar, high contrast, phantom test objects through 5 cm thick scattering media at optical depths of up to 14.6 (scattered to ballistic photon ratio ≈ 2×106). Experimental images, obtained using a 12 mm diameter beam produced by a quartz-halogen incandescent source (beam divergence angle 0.52°, beam power < 10 mW), demonstrate the advantages of this combination of broadband, incoherent source and spatial filter: lack of interference artifacts seen with laser sources, ease of changing image magnification, simple correlation between system geometry and resolution, and ease of spectral filtration to obtain multispectral images. Monte Carlo simulation with angular tracking is used to validate the experimental results and determine system tradeoffs.

This paper presents a novel optical filter called the Radial Angular Filter Array (RAFA) for real-time measurement of the angular and spectral distribution of diffuse light exiting a turbid medium. The RAFA consists of a radiallydistributed series of 48 micro-channels micro-machined into a silicon substrate. To test the device, we constructed an angle-resolved spectroscopy system by integrating a wideband light source, the RAFA, and an imaging spectrometer. The collimated broadband light source was configured to trans-illuminate a turbid sample over a wide range of wavelengths in the near infrared spectral region. The RAFA was used to collect the angular distribution of light exiting the turbid sample. The imaging spectrometer decomposed the output of the RAFA into hyperspectral images representative of scatter angle and wavelength. By scanning the RAFA and imaging spectrometer over the sample, the intensity of the scattered light was acquired as a function of location on the sample surface, wavelength, and angle relative to the surface normal. With angle resolved spectroscopy it will be possible to characterize the optical properties of turbid samples in great detail.

Optical Phase Conjugation is a non-linear optical phenomenon that generates a phase conjugate replica of an incident beam. It has been widely used to suppress the effects of aberrations in optical systems such as resonators or imagetransmitting optical fibers. In this work, the possibility of using optical phase conjugation as a means of suppressing the effect of scattering in turbid media is analyzed, with the final aim to apply it to biological tissues. Firstly, light propagation through a slab representing a turbid sample was calculated by solving Maxwell's equations with the Finite-Difference Time-Domain method, in order to preserve all the information about the phase and coherence of the wavefront. The non-linear process that takes place within the phase conjugation mirror is described by coupledwave theory. A set of simulations was performed, and the results confirm the feasibility of using this effect to compensate the effect of scattering in turbid media. Subsequently, an experimental set-up was performed. In order to obtain a phase conjugation mirror, degenerate fourwave mixing was achieved by a real-time volume holography configuration. The pulsed laser source was a Nd3+:YAG laser at its second-harmonic (532nm). An ethanol solution of Rhodamine 6G was used as a non-linear medium. A lipidbased scattering sample was obtained by a solution of homogenized milk and distilled water, which provided us with an appropriate tissue phantom. The experimental results demonstrate scattering suppression, and constitute some preliminary measurements of an effect with a promising potential for a wide range of applications.

Researchers have been using simple optics to image optically induced fluorescence in tissues. We now apply the Angular Domain Imaging technique using a Spatiofrequency filter which accepts only photons within a small deviation angle from its original trajectory to image a fluorescing medium beneath a scattering layer. A Rhodamine 6 G dye fluorescing layer or fluorescence slides, under an Intralipid scattering medium was used. By applying ADI with acceptance angle of 0.17°, the structures are distinguishable at low scattering depth depending of the emission wavelength of the fluorescence source. It was established previously that as the acceptance angle increases, the amount of scattered light/noise in the images increases, however, the resolution also deteriorates. Simulations using a Monte-Carlo program are done for both angular filters, Spatiofrequency filter and Linear Collimating Array. Due to the additional positional filtration on top of the angular filtration with Linear Collimating Array, collimators with aspect ratio as low as 10:1 can improve the quality of the fluorescence images significantly in both contrast ratio and resolution.

The near-infrared (NIR) optical properties of human retinal pigmented epithelial (RPE) cells and rare earth nanopowders were studied using a double-integrating sphere setup. The Kubelka-Munk and Inverse Adding-Doubling techniques were applied to obtain absorption and scattering coefficients. These are compared with the coefficients obtained through the Representative Layer Theory described by the Dahm equation. Retinal pigmented epithelial monolayers were cultured from an ARPE19 line in thin cell culture windows, and the nanopowders were pressed into samples of varying thickness. Samples were optically characterized as a function of wavelength. A brief discussion of the shortcomings of existing techniques for computing optical properties when applied to physically thin samples is provided, followed by a comparison between the optical properties of the samples returned by the different techniques.

The clinical outcome of photodynamic therapy (PDT) may be improved by the accurate knowledge about the light distribution within the tissue. Optical properties [absorption coefficient (μ_a), scattering coefficient (μ_s), anisotropy factor (g), refractive index, etc.] of tissues help us realizing a light propagation through the tissue. The aim of this study is acquisition of the knowledge of light propagation within tissue with the optical property of mouse tumor tissue performed PDT. We evaluated the optical property of mouse tumor tissue before and after PDT using the double integrating sphere setup and algorithms based on the inverse Monte Carlo method in the wavelength range from 350 to 1000 nm. During PDT, the reduced scattering coefficient spectra were decreased entirely after 5 and 10 min irradiation. 1, 2, 7 days after PDT, the absorption coefficient was increased in the wavelength range from 400 to 660 nm. And, the reduced scattering coefficient at the wavelength of 664 nm was increased with the passage of time. These results are used for medical diagnostic applications for the quantitative assessment of the PDT effect. 7 days after PDT, the reduced scattering coefficient at the wavelength of 664 nm was increased significantly from 0.64 mm^-1 to 1.24 mm^-1, which results in the optical penetration depth decreased from 1.49 mm to 0.84 mm, respectively. To ensure the effective procedure, an adjustment of the laser parameter for the decreasing penetration depth is recommended for a second PDT.

A study of retinal damage thresholds in non-human primates (NHP) in the near-infrared (NIR) wavelengths of 1110, 1130, 1150, and 1319 nm has recently been reported. The progression of damage in retinal areas that received exposures below, greater than, and at threshold values for each respective wavelength are compared. Subjects were imaged using an Adaptive Optics (AO) enhanced Spectral Domain Optical Coherence Tomographer (SD-OCT) a year post laser exposure to examine damage characteristics and localization. The subject's retinas within the study exhibited a delayed response to NIR exposures in that many of the lesions that were not visible at the 1-hour observation period continued to grow in size over the 24-hour period and or became visible. Thermal lensing is believed to play a significant role in the formation or retinal lesions in the NIR and may explain the delayed response.

Fluorescent molecular probes offer a potential for early cancer detection. Indocyanine green (ICG) is an FDAapproved near-infrared (NIR) fluorescent dye used in ophthalmic angiography and assessment of cardiac and hepatic functions. However, clinical applications of ICG remain very limited due to its rapid clearance from vascular circulation, unstable optical properties, non-specific interactions with plasma proteins, and inability for localized targeting. To overcome these limitations, we have encapsulated ICG within nanoconstructs composed of poly(allylamine) hydrochloride and disodium hydrogen phosphate salt. To understand the effects of coating materials on the cellular uptake of the nanocapsules, we have measured the uptake of ICG-loaded nanocapsules (ICG-NCs) with various coating materials by HeLa cancerous cervical epithelial cells in-vitro. Results of this study provide important information for the choice of appropriate coating materials that will result in maximal uptake of ICG-NCs in optical and phototherapy of cancerous tissue.

Blood vessels, especially in the brain, dynamically change the diameters over time to provide sufficient blood supply where needed. At present, there is no technique that allows noninvasive control of vascular diameter in vivo. Here we report that label-free irradiation of femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to laser irradiation, cultured vascular smooth muscle cell showed a rapid increase in calcium concentration followed by the cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused localized vascular contraction followed by recovery. Nonlinear nature of the pulsed laser allowed highly specific targeting of subcortical vessels without affecting the surrounding region. We propose that femtosecond pulsed laser irradiation will be a useful experimental tool in the field of vascular biology.

As the application and commercial use of millimeter- and submillimeter-wavelength radiation become more widespread, there is a growing need to understand and quantify both the coupling mechanisms and the impact of this long wavelength energy on biological function. Independent of the health impact of high doses of radio frequency (RF) energy on full organisms, which has been extensively investigated, there exists the potential for more subtle effects, which can best be quantified in studies which examine real-time changes in cellular functions as RF energy is applied. In this paper we present the first real time examination of RF induced changes in cellular activity at absorbed power levels well below the existing safe exposure limits. Fluorescence microscopy imaging of immortalized epithelial and neuronal cells in vitro indicate increased cellular membrane permeability and nanoporation after short term exposure to modest levels (10-50 mW/cm²) of RF power at 60 GHz. Sensitive patch clamp measurements on pyramidal neurons in cortical slices of neonatal rats showed a dramatic increase in cellular membrane permeability resulting either in suppression or facilitation of neuronal activity during exposure to sub-μW/cm² of RF power at 60 GHz. Non-invasive modulation of neuronal activity could prove useful in a variety of health applications from suppression of peripheral neuropathic pain to treatment of central neurological disorders.

The terahertz (THz) region has been shown to have considerable application potential for spectroscopic imaging, nondestructive imaging through nonpolar, nonmetallic materials and imaging of biological materials. These applications have all been possible due to the recent progress in THz sources, detectors and measurement techniques. However, only moderate progress has been made in developing passive and active devices to control and manipulate THz radiation, which can enhance current imaging capabilities. One promising approach for implementing passive and active devices at THz frequencies are metamaterials - composite materials designed to have specific electromagnetic properties not found in naturally occurring materials. The most common implementation utilizes a metallic resonant particle periodically distributed in an insulator matrix where the periodicity is significantly smaller than the wavelength of operation. We have designed and implemented three metamaterial based devices with potential applications to THz imaging. We present an electrically-driven active metamaterial which operates as an external modulator for a ~2.8 THz CW quantum cascade laser. We obtained a modulation depth of ~60%. We also demonstrate a polarization sensitive metamaterial which can be used as a continuously variable attenuator or as a wave plate. The latter may be useful for the development of THz phase contrast imaging.

Terahertz (THz) radiation is increasingly being used in biomedical imaging and spectroscopy applications. These techniques show tremendous promise to provide new sophisticated tools for the improved detection of skin cancer. However, despite recent efforts to develop these applications, few studies have been conducted to characterize the optical properties of skin at THz frequencies. Such information is required to better understand THz-tissue interactions, and is critical for determining the feasibility of proposed applications. In this study, we have developed and tested a THz time-domain spectroscopy system. We used this system to acquire the optical properties for fresh and frozen/thawed excised porcine skin from 0.1 to 2.0 THz. Results show that the index of refraction (n) for both frozen and fresh skin decreases with frequency. For frozen skin, n equals 2.5 at 0.1 THz and 2.0 at 2.0 THz, and for fresh skin equals 2.0 at 0.1 THz and 1.7 at 2.0 THz. Values for the absorption coefficient (μa) increase with frequency for both frozen and fresh skin. Frozen skin exhibits μa values equal to 56 cm^-1 at 0.1 THz and 550 cm^-1 at 2.0 THz, whereas fresh skin exhibits values of 56 cm^-1 at 0.1 THz and 300 cm^-1 at 2.0 THz. Assuming the optical penetration depth (δ) is inversely proportional to μa (absorption-dominated interactions), THz radiation has limited δ in skin (200 μm at 0.1 THz to 40 μm at 2.0 THz). These results suggest that applications exploiting THz radiation show the most promise for investigating superficial tissues.

In recent years, numerous security, military, and medical applications have been developed which use Terahertz (THz) radiation. These developments have heightened concerns in regards to the potential health risks that are associated with this type of radiation. To determine the cellular and molecular effects caused by THz radiation, we exposed several human cell lines to high-power THz radiation, and then we determined death thresholds and gene expression profiles. Necrotic and apoptotic death thresholds were determined for Jurkat cells using an optically-pumped molecular gas THz source (υ = 2.52 THz, H = 227 mW/cm²), MTT viability assays, and flow cytometric techniques. In addition, we used confocal microscopic techniques to demarcate lethal spatial regions in a monolayer of dermal fibroblasts exposed to THz radiation. Then, to determine if cells exhibit a THz-specific gene expression signature, we exposed dermal fibroblasts to THz radiation and analyzed their transcriptional response using microarray gene chips. We found that 60% of the Jurkat cells survived the 30-minute THz exposure, whereas only 20% survived the 40-minute exposure. The flow data confirmed these results and provided evidence that THz-induced cell death was mediated using both nectrotic and apoptotic processes. The preliminary microscopy studies provided convincing evidence warranting future efforts using these techniques. Lastly, we found that dermal fibroblasts up-regulated several genes when exposed to THz radiation. Overall, these results provide evidence for the cellular and molecular effects associated with THz radiation, and we speculate that the identified up-regulated genes may serve as excellent candidate biomarkers for THz exposures.

The biological effects associated with Terahertz (THz) radiation are not well characterized. In this study, we investigated the cellular response of human dermal fibroblasts exposed to an optically-pumped molecular gas THz laser (υ = 2.52 THz, irradiance = 84.8 mW/cm², exposure duration = 5 to 80 minutes). Computational dosimetry was conducted using finite-difference time-domain (FDTD) modeling techniques. Empirical dosimetry was conducted using infrared cameras and thermocouples. Cellular viability was assessed 24 h post-exposure using MTT calorimetric assays. Quantitative PCR was performed 4 h post-exposure to evaluate the transcriptional activation of genes involved in protein and DNA damage pathways. Comparable analyses were also performed for hyperthermic and genotoxic positive control samples. For all of the exposure durations tested, we found that greater than 95% of the cells were viable post-exposure. In addition, the exposed cells showed only minor increases (~3.5-fold) in heat shock protein expression. The empirical dosimetric data showed that the temperature of the cells increased by ~3 °C during exposure. This value was consistent with that predicted by the computational models. Interestingly, although the THz-exposed cells exhibited increases in heat shock protein expression, the magnitude of these increases was comparable to those observed in hyperthermic controls. In addition, none of the DNA repair genes tested were up-regulated in the THz-exposed cells, whereas 40-fold increases were observed in the genotoxic control cells. These results suggest that the biological effects imposed by THz radiation appear to be primarily photothermal in nature.

Several international organizations establish minimum safety standards to ensure that workers and the general population are protected against adverse health effects associated with electromagnetic radiation. Suitable standards are typically defined using published experimental data. To date, few experimental studies have been conducted at Terahertz (THz) frequencies, and as a result, current THz standards have been defined using extrapolated estimates from neighboring spectral regions. In this study, we used computational modeling and experimental approaches to determine tissue-damage thresholds at THz frequencies. For the computational modeling efforts, we used the Arrhenius damage integral to predict damage-thresholds. We determined thresholds experimentally for both long (minutes) and short (seconds) THz exposures. For the long exposure studies, we used an in-house molecular gas THz laser (υ= 1.89 THz, 189.92 mW/cm², 10 minutes) and excised porcine skin. For the short exposure studies, we used the Free Electron Laser (FEL) at Jefferson Laboratory (υ= 0.1-1.0 THz, 2.0-14.0 mW/cm², 2 seconds) and wet chamois cloths. Thresholds were determined using conventional damage score determination and probit analysis techniques, and tissue temperatures were measured using infrared thermographic techniques. We found that the FEL was ideal for tissue damage studies, while our in-house THz source was not suitable to determine tissue damage thresholds. Using experimental data, the tissue damage threshold (ED50) was determined to be 7.16 W/cm2. This value was in well agreement with that predicted using our computational models. We hope that knowledge of tissue-damage thresholds at THz frequencies helps to ensure the safe use of THz radiation.

The LASIK procedure is a well established laser based treatment in ophthalmology. Nowadays it includes a cutting of the corneal tissue bases on ultra short pulses which are focused below the tissue surface to create an optical breakdown and hence a dissection of the tissue. The energy of the laser pulse is absorbed by non-linear processes that result in an expansion of a cavitation bubble and rupturing of the tissue. Due to a reduction of the duration of treatment the current development of ultra short laser systems points to higher repetition rates. This in turn results in a probable interaction between different cavitation bubbles of adjacent optical breakdowns. While the interaction of one single laser pulse with biological tissue is analyzed reasonably well experimentally and theoretically, the interaction of several spatial and temporal following pulses is scarcely determined yet. We present a high-speed photography analysis of cavitation bubble interaction for two spatial separated laser-induced optical breakdowns varying the laser pulse energy as well as the spatial distance. Depending on a change of these parameters different kinds of interactions such as a flattening and deformation of bubble shape, asymmetric water streams and jet formation were observed. The results of this research can be used to comprehend and optimize the cutting effect of ultra short pulse laser systems with high repetition rates (> 1 MHz).

Recently we established an experimental setup for robot-assisted laser bone ablation using short-pulsed CO₂ laser. Due to the comparable low processing speed of laser bone ablation the application in surgical interventions is not yet feasible. In order to optimize this ablation process, we conducted a series of experiments to derive parameters for a discrete process model. After applying single and multiple laser pulses with varying intensity onto bone, the resulting craters were measured using a confocal microscope in 3D. The resulting ablation volumes were evaluated by applying Gaussian function fitting. We then derived a logarithmic function for the depth prediction of laser ablation on bone. In order to increase the ablation performance we conducted experiments using alternate fluids replacing the water spray: pure glycerin, glycerin/water mixture, acids and bases. Because of the higher boiling point of glycerin compared to water we had expected deeper craters through the resulting higher temperatures. Experimental results showed that glycerin or a glycerin/water mix do not have any effect on the depth of the ablation craters. Additionally applying the acid or base on to the ablation site does only show minor benefits compared to water. Furthermore we preheated the chemicals with a low energy pulse prior to the ablation pulse, which also showed no effect. However, applying a longer soaking time of the chemicals induced nearly a doubling of the ablation depth in some cases. Furthermore with this longer soaking time, carbonization at the crater margins does not occur as is observed when using conventionally water spray.

Endoscopic submucosal dissection (ESD) is accepted as a minimally invasive treatment technique for small early gastric cancers. Procedures are carried out using some specialized electrosurgical knifes with a submucosal injection solution. However it is not widely used because its procedure is difficult. The objective of this study is to develop a novel ESD method which is safe in principle and widely used by using laser techniques. In this study, we used CO₂ lasers with a wavelength of 10.6 μm for mucosal ablation. Two types of pulse, continuous wave and pulsed wave with a pulse width of 110 ns, were studied to compare their values. Porcine stomach tissues were used as a sample. Aqueous solution of sodium hyaluronate (MucoUp^R) with 50 mg/ml sodium dihydrogenphosphate is injected to a submucosal layer. As a result, ablation effect by CO₂ laser irradiation was stopped because submucosal injection solution completely absorbed CO₂ laser energy in the invasive energy condition which perforates a muscle layer without submucosal injection solution. Mucosal ablation by the combination of CO₂ Laser and a submucosal injection solution is a feasible technique for treating early gastric cancers safely because it provides a selective mucosal resection and less-invasive interaction to muscle layer.

This study evaluated the effectiveness of near-infrared laser-excited gold nanorods as the active target to selectively kill the cancer cells. The key parameters of laser and sample to be measured include the absorption coefficient, the laser fluence and irradiation time, and the temperature profiles. The optimal laser operation for the surface and volume heating was achieved by a novel pulsed-train technique using an auto-controlled laser on-off time to meet the desired temperatures. The measured temperature is an increasing function of laser fluence and irradiation time. For a fixed laser influence, GNRs solution with smaller extinction coefficients (A) provides higher volume temperature, but slower surface raising speed. This novel measured features are predicted by our theory based on a heat diffusion equation, which was solved numerically, for volume heating.

Atherosclerosis and specifically rupture of vulnerable plaques account for 23% of all deaths worldwide, far surpassing both infectious diseases and cancer. In atherosclerosis, macrophages can infiltrate plaques which are often associated with lipid deposits. Photothermal wave imaging is based on the periodic thermal modulation of a sample using intensity modulated light. Intensity modulated light enters the sample and is absorbed by targeted chromophores and generates a periodic thermal modulation. We report use of photothermal wave imaging to visualize nanoroses (taken up by macrophages via endocytosis) and lipids in atherosclerotic plaques. Two excitation wavelengths were selected to image nanoroses (800 nm) and lipids (1210 nm). Atherosclerotic plaque in a rabbit abdominal artery was irradiated (800 nm and 1210 nm separately) at a frequency of 4 Hz to generate photothermal waves. The radiometric temperature at the tissue surface was recorded by an infrared (IR) camera over a 10 second time period at the frame rate of 25.6 Hz. Extraction of images (256 × 256 pixels) at various frequencies was performed by Fourier transform at each pixel. Frequency amplitude images were obtained corresponding to 800 nm and 1210 nm laser irradiation. Computed images suggest that the distributions of both nanorose and lipid can be identified in amplitude images at a frequency of 4 Hz. Nanoroses taken up by macrophages are distributed at the edges of lipid deposits. Observation of high concentration of nanoroses in atherosclerotic plaque confirms that nanoroses are present at locations associated with lipid deposits.

An adaptive optics imaging system was used to qualitatively observe the types of aberrations induced by an infrared laser in a rhesus eye. Thermal lensing was induced with an infrared laser radiation wavelength of 1150-nm. The adaptive optics system tracked the temporal response of the aberrations at a frequency of 30 Hz for continuous-wave exposures. Results are compared against thermal lensing aberrations induced in an artificial eye.

The fluorescence properties of human tissue are known to be temperature dependent. The most apparent effect of this dependence is the inverse relationship between fluorescence and temperature. In this study, we used fluorescence and diffuse-reflectance spectroscopy to investigate the effects of temperature on fluorescence, thermal coagulation, and tissue optical properties. Human tissue from the breast and abdomen were examined in vitro, and human skin was examined in vivo using a fluorescence and diffuse-reflectance system to observe the effects of temperature on fluorescence and optical properties. Fluorescence measurements were carried out using a pulsed nitrogen laser at 337 nm for excitation and a thermal camera for temperature measurements. Thermal variation of the specimens was provided by a phosphate buffered saline solution for the in vitro experiments and an ice pack and heat lamp for the in vivo experiments. In vitro temperatures varied from 0°C to 70°C and in vivo temperatures varied from 15°C to 40°C. Optical property measurements and Monte Carlo simulations were carried out on the in vitro samples for different levels of thermal exposure. Results of both the in vivo and in vitro experiments indicate that optical properties of human tissue change at high temperatures, primarily due to increased scattering. In addition, certain internal processes may have contributed to a decrease in fluorescence with increasing temperature. Some of these effects were found to be reversible before a certain temperature threshold, while some effects of coagulation on fluorescence and optical properties were not reversible.

The effect of temperature on the fluorescence of enucleated porcine eyes and rat skin was studied. The fluorescence peak intensity was found to decrease as the tissue temperature increased. A dual-excitation, fiber-based system was used to collect fluorescence and diffuse-reflectance spectra from the samples. A thermal camera was used to determine the temperature of the tissue at the time of fluorescence measurement. The samples were mounted in a saline bath and measurements were made as the tissue temperature was increased from -20°C to 70°C. Results indicate that temperature affects several fluorescence spectra characteristics. The peak height decreased as temperature increased. At temperatures above 60°C, the peak position shifted to lower wavelengths. Heating and cooling experiments of the rat skin demonstrate the recovery of the loss in fluorescence. The diffuse reflectance spectra indicated a change in optical properties past 60°C, but prior to the denaturation temperature for collagen at 57°C, no change in optical properties was observed. Results suggest that the decrease in fluorescence is both a property of fluorescence and a result of altering optical properties.

We have been developing the novel short-term heating angioplasty in which sufficient artery lumen-dilatation was attained with thermal softening of collagen fiber in artery wall. In the present study, we investigated on the relation between the mechanical properties of heated artery and thermal denaturation fractures of arterial collagen in ex vivo. We employed Lumry-Eyring model to estimate temperature- and time-dependent thermal denaturation fractures of arterial collagen fiber during heating. We made a kinetic model of arterial collagen thermal denaturation by adjustment of K and k in this model, those were the equilibrium constant of reversible denaturation and the rate constant of irreversible denaturation. Meanwhile we demonstrated that the change of reduced scattering coefficient of whole artery wall during heating reflected the reversible denaturation of the collagen in artery wall. Based on this phenomenon, the K was determined experimentally by backscattered light intensity measurement (at 633nm) of extracted porcine carotid artery during temperature elevation and descending (25°C→80°C→25°C). We employed the value of according to our earlier report in which the time-and temperature- dependent irreversible denaturation amount of the artery collagen fiber that was assessed by the artery birefringence. Then, the time- and temperature- dependent reversible (irreversible) denaturation fraction defined as the reversible ((irreversible) denatured collagen amount) / (total collagen amount) was calculated by the model. Thermo-mechanical analysis of artery wall was performed to compare the arterial mechanical behaviors (softening, shrinkage) during heating with the calculated denaturation fraction with the model. In any artery temperature condition in 70-80°, the irreversible denaturation fraction at which the artery thermal shrinkage started was estimated to be around 20%. On the other hand, the calculated irreversible denaturation fraction remained below 5% and reversible denaturation fraction reached up to 20% while the artery softening occurred without shrinkage. We think that our model of arterial collagen thermal denaturation might be reasonable to estimate the artery mechanical properties during heating.

Laser-based gene delivery is attractive as a new method for topical gene therapy because of the high spatial controllability of laser energy. Previously, we demonstrated that an exogenous gene can be transferred to cells both in vitro and in vivo by applying nanosecond pulsed laser-induced stress waves (LISWs) or photomechanical waves (PMWs). In this study, we investigated effects of laser parameters on the propagation characteristics of LISWs in soft tissue phantoms and depth-dependent properties of gene transfection. Temporal pressure profiles of LISWs were measured with a hydrophone, showing that with a larger laser spot diameter, LISWs can be propagated more efficiently in phantoms with keeping flat wavefront. Phantoms with various thicknesses were placed on the rat dorsal skin that had been injected with plasmid DNA coding for reporter gene, and LISWs were applied from the top of the phantom. Efficient gene expression was observed in the rat skin that had interacted with LISWs propagating through a 15-mm-thick phantom. These results would be useful to determine appropriate laser parameters for gene delivery to deep-located tissue by transcutaneous application of LISWs.

Ultrashort lasers are typically utilized for tissue dissection by sequential application of tightly focused beam along a scanning pattern. Each pulse creates a small (on the order of 1μm) zone of multiphoton ionization (optical breakdown). At energies exceeding vaporization threshold cavitation bubble is formed around the focal volume. A continuous cut is formed if the rupture zones produced by separate bubbles coalesce. We present an alternative approach, in which an extended zone of tissue is cut by simultaneous application of laser energy in multiple foci. Simultaneous formation of multiple cavitation bubbles results in hydrodynamic interactions that can lead to significant extension of the rupture zone in tissue. Two simultaneously expanding bubbles compress and strain material between them, while simultaneously collapsing bubbles can produce jets towards each other. We calculated and experimentally imaged the flow dynamics of expanding and collapsing bubbles and obtained maps of tissue deformation. With the measured tissue threshold strain, the deformation map allows predicting the rupture zone as a function of maximum bubble size and distance between the bubbles. We also demonstrate an optical system producing 1 mm long dissection with a single laser pulse. A combination of a lens and an axicon produces a line of optical breakdown, with aspect ratio 250:1. The subsequent cavitation bubble has aspect ratio 100:1 at early stage of expansion. We calculated an optimal laser beam intensity profile to create axiallyuniform elongated ionization pattern.

During laser ablation of a diseased area, the surrounding tissues and organs suffer serious damage. In order to optimize laser ablation of biological tissues, it is necessary to observe the laser ablation in situ. The real-time imaging of tissue laser ablation is realized in the fusion system of the YAG ablation laser and optical coherence tomography (OCT). A swept-source OCT (SS-OCT) is combined with a YAG-laser ablation system. In this paper, we demonstrate real-time OCT imaging of tissue laser ablation. The fiber-optic swept source OCT (SS-OCT) with 25 frames / s is used for the in situ observation where tissue laser ablation is made continuously by 10-Hz YAG laser pulses. Dynamic analysis for laser ablation, therefore, is made, taking thermal effect into account.

Novel results are presented on thermocavitation in highly absorbing solutions using CW low power laser (λ=975 nm). Due to the large absorption coefficient (135 cm-1) at the laser wavelength, penetration length is only ~74μm inside the liquid and asymmetric bubbles are generated near the beam's entrance wall. We report the temporal dynamic of the cavitation bubble, which is much shorter than previously reported. We found that the amplitude of the shock wave decreases exponentially with the beam power. As shown in this work, thermocavitation is a phenomenon that has a great application potential in areas such as ultrasonic waves generation and controlled tissue ablation for use in lithotripsy.

Nanosecond long laser pulses are used in medical applications where precise tissue ablation with minimal thermal and mechanical collateral damage is required. When a laser pulse is incident on a material, optical energy will be absorbed by a combination of linear and nonlinear absorption according to both: laser light intensity and material properties. In the case of water or gels, the first results in heat generation and thermoelastic expansion; while the second results in an expanding plasma formation that launches a shock wave and a cavitation/boiling bubble. Plasma formation due to nonlinear absorption of nanosecond laser pulses is originated by a combination of multiphoton ionization and thermionic emission of free electrons, which is enhanced when the material has high linear absorption coefficient. In this work, we present measurements of pressure transients originated when 6 ns laser pulses are incident on agar gels with varying linear absorption coefficient, mechanical properties and irradiation geometry using laser radiant exposures above threshold for bubble formation. The underlying hypothesis is that pressure transients are composed of the superposition of both: shock wave originated by hot expanding plasma resulting from nonlinear absorption of optical energy and, thermoelastic expansion originated by heat generation due to linear absorption of optical energy. The objective of this work is to evaluate the relative contribution of each absorption mechanism to mechanical effects in agar gel. Real time pressure transients are recorded with PVDF piezoelectric sensors and time-resilved imaging from 50 μm to 10 mm away from focal point.

Indirect effects arising from bright artificial optical sources like temporary blinding might result in serious incidents or even accidents due to accompanying alteration of visual functions like visual acuity, contrast sensitivity and color discrimination. In order to determine the degree and duration of impairment resulting from glare, dazzle, flash-blindness and afterimages, caused by a beam from a laser or lamp product, particularly under low ambient light conditions, an investigation has been performed with the goal to improve the current knowledge as far as especially recovery duration of visual acuity is concerned. For this two different test set-ups were designed and engineered in order to be able to determine the time duration after which visual acuity returns to its previous value after temporary blinding with a laser or an LED and in addition to search for functional relations as far as wavelength, optical power and exposure duration are concerned. Instead of normal visual acuity measurement, which is the standard test done by eye care professionals, and which has been applied in order to determine the recovery time after irradiation with a high brightness LED (HB-LED) with the aid of a modified commercially available binoptometer with Landolt-C rings as optotypes, a special reading test on a computer monitor was developed for the case of laser irradiation. Two different laser were applied, one with a wavelength of 632.8 nm and the other with 532 nm. Red, green, royal blue and white HB-LEDs were used as stimulating light sources. The maximum applied optical power in a 7-mm aperture, which is equivalent to the pupil diameter of a dark adapted eye, was 0.783 mW (laser) and 3 mW (LED). The exposure durations were chosen as 0.25 s, 0.5 s, 1 s, 5 s, and 20 s in the case of laser irradiation and 0.25 s, 1 s, 5 s, and 10 s for LEDs, respecting maximum permissible exposure (MPE) and/or limit exposure levels (ELVs) in all exposure situations. The visual acuity recovery time tVA has been found to obey the dose relationship: t_VA /s ≈ 3.7•ln(energy/μJ) - 16.2 in the case of a green HB-LED in the power range 0.12 mW to 1.5 mW and for exposure durations between 1 s and 8 s. Further investigations were performed with other LED colors especially as far as threshold values for temporary blinding are concerned. The afterimage duration t_{afterimage,fv} produced by a red laser beam was determined to be: t_{afterimage,fv}/s ≈ 50.6•ln[(P•t_exp)/μJ] - 13.4, for laser output powers P between 10 μW and 30 μW with exposure durations t_exp from 0.25 s up to 10 s, when the beam hits the fovea. Additional results have been achieved with a green laser at a wavelength of 532 nm and compared with the respective values at 632.8 nm. The results of the research project suggest classifying light sources like laser and LEDs into so-called blinding groups. In total 3 different groups which reflect the obtained results and are proposed in order to fulfil the requirements of special classification and might be regarded as an appropriate assistance to perform a risk analysis.

Multiple femtosecond lasers have now been cleared for use for ophthalmic surgery, including for creation of corneal flaps in LASIK surgery. Preliminary measurements indicated that during typical surgical use, 50-60% of laser energy may pass beyond the cornea with potential effects on the iris. To further evaluate iris laser exposure during femtosecond corneal surgery, we measured the temperature increase in porcine cadaver iris in situ during direct illumination by the iFS Advanced Femtoosecond Laser (AMO Inc. Santa Ana, CA) with an infrared thermal imaging camera. To replicate the illumination geometry of the eye during the surgery, an excised porcine cadaver iris was placed 1.5 mm from the flat glass contact lens. The temperature field was observed in twenty cadaver iris at laser pulse energy levels ranging from 1 to 2 μJ (corresponding approximately to surgical energies of 2 to 4 μJ per pulse). Temperature increases up to 2.3 °C (corresponding to 2 μJ per pulse and 24 second procedure time) were observed in the cadaver iris with little variation in temperature profiles between specimens for the same laser energy illumination. For laser pulse energy and procedure time characteristic to the iFS Advanced Femtoosecond Laser the temperature increase was measured to be 1.2 °C. Our studies suggest that the magnitude of iris heating that occurs during such femtosecond laser corneal surgery is small and does not present a safety hazard to the iris.

A commercial spectrophotometer with an integrating sphere is widely used to measure the spectra of transmittance and reflectance of turbid sample, and then the optical properties can be deduced by inverse adding-doubling algorithm. Unfortunately, the accuracy of the measurement is not been elucidated completely. What's more, for the system, there still exists some other limits, i.e., the incident light is not collimated and size of light spot is too large compared with the sample port. Thus, the purpose of our study is to evaluate the accuracy of the commercial spectrophotometer with an integrating sphere for measuring optical properties of tissue phantom. Two phantom materials, Intralipid and Evans blue, and the mixture of these two, were chosen for the experiments. Mie theory was also introduced to calculate the reduced scattering coefficient according to the particle size distribution. The results show that the phantom measurement in conjunction with IAD algorithm enable the determination of scattering coefficient μ_s' to be better than 5% accuracy, absorption coefficient μ_a to be better than 10% accuracy when the optical depth of sample is between 1 and 10, and the albedo is bigger than 0.4. For scattering of samples during 1-5 mm^-1, the error of μ_s' is smaller than 4%; whereas for absorbing of samples >0.4 mm^-1, the maximum error is smaller than 8.3%. Therefore, spectrophotometer with an integrating sphere technique combined IAD algorithm is applicable for the measurements of optical properties for most tissue, and its repeatability and accuracy is good in proper scope of the optical depth and albedo.