This PDF file contains the front matter associated with SPIE Proceedings Volume 8941, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Thresholds for microcavitation of isolated bovine and porcine melanosomes were determined using single nanosecond (ns) laser pulses in the NIR (1000 – 1319 nm) wavelength regime. Average fluence thresholds for microcavitation increased non-linearly with increasing wavelength. Average fluence thresholds were also measured for 10-ns pulses at 532 nm, and found to be comparable to visible ns pulse values published in previous reports. Fluence thresholds were used to calculate melanosome absorption coefficients, which decreased with increasing wavelength. This trend was found to be comparable to the decrease in retinal pigmented epithelial (RPE) layer absorption coefficients reported over the same wavelength region. Estimated corneal total intraocular energy (TIE) values were determined and compared to the current and proposed maximum permissible exposure (MPE) safe exposure levels. Results from this study support the proposed changes to the MPE levels.

We have been developing molecular delivery systems based on photomechanical waves (PMWs), which are generated by the irradiation of a laser absorbing material with nanosecond laser pulses. This method enables highly site-specific delivery in the horizontal plane of the tissue. However, targeting in the vertical direction is a remaining challenge. In this study, we developed a novel PMW focusing device for deeper tissue targeting. A commercial optical concave lens and black natural rubber sheet (laser absorber) were attached to the top and bottom end of a cylindrical spacer, respectively, which was filled with water. A laser pulse was transmitted through the lens and water and hit the rubber sheet to induce a plasma, generating a PMW. The PMW was propagated both downward and upward. The downward wave (1st wave) was diffused, while the upward (2nd wave) wave was reflected with the concave surface of the lens and focused at a depth determined by the geometrical parameters. To attenuate the 1st wave, a small-diameter silicon sponge rubber disk was adhered just under the rubber sheet concentrically with the laser axis. With the lens of f = -40 mm, the 2nd wave was focused to a diameter of 5.7 mm at a targeted depth of 20 mm, which was well agreed with the result of calculation by ray tracing. At a laser fluence of 5.1 J/cm², peak pressure of the PMW reached ~40 MPa at the depth of 20 mm. Under this condition, we examined depth-targeted gene delivery to the rat skin.

In this work we propose a predictive model that allows the study of thermal effects produced when the optical radiation interacts with an esophageal or stomach disease with gold nanoparticles embedded. The model takes into account light distribution in the tumor tissue by means of a Monte Carlo method. Mie theory is used to obtain the gold nanoparticles optical properties and the thermal model employed is based on the bio-heat equation. The complete model was applied to two types of tumoral tissue (squamous cell carcinoma located in the esophagus and adenocarcinoma in the stomach) in order to study the thermal effects induced by the inclusion of gold nanoparticles.

The immediate pulse-to-pulse interaction becomes more and more important for future-generation high-repetition rate ophthalmic laser systems. Therefore, we investigated the interaction of two laser pulses with different spatial and temporal separation by time-resolved photography. There are various different characteristic interaction mechanisms which are divided into 11 interaction scenarios. Furthermore, the parameter range has been constricted regarding the medical application; here, the efficiency was optimized to a maximum jet velocity along the scanning axis with minimum applied pulse energy as well as unwanted side effects at the same time. In conclusion, these results are of great interest for the prospective optimization of the ophthalmic surgical process with future-generation fs-lasers.

High repetition rated femtosecond laser oscillator systems with low pulse energy are more often applied for precise and safer eye surgery. Especially, the cutting procedure in the crystalline lens is of high important for presbyopia treatment. Nevertheless, the fundamental laser tissue interaction process is not completely understood, because apparently a self-induced process takes place, were one modified region changes the focusing behavior of following laser pulses. We used a MHz repetition rate femtosecond laser system with nJ-pulse energy which were focused inside an ocular-tissue-phantom (Hydroxy-ethylmethacrylat - HEMA) to induce photodisruption. The material change, caused by the fs-pulses was measured simultaneously with a compact digital-holographic microscope. To investigate the material manipulation at different time scales, we used a continuously illuminating light source. The holographic images provide quantitative values for optical path length difference (OPL), which is equivalent to a refractive index change. This change of the optical properties may cause following pulses to obtain different focusing conditions. Time lapse measurements during the laser application were performed, which show the temporal evolution of OPL. An increase of OPL during the laser application was measured, which was followed by a decrease in OPL after laser processing. Furthermore, similar experiments were performed in distilled water and in native porcine crystalline lenses. The fs-laser cutting effects in HEMA and crystalline lens were transferable. Simultaneous measurements of the material modification during the cutting process give rise to better knowledge of treatment modalities during ocular tissue processing.

In order to understand extracellular-photosensitization reaction (PR) using talaporfin sodium, we studied comparison of oxidation dynamics of albumin and talaporfin sodium in solution system by visible and ultraviolet absorption spectrum measurements. Almost all talaporfin sodium particles may be bound to albumin in interstitial fluid, and this binding would affect the oxidation dynamics during this PR. Bovine serum albumin (BSA) is commonly used in vitro study but its binding characteristics with talaporfin sodium are different from human serum albumin (HSA). PR was operated in a solution composed of 20 μg/ml talaporfin sodium and 1.3 mg/ml HSA or BSA to simulate myocardial extracellular PR condition. Laser radiation of 662 nm was irradiated to this solution with irradiance of 0.29 W/cm². Absorption spectra of these solutions were measured during the PR. We estimated oxidized ratio by absorption difference around 240 nm before and after the PR. Talaporfin sodium was oxidized 100% with HSA and BSA by the PR of 100 J/cm² in radiant exposure. On the other hand, HSA and BSA were oxidized 60% and 94%, respectively in this radiant exposure. Q-band absorption peak of talaporfin sodium with HSA was shifted to 1 nm longer wavelength increasing radiant exposure up to 100 J/cm². This longer wavelength shift would mean binding ratio of non-oxidized talaporfin sodium to non-oxidized HSA was increased with increasing radiant exposure. Therefore it would be possible that PR with talaporfin sodium bound to HSA might present efficient PDT than PR bound to BSA.

Femtosecond lasers are widely used in everyday clinical procedures to perform minimally invasive corneal refractive surgery. The intralase femtosecond laser (AMO Corp. Santa Ana, CA) is a common example of such a laser. In the present study a numerical simulation was developed to quantify the temperature rise in the retina during femtosecond intracorneal surgery. Also, ex-vivo retinal heating due to laser irradiation was measured with an infrared thermal camera (Fluke Corp. Everett, WA) as a validation of the simulation. A computer simulation was developed using Comsol Multiphysics to calculate the temperature rise in the cadaver retina during femtosecond laser corneal surgery. The simulation showed a temperature rise of less than 0.3 degrees for realistic pulse energies for the various repetition rates. Human cadaver retinas were irradiated with a 150 kHz Intralase femtosecond laser and the temperature rise was measured withan infrared thermal camera. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser corneal surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage. The simulation predictions are in agreement with thermal measurements providing a level of experimental validation.

Retinal photocoagulation is an established treatment for various retinal diseases. The temperature development during a treatment can be monitored by applying short laser pulses in addition to the treatment laser light. The laser pulses induce temperature dependent thermoelastic pressure waves that can be detected at the cornea. When determining the temperature from the detected pressure waves, the static tissue parameters are assumed to be equal to their mean value that can be found in literature everywhere. However, this is unlikely in a treatment, as the tissue parameters vary from one irradiation site to another. In order to investigate the inaccuracies that are introduced by the assumption of ideal conditions, a numerical model was devised to examine the temperature development during the treatment as well as the formation and propagation of the ultrasonic waves. Using the model, it is possible to determine the peak temperature during retinal photocoagulation from the measured signal, and to investigate the behaviour of the temperature profile and the accuracy of the temperature determination under varying conditions such as changes in the irradiation beam profile. It is shown that there is an error of 15% in determining the peak temperature, when the irradiation beam profile changes from a top hat profile to a gaussian profile. Furthermore, the model was extended in order to incorporate the photoacoustic pressure generation and wave propagation. It was shown that for an irradiation pulse duration of 75 ns there is a difference in pressure amplitude of a factor 2 bet between a top hat and a gaussian shaped irradiation profile due to the difference in energy deposition in the fundus layers.

It has been shown that exposure of live neurons to a low-intensity pulsed infrared light can be used to excite action potentials. Infrared pulsed laser coupled to an optical fiber can be utilized to create a rapid localized increase in temperature in the vicinity of the cell. The resulting temperature gradient leads to an increase in membrane fluidity and permeability, causing depolarization of the target cell. In order to characterize the fluidity of the cell membrane at various temperatures with and without pulsed IR light exposure, we used a polarity-sensitive fluorescent probe di-4- ANEPPDHQ. This dye exhibits a fluorescent shift between the disordered and ordered phases of the membrane, and can be used to quantitatively evaluate the state of the membrane by calculating the generalized polarization (GP) value. Using high-speed imaging of cells exposed to a IR light of varying pulse width, it was determined that a longer pulse width leads to a greater change in the GP value. Comparison of GP values of cells at different ambient temperatures without the pulsed IR light exposure and cells exposed to pulsed IR light indicated that a rapid temperature gradient caused by the exposure to pulsed light induces a larger change in GP value than the ambient temperature increase alone, indicating a greater disruption of membrane fluidity and permeability.

An artificially pigmented retinal pigment epithelial (RPE) cell model was used to study the damage rates for exposure to 1, 10, 100, and 1,000 230-μs laser pulses at 532 nm, at two different concentrations of melanosome particles (MPs) per cell. Multiple pulses were delivered at pulse repetition rates of 50 and 99 pulses per second. Standard fluorescence viability indicator dyes and the method of microthermography were used to assess damage and thermal responses, respectively. Although frame rate during microthermography was more than five times slower than the duration of laser pulses, thermal information was useful in refining the BTEC computational model for simulating high-resolution thermal responses by the pigmented cells. When we temporally sampled the thermal model output at a rate similar to our microthermography, the resulting thermal profiles for multiple pulses resembled the thermal experimental profiles. Complementary to the thermal simulations, our computer-generated thresholds were in good agreement with the in vitro data. Findings are examined within the context of common exposure limit definitions in the national and international laser safety standards.

Vaporization and coagulation are two fundamental processes that can be performed during laser-tissue ablation. We demonstrated a method allowing quasi-dynamically observing of the cross-sectional images of tissue response during ablation. The results showed that coagulation depth is relatively constant during vaporization, which supports the excellent hemostasis of green laser benign prostate hyperplasia (BPH) treatment. We also verified a new technology for real-time, in situ tissue temperature monitoring, which may be promising for in vivo tissue vaporization degree feedback during laser ablation to improve the vaporization efficiency and avoid complications.

We studied the relations between the time history of smooth muscle cells (SMCs) death rate and heating condition in vitro to clarify cell death mechanism in heating angioplasty, in particular under the condition in which intimal hyperplasia growth had been prevented in vivo swine experiment. A flow heating system on the microscope stage was used for the SMCs death rate measurement during or after the heating. The cells were loaded step-heating by heated flow using a heater equipped in a Photo-thermo dynamic balloon. The heating temperature was set to 37, 50-60°C. The SMCs death rate was calculated by a division of PI stained cell number by Hoechst33342 stained cell number. The SMCs death rate increased 5-10% linearly during 20 s with the heating. The SMCs death rate increased with duration up to 15 min after 5 s heating. Because fragmented nuclei were observed from approximately 5 min after the heating, we defined that acute necrosis and late necrosis were corresponded to within 5 min after the heating and over 5 min after the heating, respectively. This late necrosis is probably corresponding to apoptosis. The ratio of necrotic interaction divided the acute necrosis rate by the late necrosis was calculated based on this consideration as 1.3 under the particular condition in which intimal hyperplasia growth was prevented in vivo previous porcine experiment. We think that necrotic interaction rate is larger than expected rate to obtain intimal hyperplasia suppression.

The pancreas is a deeply seated organ requiring endoscopically, or radiologically guided biopsies for tissue diagnosis. Current approaches include either fine needle aspiration biopsy (FNA) for cytologic evaluation, or core needle biopsies (CBs), which comprise of tissue cores (L = 1-2 cm, D = 0.4-2.0 mm) for examination by brightfield microscopy. Between procurement and visualization, biospecimens must be processed, sectioned and mounted on glass slides for 2D visualization. Optical information about the native tissue state can be lost with each procedural step and a pathologist cannot appreciate 3D organization from 2D observations of tissue sections 1-8 μm in thickness. Therefore, how might histological disease assessment improve if entire, intact CBs could be imaged in both brightfield and 3D? CBs are mechanically delicate; therefore, a simple device was made to cut intact, simulated CBs (L = 1-2 cm, D = 0.2-0.8 mm) from porcine pancreas. After CBs were laid flat in a chamber, z-stack images at 20x and 40x were acquired through the sample with and without the application of an optical clearing agent (FocusClear®). Intensity of transmitted light increased by 5-15x and islet structures unique to pancreas were clearly visualized 250-300 μm beneath the tissue surface. CBs were then placed in index matching square capillary tubes filled with FocusClear® and a standard optical clearing agent. Brightfield z-stack images were then acquired to present 3D visualization of the CB to the pathologist.

A focused nanosecond laser pulse produces optical damage to subsurface targets when its intensity is high enough to overcome the required threshold irradiance. However, when the material is highly scattering, the laser pulse irradiance decreases as it propagates through the sample because the temporal pulse profile is stretched due to multiple scattering events. The objective of this work is to determine the transfer function associated to an integrating sphere measurement involving turbid media samples. Integrating spheres are used to measure the total diffuse reflectance and transmittance of homogeneous turbid media samples to retrieve its absorption and scattering coefficients. Reflectance and transmittance measurements, being static properties, are not affected by multiple reflections of light inside the integrating spheres. However, for a time-dependent measurement, such as the temporal profile of a short laser pulse propagating through a turbid medium, the light reflection and multiple scattering events inside the sphere contributes to an additional stretching deformation of the measured temporal pulse profile, which complicates the interpretation of the measurements. In this work we use integrating spheres to analyze the effect of a turbid media on the propagation of a nanosecond laser pulse.

A system integrating high density diffuse optical imaging with adaptive optics using MEMS for deep tissue interaction is presented. In this system, a laser source is scanned over a high density fiber bundle using Digital Micromirror Device (DMD) and channeled to a tissue phantom. Backscatter is then collected from the tissue phantom by a high density fiber array of different fiber type and channeled to CMOS sensor for image acquisition. Intensity focus is directly verified using a second CMOS sensor which measures intensity transmitted though the tissue phantom. A set of training patterns are displayed on the DMD and backscatter is numerically fit to the transmission intensity. After the training patterns are displayed, adaptive focus is performed using only the backscatter and fitting functions. Additionally, tissue reconstruction and prediction of interference focusing by photoacoustic and optical tomographic methods is discussed. Finally, potential NIR applications such as in-vivo adaptive neural photostimulation and cancer targeting are discussed.

Finite difference time domain (FDTD) analysis has been formulated for predicting time-resolved reflectance from an adult head model with brain grooves containing a non-scattering layer. Mean delay (MD) dependences on source detector separation (d) and time-resolved reflectance calculated using the FDTD analysis were compared with in vivo experiments of human heads. It is shown that the theoretical and experimental MD dependences on d and the time-resolved reflectance are well predicted by FDTD analysis. These results have shown that tomographic imaging of brain activities is promising, which improves depth sensitivities by enhancing the contribution of late photons in time-resolved systems.

The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA’s enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein’s enzymatic and signaling capabilities.

We studied the immediate response of myocardial cells by continuous observation using confocal microscope against oxidation stress by extracellular photosensitization reaction using Talaporfin sodium for tachyarrhythmia treatment application. Immediate response in order from several seconds to several minutes is required for the arrhythmia treatment since operators should judge the therapeutic effect during the tachyarrhythmia ablation procedure. To understand the immediate response of myocardial cells, we measured the intracellular Ca²⁺ concentration using fluo-4 AM during and after the extracellular photosensitization reaction. Talaporfin sodium concentration was varied 10-30 μg/ml. A red diode laser of 663 nm in wavelength was irradiated under the microscope with the radiant exposure of 40 J/cm² and irradiance of 0.29 W/cm². We observed the fluorescence image of fluo-4 AM each 400 ms during until 10 min after the photosensitization reaction. The myocardial cell beatings were stopped about 2 s after the beginning of the laser irradiation. The blebs were formed with the Ca²⁺ inflow. The intracellular Ca²⁺ was re-decreased after the bleb formation and then the cell necrosis was induced. The cell lethality 10 min after the laser irradiation was less than bleb formation ratio. The time response of the cell necrosis was shortened with the photosensitizer concentration increasing and the minimum average value was 209 s in the case of the 30 μg/ml in photosensitizer concentration and 40 J/cm² in the radiant exposure. We think this extracellular photosensitization reaction may be applicable to tachyarrhythmia treatment in terms of its immediate response.

We studied photosensitization reaction progress in a cell culture well by oxygen partial pressure distribution measurement along the well depth direction with a high concentration of talaporfin sodium solution. The talaporfin sodium solution of 20 μg/ml in concentration with 2.8 mm thickness in the well was irradiated from the well bottom by 663 nm excitation laser with 0.29 W/cm². A small Clark-type oxygen electrode was used to measure oxygen partial pressure during the photosensitization reaction with approximately 200 μm resolution. Corrections against solution temperature change and direct light irradiation were applied to the electrode output. The oxygen partial pressures at various depths were decreased uniformly from the atmospheric oxygen partial pressure with the photosensitization reaction progress up to the irradiation of 1.0 J/cm² in radiant exposure. In the case of photosensitization reaction over 1.0 J/cm² in radiant exposure, the oxygen partial pressure distribution along the well depth was non-uniform. In the case of photosensitization reaction with 40 J/cm² in radiant exposure in the solution without cells, there was pressure gradient of 2.8×10⁴ mmHg/m from 0.5 to 1.0 mm in depth from the solution surface. In this case, there was no pressure gradient near the bottom of the well. In contrast, with myocardial cells at the bottom, oxygen partial pressure gradient of 7.5×10³ mmHg/m from 1.5 to 2.0 mm in the depth was appeared after irradiation with 40 J/cm² in radiant exposure. Consequently, we found that oxygen partial pressure distribution along the depth in the well with high concentration of talaporfin sodium solution was dynamically changed with time of the photosensitization reaction using the laser irradiation from the bottom. We think this dynamic pressure change in the well might be useful to understand the photosensitization reaction progress in the well experiment system in vitro corresponding to the extracellular PDT.

The optical properties of pigmented lesions have been studied using diffuse reflectance spectroscopy in a noninvasive configuration on optically thick samples such as skin in vivo. However, it is difficult to un-mix the effects of absorption and scattering with diffuse reflectance spectroscopy techniques due to the complex anatomical distributions of absorbing and scattering biomolecules. We present a device and technique that enables absorption and scattering measurements of tissue volumes much smaller than the optical mean-free path. Because these measurements are taken on fresh-frozen sections, they are direct measurements of the optical properties of tissue, albeit in a different hydration state than in vivo tissue. Our results on lesions from 20 patients including melanomas and nevi show the absorption spectrum of melanin in melanocytes and basal keratinocytes. Our samples consisted of fresh frozen sections that were unstained. Fitting the spectrum as an exponential decay between 500 and 1100 nm [mua = A*exp(-B*(lambda-C)) + D], we report on the fit parameters of and their variation due to biological heterogeneity as A = 4.20e⁴ +/- 1.57e⁵ [1/cm], B = 4.57e^-3 +/- 1.62e^-3 [1/nm], C = 210 +/- 510 [nm] , D = 613 +/- 534 [1/cm]. The variability in these results is likely due to highly heterogeneous distributions of eumelanin and pheomelanin.

The development of new cancer treatments and the early prediction of their therapeutic potential are often made difficult by the lack of predictive pharmacological models. The 3D multicellular tumor spheroid (MCTS) model offers a level of complexity that recapitulates the three-dimensional organization of a tumor and appears to be fairly predictive of therapeutic efficiency. The use of spheroids in large-scale automated screening was recently reported to link the power of a high throughput analysis to the predictability of a 3D cell model. The spheroid has a radial symmetry; this simple geometry allows establishing a direct correlation between structure and function. The outmost layers of MCTS are composed of proliferating cells and form structurally uniform domain with an approximate thickness of 100 microns. The innermost layers are composed of quiescent cells. Finally, cells in the center of the spheroid can form a necrotic core. This latest region is structurally heterogeneous and is poorly characterized. These features make the spheroid a model of choice and a paradigm to study the optical properties of various epithelial tissues. In this study, we used an in-vitro optical technique for label-free characterization of multicellular systems based on the index- mismatch induced spherical aberrations. We achieve to monitor and characterize the optical properties of MCTS. This new and original approach might be of major interest for the development of innovative screening strategies dedicated to the identification of anticancer drugs.

Pulsed photothermal radiometry (PPTR) allows noninvasive measurement of laser-induced temperature depth profiles. The obtained profiles provide information on depth distribution of absorbing chromophores, such as melanin and hemoglobin. We apply this technique to objectively characterize mass diffusion and decomposition rate of extravasated hemoglobin during the bruise healing process. In present study, we introduce objective fitting of PPTR data obtained over the course of the bruise healing process. By applying Monte Carlo simulation of laser energy deposition and simulation of the corresponding PPTR signal, quantitative analysis of underlying bruise healing processes is possible. Introduction of objective fitting enables an objective comparison between the simulated and experimental PPTR signals. In this manner, we avoid reconstruction of laser-induced depth profiles and thus inherent loss of information in the process. This approach enables us to determine the value of hemoglobin mass diffusivity, which is controversial in existing literature. Such information will be a valuable addition to existing bruise age determination techniques.

The study highlights the effect of different modes of in vivo laser irradiation of mice using a PFL8LA laser with λ = 1560 nm, pulse duration of 1,4•10^-12 s, peak power of 3,72•10³ W and average output power of 20•10^-3 W on the lipid peroxidation parameters: conjugated dienes, ketodienes and conjugated trienes, malondialdehyde, Schiff bases and the activity of antioxidant enzymes - catalase, glutathione -S-transferase and superoxide dismutase in erythrocytes and plasma of mice. Two groups of mice received a total dose of 3.8 J/cm² per group, but the 1st group was irradiated only once, while the 2nd – four times. Significant differences in the parameters of the 1st and 2nd groups indicate different effects of the irradiation modes on redox-dependent processes in red blood cells of mice.

The scattering coefficient, μ_s, the anisotropy factor, g, the scattering phase function, p(θ), and the angular dependence of scattering intensity distributions of human cancerous and normal prostate tissues were systematically investigated as a function of wavelength, scattering angle and scattering particle size using Mie theory and experimental parameters. The Matlab-based codes using Mie theory for both spherical and cylindrical models were developed and applied for studying the light propagation and the key scattering properties of the prostate tissues. The optical and structural parameters of tissue such as the index of refraction of cytoplasm, size of nuclei, and the diameter of the nucleoli for cancerous and normal human prostate tissues obtained from the previous biological, biomedical and bio-optic studies were used for Mie theory simulation and calculation. The wavelength dependence of scattering coefficient and anisotropy factor were investigated in the wide spectral range from 300 nm to 1200 nm. The scattering particle size dependence of μ_s, g, and scattering angular distributions were studied for cancerous and normal prostate tissues. The results show that cancerous prostate tissue containing larger size scattering particles has more contribution to the forward scattering in comparison with the normal prostate tissue. In addition to the conventional simulation model that approximately considers the scattering particle as sphere, the cylinder model which is more suitable for fiber-like tissue frame components such as collagen and elastin was used for developing a computation code to study angular dependence of scattering in prostate tissues. To the best of our knowledge, this is the first study to deal with both spherical and cylindrical scattering particles in prostate tissues.

This study describes the propagation of direct and diffuse light through coral tissue and how changes in the directional quality of light affect photosynthesis. Scalar irradiance microsensors were used in vivo to measure tissue light propagation of incident collimated and diffuse irradiance. O₂ microsensors were used to estimate changes in local O₂ evolution. The results show that the directional quality of incident irradiance affects both coral optics and photosynthesis. Collimated irradiance is enhanced at the coral surface while diffuse irradiance is enhanced at the coral skeleton. Coral O₂ evolution is enhanced under collimated compared to diffuse light. It is concluded that the directional quality of light is an important and hitherto ignored parameter in coral photosynthesis.

The recent effort leads to reliable imaging techniques which can help to a surgeon during operations. The fluorescence spectroscopy was selected as very useful online in vivo imaging method to organics and biological materials analysis. The presented work scopes to a laser induced fluorescence spectroscopy technique to detect tissue local necrosis in small intestine surgery. In first experiments, we tested tissue auto-fluorescence technique but a signal-to-noise ratio didn’t express significant results. Then we applied a contrast dye - IndoCyanine Green (ICG) which absorbs and emits wavelengths in the near IR. We arranged the pilot experimental setup based on highly coherent extended cavity diode laser (ECDL) used for stimulating of some critical areas of the small intestine tissue with injected ICG dye. We demonstrated the distribution of the ICG exciter with the first file of shots of small intestine tissue of a rabbit that was captured by high sensitivity fluorescent cam.

The THz impulse radar is an “RF-inspired” sensor system that has performed remarkably well since its initial development nearly six years ago. It was developed for ex vivo skin-burn imaging, and has since shown great promise in the sensitive detection of hydration levels in soft tissues of several types, such as in vivo corneal and burn samples. An intriguing aspect of the impulse radar is its hybrid architecture which combines the high-peak-power of photoconductive switches with the high-responsivity and -bandwidth (RF and video) of Schottky-diode rectifiers. The result is a very sensitive sensor system in which the post-detection signal-to-noise ratio depends super-linearly on average signal power up to a point where the diode is “turned on” in the forward direction, and then behaves quasi-linearly beyond that point. This paper reports the first nonlinear systems analysis done on the impulse radar using MATLAB.

This article provides an update to recent reviews of the Frohlich hypothesis that biological organisation is facilitated by the creation of coherent excited states driven by a flow of free energy provided by metabolic processes and mediated by molecular motions in the terahertz range. Sources of intense terahertz radiation have the potential to test this hypothesis since if it is true the growth and development of sensitive systems such as stem cells should be influenced by irradiation with intense terahertz radiation. A brief survey of recent work shows that it is not yet possible to make an assessment of the validity of the Frohlich hypothesis. Under some conditions a variety of cell types respond to irradiation with intense THz radiation in ways that involve changes in the activity of their DNA. In other experiments very intense and prolonged THz radiation has no measureable effect on the behavior of very sensitive systems such as stem cells. The wide variation in experimental conditions makes it impossible to draw any conclusions as to characteristics of THz radiation that will induce a response in living cells. It is possible that in environments suitable for their maintenance and growth cells are capable of compensating for any effects caused by exposure to THz radiation up to some currently unknown level of THz peak power.

The growing experimental evidence suggests that broadband, picosecond-duration THz pulses may influence biological systems and functions. While the mechanisms by which THz pulse-induced biological effects are not yet known, experiments using in vitro cell cultures, tissue models, as well as recent in vivo studies have demonstrated that THz pulses can elicit cellular and molecular changes in exposed cells and tissues in the absence of thermal effects. Recently, we demonstrated that intense, picosecond THz pulses induce phosphorylation of H2AX, indicative of DNA damage, and at the same time activate DNA damage response in human skin tissues. We also find that intense THz pulses have a profound impact on global gene expression in human skin. Many of the affected genes have important functions in epidermal differentiation and have been implicated in skin cancer and inflammatory skin conditions. The observed THzinduced changes in expression of these genes are in many cases opposite to disease-related changes, suggesting possible therapeutic applications of intense THz pulses.

Terahertz (THz) imaging and sensing technologies are increasingly being used at international airports for security screening purposes and at major medical centers for cancer and burn diagnosis. The emergence of new THz applications has directly resulted in an increased interest regarding the biological effects associated with this frequency range. Knowledge of THz biological effects is also desired for the safe use of THz systems, identification of health hazards, and development of empirically-based safety standards. In this study, we developed a state-of-the-art exposure chamber that allowed for highly controlled and reproducible studies of THz biological effects. This innovative system incorporated an industry grade cell incubator system that permitted a highly controlled exposure environment, where temperatures could be maintained at 37 °C ± 0.1 °C, carbon dioxide (CO₂) levels at 5% ± 0.1%, and relative humidity (RH) levels at 95% ± 1%. To maximize the THz power transmitted to the cell culture region inside the humid incubator, a secondary custom micro-chamber was fabricated and incorporated into the system. This micro-chamber shields the THz beam from the incubator environment and could be nitrogen-purged to eliminate water absorption effects. Additionally, a microscope that allowed for real-time visualization of the live cells before, during, and after THz exposure was integrated into the exposure system.

Despite 30 years of research, the mechanism behind the induced breakdown of plasma membranes by electrical pulses, termed electroporation, remains unknown. Current theories treat the interaction between the electrical field and the membrane as an entirely electrical event pointing to multiple plausible mechanisms. By investigating the biophysical interaction between plasma membranes and nanosecond electrical pulses (nsEP), we may have identified a non-electric field driven mechanism, previously unstudied in nsEP, which could be responsible for nanoporation of plasma membranes. In this investigation, we use a non-contact optical technique, termed probe beam deflection technique (PBDT), to characterize acoustic shockwaves generated by nsEP traveling through tungsten wire electrodes. We conclude these acoustic shockwaves are the result of the nsEP exposure imparting electrohydraulic forces on the buffer solution. When these acoustic shockwaves occur in close proximity to lipid bilayer membranes, it is possible that they impart a sufficient amount of mechanical stress to cause poration of that membrane. This research establishes for the first time that nsEP discharged in an aqueous medium generate measureable pressure waves of a magnitude capable of mechanical deformation and possibly damage to plasma membranes. These findings provide a new insight into the longunanswered question of how electric fields cause the breakdown of plasma membranes.

Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to orientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di- 4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.

While generally inducing minimal heating in many biomedical applications, electric fields may still induce significant temperature gradients, particularly for pulses of short duration and AC (sinusoidal) fields of high frequency, such as microwaves. This paper extends a recent analysis of temperature gradients across a biological cell and membrane for single pulses [(A. L. Garner, et al., J. Appl. Phys. 113, 214701 (2013).] to multiple pulses or AC fields where the time between the two pulses, or the period for AC signals, is shorter than the thermal diffusion time. We calculate profiles of the induced temperature changes and gradients across a biological cell for AC wave of different frequencies and show that the location of the peak temperature and gradient shifts toward the center of the cell during subsequent half-waves. Higher frequency fields induce higher temperature gradients with the temperature gradient shifts toward the center of the cell for subsequent cycles.

Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell’s ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line’s higher tolerance of nsPEF exposure.

The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few μL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.

Previously, it was demonstrated that small nanometer-sized pores (nanopores) are preferentially formed after exposure to nanosecond pulsed electric fields (nsPEF). We have reported that nanoporation of the plasma membrane directly affects the phospholipids of the cell membrane, ultimately culminating in phosphatidylinositol_4,5- bisphosphate (PIP₂) intracellular signaling. PIP₂, located within the internal layer of the plasma membrane, plays a critical role as a regulator of ion transport proteins, a source of second messenger compounds, and an anchor for cytoskeletal elements. In this proceeding, we present data that demonstrates that nsPEFs initiate electric field dose-dependent PIP₂ hydrolysis and/or depletion from the plasma membrane through the observation of the accumulation of inositol_1,4,5-trisphosphate (IP3) in the cytoplasm and the increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. The phosphoinositide signaling cascade presented here involves activation of phospholipase C (PLC) and protein kinase C (PKC), which are responsible for a multitude of biological effects after nsPEF exposure. These results expand our current knowledge of nsPEF induced physiological effects, and serve as a basis for development of novel tools for drug independent stimulation or modulation of different cellular functions.