In this work, preliminary results of a method to calculate anisotropy constant of scattering phase function (g) of a homogeneous, semi-infinite slab are shown. The method relies on measuring steady-state diffuse reflectance from the medium. In order to test the method, Monte-Carlo simulation of photon propagation in turbid media is utilized. Simulation depends on capturing diffuse-reflected photon packets which are injected perpendicularly by a point source in a continuous-wave manner. Photon packets are captured by a hemi-sphere like detector residing on the upper plane of the slab a few centimeters away from the source. The surface of the detector is divided into 120 sub-regions with equal area. Exit angles are converted into spherical coordinates. Therefore, the intersection of a particular photon packet with the detector surface is evaluated and the weight of photon packet is added to its corresponding sub-area. This process is repeated for all packets leaving the medium through the base of the hemisphere in each run. To evaluate the effect of anisotropy constant of the phase function on hemispheric weight accumulation the following simulations are performed. Optical properties of the medium are chosen as 10 cm-1 for scattering coefficient, 0.1 cm-1 for absorption coefficient, and 1.0 for refractive index. Detector is a non-scattering and non-absorbing medium whose refractive index is 1.0. Twenty millions of packets are used in the simulations. All optical properties except g are kept constant, while g is chosen to be 0.30, 0.50, 0.70, 0.75 and 0.80 for different runs.

In a previous study, we presented a new technique for representation of the shape of a scattering surface. A sensor based on two parallel fiber arrays yielded a source-detector intensity matrix (SDIM). In that study, it was shown that convex and concave polyacetal plastic (Delrin) surfaces could be accurately distinguished using the proposed technique. A simplified simulation model for calculating the SDIM was used, assuming that backscattered light was generated by Lambertian sources in the illuminated surface. These simulations showed discrepancies compared to measurements, probably due to the absence of light scattering in the model. Here, we will present an improved model, based on the Monte Carlo technique for light transport in turbid media. The optical properties of the Delrin phantoms were estimated by means of different measurement techniques. The optical properties and the geometry of the Delrin phantoms were implemented in the model along with the spatial distribution of the source and detector fibers of the sensor. The SDIM was extracted from backscattered photons exiting the turbid medium from the curved surface. The SDIM:s obtained with the Monte Carlo model, showed a much closer agreement with the measurements than those obtained with the Lambertian model. The small discrepancies observed are probably due to spatially varying optical properties of the plastic phantoms. Measurements, using the previously described sensor, of the SDIM from Delrin pieces with convex and concave surfaces, are compared to the SDIM extracted from simulations using the Monte Carlo model.

Resonance effects can occur upon laser absorption by micro and nanoparticles when a train of pulses is used. The pressure generated by the train of pulses may be significantly different than the pressure generated by a single pulse with the same total energy. For pulsed lasers with a gap duration between pulses that is an integer multiple of the characteristic oscillation time of the absorber, constructive interference occurs and the pressure is increased. For pulsed lasers with a gap duration between pulses that is an half integer multiple of the characteristic oscillation time of the absorber, destructive interference occurs and the pressure is significantly decreased. We present numerical computations showing the manifestation of this effect in gold particles with a radius of 100 nm. The resonance effects have implications for damage thresholds and therapeutic applications of laser radiation.

We present initial results showing chaotic behavior in the pressure signals generated by laser absorption by a microparticle. Specifically, the system of a melanosome immersed in water is investigated. We describe how the system manifests chaos, and the implications for causing damage to the surrounding material. We also find that a characteristic acoustic time of the absorber, the time it takes a sound wave to traverse the absorber, known as the stress confinement time, defines an important time scale for laser pulse duration. For pulse durations shorter than the stress confinement time, the pressure response is periodic, while for pulse durations greater than the stress confinement time, chaotic pressure transients are observed.

Phase function is to determine the photon propagation direction change for each scattering step in Monte Carlo simulation. Henyey Greenstein function is widely used in the Monte Carlo program. In this study, we implement 3 different phase functions: Henyey Greenstein, Double Henyey Greenstein and Mie-Theory-Generated phase function. The results show that when light source is collimated beam or focused beam, there is remarkable difference of reflectance flux in the central region of incident light.

Monte Carlo fluorescence model has been developed to estimate the autofluorescent spectra associated with the progression of the Exo-Cervical Intraepithelial Neoplasm (CIN). We used double integrating spheres system and a tunable light source system, 380 to 600 nm, to measure the reflection and transmission spectra of a 50 μm thick tissue, and used Inverse Adding-Doubling (IAD) method to estimate the absorption (μa) and scattering (μs) coefficients. Human cervical tissue samples were sliced vertically (longitudinal) by the frozen section method. The results show that the absorption and scattering coefficients of cervical neoplasia are 2~3 times higher than normal tissues. We applied Monte Carlo method to estimate photon distribution and fluorescence emission in the tissue. By combining the intrinsic fluorescence information (collagen, NADH, and FAD), the anatomical information of the epithelium, CIN, stroma layers, and the fluorescence escape function, the autofluorescence spectra of CIN at different development stages were obtained.We have observed that the progression of the CIN results in gradually decreasing of the autofluorescence intensity of collagen peak intensity. In addition, the existence of the CIN layer formeda barrier that blocks the autofluorescence escaping from the stroma layer due to the strong extinction(scattering and absorption) of the CIN layer. To our knowledge, this is the first study measuring the CIN optical properties in the visible range; it also successfully demonstrates the fluorescence model forestimating autofluorescence spectra of cervical tissue associated with the progression of the CIN tissue;this model is very important in assisting the CIN diagnosis and treatment in clinical medicine.

Living cells and transparent specimen can be observed by the Differential Interference Contrast (DIC) microscopy. In this paper, the morphologic changes of mouse skin in vivo treated in several configurations by intense pulse light (IPL) were observed by a DIC microscopy. The differences of the images before and after the IPL irradiation were obtained and the mechanism was analyzed. In addition, the collagen recombination after the irradiation of IPL in skin was acquired.

A series of experiments were conducted in vivo on female Yucatan mini-pigs to determine the ED₅₀ damage thresholds for 2000 nm continuous wave laser irradiation. These results provide new information for refinement of Maximum Permissible Exposure (MPE). The study employed Gaussian laser beam exposures with spot diameters (1/e²) of 4.83 mm, 9.65 mm and 14.65 mm and exposure durations of 0.25 s, 0.5 s, 1.0 s and 2.5 seconds as a function of laser power. The effect of each irradiation was evaluated within one minute after irradiation and the final determination was made at 48 hours post exposure. Probit analysis was conducted to estimate the dose for 50% probability of laser-induced damage (ED₅₀) defined as persistent redness at the site of irradiation for the mini-pig skin after 48 hours. Histopathologic procedures were used to determine the mechanisms of the laser effects in the skin and map the extent and severity of the lesions. The thresholds study shows that consideration for lowering the current Maximum Permissible Exposure (MPE) limits should be explored as the laser beam diameter becomes larger than 3.5 mm. Based on the limited experimental data, the duration and size dependences of the ED₅₀ damage thresholds could be described by an empirical equation: (Equation available in manuscript.)

An optical phantom was designed to physically and optically resemble human tissue, in an effort to provide an alternative for detecting visual damage resulting from inadvertent exposure to infrared lasers. The phantom was exposed to a 1540-nm, Erbium:Glass, Q-switched laser with a beam diameter of 5 mm for 30 ns at varying power levels. Various materials were tested for use in the phantom; including agar, ballistic media, and silicone rubber. The samples were analyzed for damage lesions immediately after exposure and the Minimum Visible Lesion - Estimated Dose 50% (MVL-ED₅₀ ) thresholds were determined from the data. In addition, any visible damage was evaluated for similarity to human tissue damage to determine if the phantom tissue would be a suitable substitute for in vivo exposures.

Skin damage thresholds were measured and compared with theoretical predictions using a skin thermal model for near-IR laser pulses at 1318 nm and 1540 nm. For the 1318-nm data, a Q-switched, 50-ns pulse with a spot size of 5 mm was applied to porcine skin and the damage thresholds were determined at 1 hour and 24 hours postexposure using Probit analysis. The same analysis was conducted for a Q-switched, 30-ns pulse at 1540 nm with a spot size of 5 mm. The Yucatan mini-pig was used as the skin model for human skin due to its similarity to pigmented human skin. The ED₅₀ for these skin exposures at 24 hours postexposure was 10.5 J/cm² for the 1318-nm exposures, and 6.1 J/cm² for the 1540-nm exposures. These results were compared to thermal model predictions. We show that the thermal model fails to account for the ED₅₀ values observed. A brief discussion of the possible causes of this discrepancy is presented. These thresholds are also compared with previously published skin minimum visible lesion (MVL) thresholds and with the ANSI Standard's MPE for 1318-nm lasers at 50 ns and 1540-nm lasers at 30 ns.

Laser Doppler perfusion monitors are effect tools in understanding blood flow in many different types of biological studies. Because the low-intensity lasers used in Doppler perfusion measurements must interact with moving blood cells, the depth of probe-able tissue is limited to the volume of tissue within the hemisphere of radius ~1mm from the probe tip. In addition, heterogeneities in surface perfusion make precise probe placement very important if one is comparing successive measurements. Consequently, useful tissue perfusion measurements have been difficult to obtain, especially in deep tissues. In this study, a new method was developed for monitoring deep-tissue blood perfusion directionally with the Laserflo laser Doppler perfusion probe. The probe was inserted just under the skin superficially to a rat prostatic tumor through the shaft of a 16-gauge needle, which was modified to allow the probe to be exposed without extending beyond the beveled needle tip. Perfusion measurements of the tumor surface or the skin were made by rotating the bevel to face either inside or outside. Using this technique, tumor tissue can be differentiated from either skin or muscle. To study the responses of tumor to light stimulation, an 805nm biomedical treatment laser was used to irradiate the tumor. The perfusion of the tumor surface was shown to decrease slightly with short treatment laser applications (1W for 30 seconds or 1 minute). After a longer treatment session (5 minutes), the perfusion of the tumor tissue increased significantly. However, with an even longer (10 minutes) treatment, the perfusion of the tumor surface was shown to decrease once again. This trend indicates that before laser heating becomes significant, the perfusion decreases for as yet poorly understood reasons. When laser heating becomes significant, after the five-minute session, the perfusion increases dramatically, corresponding to the expected dilation of blood vessels during tissue heating. After further treatment, we observe decreased perfusion again corresponding to the point, at which tissue temperatures increase to tissue- and blood vessel- damaging levels, causing a decrease in perfusion.

The phenomenon of thermal lensing was investigated in water using a Z-scan method and corresponding first-order mathematical models. Data from first-order thermal lensing models and ABCD beam propagation methods were used to simulate the non-linear absorption of water held in a thin sample cuvette for a Z-scan optical set up of CW cases at 1313 nm. The single beam closed aperture Z-scan was then used to determine the non-linear absorption at 1313 nm for water in 10 mm and 2 mm cuvettes at 48.00, 16.80, 9.80 and 2.83 mW then compared to the first-order model data. The results from the closed aperture Z-scan were also used to back calculate the spot size in the far field for comparison to the model's prediction of the beam's temporal response. Experimental Z-scan data were found not to correlate strongly with our first-order model suggesting the need for higher order models to successfully predict spot size in absorbing media inside the Rayleigh range.

Non-contact laser osteotomy brings new opportunities in maxillofacial and other surgical fields, since it allows very precise pre-programmed incisions of arbitrary geometries. Laser osteotomy is however difficult, because bone is a tough composite material, which is at the same time sensitive to a temperature increase. Besides thermal side effects, practical laser applicability was limited until now because of very low cutting rates and limited incision depths. We discuss how to overcome these disadvantages by means of an optimal arrangement of thermo-mechanical ablation with a pulsed CO₂ laser and with a water-spray as an assisting media. To the arrangement belong optimal duration, intensity and energy density of the laser pulses, as well as a multi-pass cutting procedure. We show that effective ablation of hard tissue with minor thermal damage is possible with relatively long CO₂ laser pulses of 80 μs duration and average laser power up to 40 - 50 W. To overcome the depth limit we have developed a special scanning technique, which allows cutting of massive multilayer bones with a feasible rate.

The reflectance and absorption of the skin plays a vital role in determining how much radiation will be absorbed by human tissue. Any substance covering the skin would change the way radiation is reflected and absorbed and thus the extent of thermal injury. Hairless guinea pigs (cavia porcellus) in vivo were used to evaluate how the minimum visible lesion threshold for single-pulse laser exposure is changed with a topical agent applied to the skin. The ED₅₀ for visible lesions due to an Er: glass laser at 1540-nm with a pulse width of 50-ns was determined, and the results were compared with model predictions using a skin thermal model. The ED50 is compared with the damage threshold of skin coated with a highly absorbing topical cream at 1540 nm to determine its effect on damage pathology and threshold. The ED₅₀ for the guinea pig was then compared to similar studies using Yucatan minipigs and Yorkshire pigs at 1540-nm and nanosecond pulse duration.^1,2 The damage threshold at 24-hours of a Yorkshire pig for a 2.5-3.5-mm diameter beam for 100 ns was 3.2 Jcm^-2; very similar to our ED₅₀ of 3.00 Jcm^-2 for the hairless guinea pigs.

In this study, the efficacy and optimal settings of the CO₂, Diode and cw Thulium laser systems were compared for various clinical applications in ENT, Lung and Neurosurgery. The experiments were performed using a specially developed setup, based on color Schlieren techniques, which enable real-time imaging of dynamic temperature gradients complimented with thermocouple measurements in a transparent tissue model in air and water. The CO₂ and cw Thulium laser are both efficient in superficial tissue ablation with minimal coagulation depth. The cw Thulium laser, however, is fiber delivered and can also be used in a water. The Diode laser has a relatively deep coagulation effect. The ablation efficacy was enhanced by coating the fiber tip with carbon particles. Our thermal imaging technique was useful to develop new strategies making use of the advantages and overcoming the drawbacks of laser systems. The CO₂, Diode and cw Thulium laser can be applied for similar clinical procedures using the optimal strategy and settings for each laser type. The cw 2 μm Thulium laser shows to be a versatile laser system for a broad range of applications both in air and water.

The signal from a confocal measurement as the focal volume is scanned down into a tissue yields an exponential decay versus depth (z_focus), signal = rho exp(-mu z_focus), where rho [dimensionless] is the local reflectivity and mu [1/cm] is an attenuation coefficient. A simple theory for how rho and mu depend on the optical properties of scattering (mu_s) and anisotropy (g) is presented. Experimental measurements on 5 tissue types from mice (white and gray matter of brain, skin, liver, muscle) as well as 0.1-um-dia. polystyrene microspheres are presented. The tissues have similar mu_s values (about 500 [1/cm]) but variable g values (0.8-0.99). Anisotropy appears to be the primary mechanism of contrast for confocal measurements such as reflectance-mode confocal scanning laser microscopy (rCLSM) and optical coherence tomography (OCT). While fluorescence imaging depends on fluorophores, and absorption imaging depends on chromophores, the results of this study suggest that contrast of confocal imaging of biological tissues depends primarily on anisotropy.

Vascularization (hemoglobin concentration) is the main intrinsic contrast in near infrared optics and may be considered as the background heterogeneity or biological noise. This noise causes complexity of the assessment of boundary measurements and uncertainty of the detectability of an optical anomaly in the medium. We have investigated the influence of this biological noise on boundary measurements with its varying degrees. We have studied an infinite-slab medium hosting an absorbing anomaly in transmission mode. In order to model this optical heterogeneity in the volume of interest we have employed a spatially random distribution pattern of absorption and observed that the amplitude of measured signals at air-tissue boundary is even decreased by 26% for low contrast heterogeneity having 40% volume fraction of heterogeneity. Furthermore optical signal measured along the detectors has been flattened as the amount of heterogeneity in the medium increases. As a measure of flatness, we have defined the contrast of the measured signals along the detectors and observed that the contrast decreases approximately linearly with an increasing heterogeneity. It is 0.79 for the heterogeneity having 40% volume fraction (VF), 0.82 for VF = %20 whereas it is 0.85 for VF = 0% suggesting that as heterogeneity (aka biological noise) increases, the detectability of an inhomogeneity diminishes.

A novel method for measuring the optical properties of highly absorbing and scattering biological media is described. The method combines frequency-domain photothermal radiometry (FD-PTR) with spatially resolved diffuse reflectance (SR-DR) techniques aimed at improving sensitivity on the determination of both scattering and absorption coefficients. Simulation results with Monte-Carlo and Diffusion Theory approaches that assess the scope and feasibility of the method are presented. An optical fiber probe for SR-DR measurements was constructed for operations at small source-detector separations and an FD-PTR system was adapted for quasi-simultaneous operation with the probe. Several experiments on epoxy phantoms that illustrate the validity and potential of the method are presented.

Near infrared spectroscopy (NIRS) is now a commonly accepted method for the measurement of oxidative metabolism of brain and muscle tissues. Nevertheless, this technology suffers from the error caused by the homogeneous single layer assumption in the calculations of concentration changes of light absorber chromophores using either diffusion theory or modified Beer-Lambert law. Underestimation of muscle oxidative metabolism for muscles having thicker fat layer above is a particular case. Due to this uncertainty, statistical analysis can be problematic in reviewing the results across subjects having different fat thicknesses for muscle studies. In this study, partial pathlength method with two detectors based on modified Beer-Lambert law extended for heterogeneous medium with homogeneous layered regions is investigated. Using Monte Carlo simulations, comparison between this technique and single homogeneous layer assumption is done. Optical coefficients of fat and muscle layers are chosen typical for muscle tissue measurements. In the simulations, change of absorption coefficient in muscle layer was made much bigger than in fat layer. It has been found that for 2-detector partial pathlength based method, fat and muscle layer absorption coefficient change estimates are better than the homogeneous medium based modified Beer-Lambert law estimates in all simulated cases.

The aim of this study was to estimate optical properties (μ_a, μ_s, μ_t, μ_s', τ, α, g) of native and coagulated (at 45°C, 60°C, 80°C) lamb brain tissues in visible and near-infrared spectral range in vitro. Optical properties of cerebellum, brainstem, cortical (grey matter), and sub-cortical regions (white matter) of frontal lobe tissues of lamb brain were estimated during this study. Diffused transmittance (Td), diffused reflectance (Rd), total reflectance (Rt) and total transmittance (Tt) were measured with single integrating sphere method. Data were processed with software (CAL-g3) developed in Biophotonics Laboratory in the Institute of Biomedical Engineering, Bogazici University. As a result, it was stated that both μ_a and μ_s values of tissues increased as temperature increases. Also scattering coefficients decreased with the increasing wavelength for all tissue types due to increase in Mie scattering.

The goal of this study was to differentiate the parts of lamb brain according to elastic scattering spectroscopy and detect the optical alterations due to coagulation. Cells and tissues are not uniform and have complex structures and shapes. They can be referred to as scattering particles. The process of scattering depends on the light wavelength and on the scattering medium properties; especially on the size and the density of the medium. When elastic scattering spectroscopy (ESS) is employed, the morphological alterations of tissues can be detected using spectral measurements of the elastic scattered light over a wide range of wavelengths. In this study firstly, the slopes of ESS spectra were used to differentiate the parts of lamb brains (brainstem, cerebellum, gray matter, white matter) in vitro in the range of 450 - 750 nm. Secondly, tissues were coagulated at different temperatures (45, 60, and 80 °C) and ESS spectra were taken from native and coagulated tissues. It was observed that as the coagulation temperature increased, the slope of the elastic scattering spectra decreased. Thus, optical properties of tissues were changed with respect to the change in nuclear to cytoplasmic ratio due to the water loss. Results showed that the slopes of ESS spectra in the visible range revealed valuable information about the morphological changes caused by coagulation.

Steady state laser light propagation in diffuse media such as biological cells generally provide bulk parameter information, such as the mean free path and absorption, via the transmission profile. The accompanying optical speckle can be analyzed as a random spatial data series and its fractal dimension can be used to further classify biological media that show similar mean free path and absorption properties, such as those obtained from a single population. A population of yeast cells can be separated into different portions by centrifuge, and microscope analysis can be used to provide the population statistics. Fractal analysis of the speckle suggests that lower fractal dimension is associated with higher cell packing density. The spatial intensity correlation revealed that the higher cell packing gives rise to higher refractive index. A calibration sample system that behaves similar as the yeast samples in fractal dimension, spatial intensity correlation and diffusion was selected. Porous silicate slabs with different refractive index values controlled by water content were used for system calibration. The porous glass as well as the yeast random spatial data series fractal dimension was found to depend on the imaging resolution. The fractal method was also applied to fission yeast single cell fluorescent data as well as aging yeast optical data; and consistency was demonstrated. It is concluded that fractal analysis can be a high sensitivity tool for relative comparison of cell structure but that additional diffusion measurements are necessary for determining the optimal image resolution. Practical application to dental plaque bio-film and cam-pill endoscope images was also demonstrated.

Thermal treatment induced collagen shrinkage has a great number of applications in medical practice. Clinically, the there is lack of reliable non-invasive methods to quantify the shrinkage. Overt treatment by heat application can lead to devastating results. We investigate the serial changes of collagen shrinkage by thermal treatment of rat tail tendons. The change in length is correlated with the finding in second harmonic generation microscopy and histology. Rat tail tendon shortens progressively during initial thermal treatment. After a certain point in time, the length then remains almost constant despite further thermal treatment. The intensity of second harmonic generation signals also progressively decreases initially and then remains merely detectable upon further thermal treatment. It prompts us to develop a mathematic model to quantify the dependence of collagen shrinkage on changes of SHG intensity. Our results show that SHG intensity can be used to predict the degree of collagen shrinkage during thermal treatment for biomedical applications.

The characteristic lengths of light linear and circular depolarization in turbid media are analyzed from random walk of vector photons. Analytical expressions are derived for polydisperse scatterers. The characteristic lengths of light depolarization computed using this model explained the different light depolarization behaviors observed in tissue and phantoms.

We demonstrated the capability of a photoacoustic method for viscoelastic measurement. The measurement method has already proved to be useful for evaluation of regenerative medicine of articular cartilage. However, characterization of the extracellular matrix as well as determination of the viscoelastic property should be carried out for evaluation of regenerative medicine because the extracellular matrix plays an important role. We therefore developed a method for characterization of the extracellular matrix that can be performed simultaneously with the photoacoustic measurement. Since collagen molecules, which are the major contents of the cartilage extracellular matrix, are well known as endogenous fluorescent molecules, it is possible that fluorescence measurement will enable characterization of the extracellular matrix. Third harmonic Q-switched Nd:YAG laser pulses were used as an excitation light source. The time-resolved fluorescence spectroscopy was obtained by using a photonic multi-channel analyzer. Tissue-engineered cartilages cultured under different conditions for various periods were used as samples. Different culture conditions resulted in different extracellular matrix formations. There were significant differences in the measured fluorescent parameters among the culture conditions of cartilage because chondrocytes produce a specific extracellular matrix depending on its culture condition. The specific extracellular matrix contained a specific type of collagen such as collagen type I or type II, which each have specific fluorescent features. Thus, the fluorescent parameters enabled characterization of synthesis of cartilage-associated extracellular matrix. Therefore, the combination of fluorescence and photoacoustic measurement is expected to become a useful evaluation method in regenerative medicine.

Various shape bubbles were generated by changing holmium-yttrium-aluminum-garnet (Ho:YAG) laser irradiation parameters. Intensive pressure waves induced by their bubble collapse were measured. The Ho:YAG laser-induced bubble in water-containing liquid had been reported by many authors regarding its shape and generated collapse pressure. However, controllability of the bubble shape and generated collapse pressure with various irradiation parameters has been still unclear. In our experiments, we changed the core diameter of optical fiber (400μm or 600μm), laser pulsewidth (FWHM 100-300μs or 50-120μs, depends on laser output energy), and positions of the optical fiber tip in a sheath. The bubble shapes were observed with the time resolved flashlamp photography. The expansion and contraction rates of the bubble volume were determined by the obtained bubble shapes. The collapse pressure was measured with a small diameter (0.5mm) calibrated hydrophone. The long Ho:YAG laser pulse irradiation made long shape bubble so-called "pear shaped" bubble. This pear shaped bubble generated low collapse pressure comparing to the spherical shape bubble which was generated by the short pulsewidth. Using the constant laser pulse energy, we obtained large volume bubbles with high collapse pressure by the optical fiber of 600μm core diameter. When the optical fiber tip was located in the sheath, the bubble expanded to the lateral direction, and then the high collapse pressure was observed along the lateral direction. Therefore, we could arrange the bubble shape by changing the irradiation parameters. We discussed the proper bubble shape for various intra-vascular applications.

Background/Objective: The traditional method of stimulating neural activity has been based on electrical methods and remains the gold standard to date despite inherent limitations. We have previously shown a new paradigm to in vivo neural activation based on pulsed infrared light, which provides a contact-free, spatially selective, artifact-free method without incurring tissue damage that may have significant advantages over electrical stimulation in a variety of diagnostic and therapeutic applications. The goal of this study was to investigate the physical mechanism of this phenomenon, which we propose is a photo-thermal effect from transient tissue temperature changes resulting in direct or indirect activation of transmembrane ion channels causing propagation of the action potential. Methods: Rat sciatic nerve preparation was stimulated in vivo with the Holmium:YAG laser (2.12μm), Free Electron Laser (2.1μm), Alexandrite laser (690nm), and the prototype for a solid state commercial laser nerve stimulator built by Aculight (1.87μm) to determine contributions of photobiological responses from laser tissue interactions, including temperature, pressure, electric field, and photochemistry, underlying the biophysical mechanism of stimulation. Single point temperature measurements were made with a microthermocouple adjacent to the excitation site, while an infrared camera was used for 2-D radiometry of the irradiated surface. Displacement from laser-induced pressure waves or thermoelastic expansion was measured using a PS-OCT system. Results: Results exclude a direct photochemical, electric field, or pressure wave effect as the mechanism of optical stimulation. Measurements show relative small contributions from thermoelastic expansion (300 nm) with the laser parameters used for nerve stimulation. The maximum change in tissue temperature is about 9°C (average increase of 3.66 °C) at stimulation threshold radiant exposures. Conclusion: Neural activation with pulsed laser-light occurs by a transient thermally induced mechanism. Future experiments will reveal if this effect is through direct membrane interaction or facilitated through an indirect effect leading to membrane depolarization.

Limited optical interrogation depth prevents studies of arrhythmias in 3D tissue. Our experiments with single photon excitation and computer simulations with single and two-photon excitation show that the addition of a fluorescence absorber can modify the region of tissue interrogated with transillumination. Experiments were performed with a pair of 0.1 cm thick rabbit cardiac slices. The slices were excited with 488 nm single photon laser excitation while light from opposite side was collected for transillumination. One slice was stained with di-4-ANEPPS while the other was not. In different measurements, both slices were also stained with the red light absorbing dye, blue 1. In our models fluorescent photons were launched from specific regions of the tissue to mimic staining of different layers with di-4-ANEPPS. In additional simulations the fluorescence absorption coefficient was increased 3-fold. In our experiments with the di-4-ANEPPS slice facing away from the laser, measured light intensity was 68% of the value found with di-4-ANEPPS slice facing the laser while it was 21% in the model. This reduction in intensity from the deeper layer became less pronounced after addition of the red absorber. Then with di-4-ANEPPS slice facing away from the laser the measured light intensity was 81% of the value found with di-4-ANEPPS slice facing the laser in experiments while it was 34% in the model. This indicates deepening of the interrogated region by addition of red absorbing dye. In computer simulations using two-photon excitation at 1064 nm, increasing the fluorescence absorption further deepend the interrogated region compared with one photon excitation at 488 nm.

This paper presents numerical simulations predicting the time-resolved reflectance and autofluorescence of human skin exposed to a pulse of collimated light at 337 nm and pulse width of 1 ns. Moreover, the feasibility of using an embedded time-resolved fluorescence sensor for monitoring glucose concentration is also studied. Skin is modeled as a multilayer medium with each layer having its own optical properties and fluorophore absorption coefficients, lifetimes and quantum yields. The intensity distributions of excitation and fluorescent light in skin are then determined by solving the transient radiative transfer equation using the modified method of characteristics. In both cases, the fluorophore lifetimes are recovered from the simulated fluorescence decays and compared with the actual lifetimes used in the simulations. It was found that the fluorescence lifetime of the fluorophore contributing the least to the fluorescence signal could not be recovered while the other lifetimes could be recovered within 2.5% of input values. Such simulations could be valuable in interpreting data from time-resolved fluorescence experiments on healthy and diseased tissue as well as in designing and testing the feasibility of various optical sensors for biomedical diagnostics.

Phosphorylation and dephosphorylation are considered to be important reactions that control the active and inactive factors of proteins. In regenerative medicine of the osteoconnective tissue (a tendon, a ligament), it has been reported that the biomaterial possessing phosphate groups promote formation of HAP, the main component of hard tissues. The noncontact measurement of phosphate groups and low-destructive controlling of phosphate groups allow for the accurate regeneration of the osteoconnective tissue, and the validation. Our objective is to propose the nondestructive controlling and measuring method of phosphorylation for regenerative medicine. In this study, as the indirect quantitative analysis of phosphate groups, we examine the correlation between the mid-infrared absorbance ratio and the ratio of phosphate groups introduction theoretically calculated from a colorimetric determination method. And the noncontact controlling method of the quantities of phosphate groups, we examine the selective and low-destructive bond cutting of phosphate groups in the phosphogelatin using a mid-infrared laser.

We investigate the size of transient artificial pores as a function of the incident laser intensity in femtosecond nearinfrared laser opto-injection into single living Bovine aortic endothelial cells (BAECs). Molecules ranging in size from 457 dalton to 500kD were used in size exclusion experiments. We found that the threshold laser intensity for pore creation was in dependence of the size of the molecule.

Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein, we report on the application of femtosecond laser pulses for cellular manipulation, and the generation of optical pores for cytoplasmic delivery of non-reducing cryoprotectants. Under precise laser focusing, we demonstrate membrane surgery on live mammalian cells, and ablation of focal adhesions adjoining fibroblast cells. In both studies, the morphology of the cell post-laser treatment was maintained with no visible collapse or disassociation. When mammalian cells were suspended in a hyperosmotic cryoprotectant solution, focused femtosecond laser pulses were used to transiently permeabilize live cells for sucrose uptake. To verify the cytoplasmic uptake, the volumetric response of cells in 0.2, 0.3, 0.4, and 0.5 M cryoprotective sucrose was measured using video microscopy. From membrane integrity assays, we determined that optimal cell survival of 91.5 ± 8% is achieved using 0.2 M sucrose, with a decline in survival at higher concentrations. Using diffusion analysis for a porous membrane, the intracellular accumulation of cryoprotective sucrose was theoretically determined. At a diffusion length of 10 um, > 70% of the extracellular osmolarity was estimated to be intracellularly delivered following closure of the transient pore. We anticipate that our study will have important applications for biopresevation, and profound implications for surgery and cell-isolation.

Artificially pigmented hTERT-RPE1 cells were exposed to a mode-locked or continuous wave (CW) laser at 458 nm for one hour in a modified culture incubator. Exposure conditions were selected to give greatest likelihood of damage due to a photochemical mechanism, with interest in possible differences between CW and mode-locked damage thresholds. After post-exposure-recovery (PER) for either 1-hour or 24-hour, cells were concurrently stained with annexin V and 6-CFDA to determine if they had undergone necrosis or apoptosis. Alternatively, cells were stained with Ethidium Homodimer (EthD-1) and Calcein AM to determine if they had undergone necrosis following 1-hour and 24-hours PER. Preliminary results indicate that laser exposure induced some apoptosis following 1-hour PER, with irradiance required for apoptosis being lower than that for necrosis with mode-locked exposure conditions. Probit analysis yielded necrosis thresholds for cell culture following 1-hour PER using data compiled from both dye sets. CW exposures resulted in a lower threshold than mode-locked exposures for necrosis following 1-hour PER. A thermal model provided the predicted temperature rise in cell culture due to laser exposure. The thermal model validates our choice of laser parameters to obtain photochemical damage. Data following 24-hours PER were inconclusive. Considerations of cell migration are included in the interpretation of data and further improvements to methods when using live cell assays are recommended.

A lot of work has been done in the area of laser sterilization using UV lasers whereas this area is not much explored using an IR laser. In this study the cells were catapulted from glass or oxidized silicon substrates by a nanosecond IR CO₂ laser. Removal of cells and bacteria was achieved under the micron thick liquid layer pre-deposited on the substrates and lifted off together with biological species at laser fluences exceeding the corresponding boiling thresholds for the liquids used. Catapulting with front-side laser illumination is studied

To precisely control the position of multiple types of cells in a coculture for the study of cell-cell interactions, we have developed a laser micropatterning technique. The technique employs the optical forces generated by a weakly focused laser beam. In the beam's focal region, the optical force draws microparticles, such as cells, into the center of the beam, propels them along the beam axis, and guides them onto a target surface. Specific patterns are created through computercontrolled micromanipulation of the substrate relative to the laser beam. Preliminary data have demonstrated cell viability after laser guidance. This project was designed to systematically vary the controllable laser parameters, namely, intensity and exposure time of the laser on single cells, and thus determine the laser parameters that allow negligible cell damage with functional cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrain neurons were cultured in 35 mm petri dishes. Control and test cells were selected one hour after cell placement to allow cell attachment. Test cells were subjected to the laser at the focal region. The experimental parameters were chosen as: wavelength - 800 nm, intensities - 100 mW, 200 mW, and 300 mW, and exposure times - 10 s and 60 s. Results were analyzed based on neurite outgrowth and the Live/Dead assay (Viability/Cytoxicity kit from Molecular Probes). No statistical difference (p >> 0.1, student t-test) in viability or function was found between the control neurons and those exposed to the laser. This confirms that laser guidance seems to be a promising method for cellular manipulation.

The uniaxial orientation and bundle formation of collagen fibres determine the mechanical properties of tendons. Thus the particular challenge of tendon tissue engineering is to build the tissue with a highly organized structure of collagen fibres. Ultimately the engineered construct will be used as autologous grafts in tendon surgery, withstanding physiological loading. We grew pig tenocytes in porous chitosan scaffolds with multiple microchannels of 250-500 μm. The cell proliferation and production of extra-cellular matrix (ECM) within the scaffolds have been successfully monitored by Optical Coherence Tomography (OCT), a bench-top OCT system equipped with a broadband light source centred at 1300 nm. Under sterile condition, the measurements were performed on-line and in a non-destructive manner. In addition, a novel method based on OCT imaging, which calculates the occupation ratio of the microchannel derived from the scattered intensity has been developed. It is confirmed that the occupation ratio is correlated to cell proliferation and ECM production in the scaffolds. Thus this method has been utilised to assess the effect of different culture conditions on the tissue formation. The use of a perfusion bioreactor has resulted in a significantly (p<1e^-3) higher cell proliferation and matrix production.

Cancer is a world-wide health problem associated with an increasing death rate. The mechanisms of how normal cells transform into cancer cells are not fully understood. Intensive investigations have been undertaken to identify genes whose unregulated expression are involved in this process. In this study, we have grown, on collagen gel, adherent mouse embryo fibroblasts (MEFs) knocked out for Cyl-1 (MEF^Cyl1-/-) which have been transfected with the human proto-oncogene cyclin D1 (CCND1) under the control of an inducible expression system. CCND1 expression can be regulated in the fibroblasts via the presence of an inducer, isopropyl β-D-Thiogalactopyranoside (IPTG). In the absence of IPTG, CCND1 expression is silenced. The migration ability of the resultant cells on the collagen gel has been monitored by complementary optical techniques: the conventional light microscopy; optical coherence tomography and Fourier Transform Infrared Microspcopic Spectroscopy (FTIR) using Synchrotron beam source. It is found that the cells expressing CCND1 exhibited cell invasion morphology and had different matrix compositions near the cell layer in comparison to the cells not expressing CCND1. The results from this study are consistent with published findings that expression of CCND1 has oncogenic potential and is involved in cell invasion in vitro. Application of complementary optical techniques proves to be an efficient way obtaining morphological and composition information of cell invasion.

The ability to image tissue engineering products without damaging histological procedures is important for the understanding of the dynamics of tissue reorganization and formation. In this work, we test the ability of multiphoton autofluorescence and second harmonic generation microscopy to image engineered tissues following chrondrogenic induction. The system we used is human bone marrow stem cells seeded in the scaffold polyglycolic acid (PGA). Our results show that autofluorescence can be used to image cells while second harmonic generation signal can be used to visualize the synthesis of extracellular matrix. This approach demonstrates the ability of multiphoton imaging in the study of tissue engineering products.

Coherence or Time Domain Optical tomography within highly scattering media observes the shortest path photons over the dominant randomly scattered background light. Angular Domain Imaging employs micromachined collimators detecting photons within small angles of aligned laser light sources. These angular filters are micromachined silicon collimator channels 51 microns wide by 10 mm long on 102 micron spacing, giving an acceptance angle of 0.29 degrees at a CMOS detector array. Phantom test objects were observed in scattering media 5 cm thick at effective scattered to ballistic ratios from 1:1 to greater than 1E8:1. Line and space test objects detection limits are set by detector pixel size (5.5 microns) not collimator hole spacing. To maximize the ballistic/quasi-ballistic photons observed, a line of light aligned with the collimator holes increases detectability by reducing the amount of scattered background light. A Cylindrical Spherical Cylindrical beam expander/shrinker creates a 16 mm by 0.35 mm line of light. Best results occur when the scattering medium, collimator and detector are within 3X the Rayleigh Range of the beam's narrow vertical axis, allowing imaging of 51 micron lines/spaces at 3E8:1 scattering ratios. Restricting the light to a 1 mm line extends this to 8E9:1. Carbon coating the SMCA to reduce reflectivity shows that at high scattering levels absorbing walls will reduce background light, improving contrast. ADI has also been shown to work when the illumination is unaligned with the detector. This allows for side illumination with detection of structures at depths of 3mm with a scattering ratio of 1E6:1.

Excised bovine retinas were used as model for threshold determination of laser induced thermal damage in the pulse regime of 1 ms to 655 ms for a range of laser spot size diameters. The thresholds as determined by fluorescence viability staining compare very well with the prediction of thermal damage models. Both models compare well with published and new Rhesus monkey threshold data. A distinctive dependence of the threshold on laser spot size diameter for different pulse duration was found which indicates that current (ICNIRP, ANSI and IEC) laser exposure limits for large spots can be increased in this pulse duration regime. A time dependent α_max is proposed which only for the case of long exposure durations has the current value of 100 mrad, but decreases to smaller angles for short exposure durations, effectively increasing the permissible exposure level. An explanation based on intra-retinal scattering is offered for the unexpected spot size dependence for spot diameters less than about 80 µm. The time dependence and nature of damage is discussed for pulse durations shorter than 1 ms where bubble induced damage seems to lead to a threshold a factor of 10 lower than the thermally induced threshold, resulting in the need to lower the MPE values for this condition. Possible changes of the MPE values are offered and discussed.

Recent studies have determined that photochemical oxidation in cultured cells can be detected at peak irradiances as low as 8.5x10⁸ W cm^-2 (87-fs pulse). Fluorescent dyes, such as CM-H₂DCFDA, enable us to quantify the oxidation response of cells to mode-locked near-infrared (NIR) laser exposure. Using a modified confocal microscope, we characterize the time-dependent 2-photon induced fluorescence generated from a given NIR laser exposure. When cultured cells were then pre-loaded with antioxidants, ascorbic acid or N-acetyl-L-cysteine (NAC), they inhibit nonlinear oxidation with different efficiencies, providing insight regarding mechanisms of damage.