This PDF file contains the front matter associated with SPIE Proceedings Volume 6854, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Numerous medical procedures utilize pulsed lasers to remove unwanted biological tissue. Mid-infrared wavelengths which preferentially target protein absorption bands ablate tissue more efficiently than wavelengths targeting water absorption. However, the mechanism responsible for this finding has not been established. In this report, we combine optical imaging and conventional techniques to assess lethal and sublethal collateral damage after ablative surgery with a Free Electron Laser (FEL). Heat shock protein expression was used to evaluate tissue damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and GFP expression (hsp70A1-L2G). To examine wavelength-dependence in the mid-IR, laser surgery was conducted on the hsp70A1-L2G mouse model using wavelengths targeting protein (amide II band, 6.45 μm), both water and protein (amide I band, 6.10 μm); and water (2.94 μm). Hsp70-driven luciferase activity was used as a quantitative biomarker for intracellular damage, and histological analyses were conducted to measure the depth of thermal damage. For all of the wavelengths tested, the bioluminescent data showed that the magnitude of hsp70 expression was dose-dependent. Tissues treated at 6.45 µm had approximately 2x higher hsp70 expression than tissues treated at 6.10 μm. Histology showed that immediate tissue injury at the 6.45 μm wavelength was ~2x deeper than at 6.10 μm. The 6.10 μm wavelength generated the least amount of epidermal hyperplasia. Overall, the data suggests that 6.10 μm is a superior wavelength for cutaneous laser ablation procedures.

The zebrafish (Danio rerio) is an attractive model system that has received wide attention for its usefulness in the study of development and disease. This organism represents a closer analog to humans than the common invetebrates Drosophila melanogaster and Caenorhabditis elegans, making this species an ideal model for human health research. Non-invasive manipulation of the zebrafish has been challenging, owing to the outer proteinaceous membrane and multiple embryonic barriers. A novel tool capable of manipulating early cleavage stage embryonic cells would be important for future advancements in medial research and the aquaculture industry. Herein, we demonstrate the laser surgery of early cleavage stage (2-cell) blastomere cells using a range of average laser powers and beam dwell times. Since the novelty of this manipulation tool depends on its non-invasive application, we examined short- and long-term laser-induced developmental defects following embryonic surgery. Laser-manipulated embryos were reared to 2 and 7 days post-fertilization and compared to control embryos at the same developmental stages. Morphological analysis was performed using light microscopy and scanning electron microscopy. Developmental features that were examined included the antero- and dorsal-lateral whole body views of the larvae, the olfactory pit, dorsal, ventral and pectoral fins, notochord, pectoral fin buds, otic capsule, otic vesicle, neuromast patterning, and kinocilia of the olfactory pit rim and cristae of the lateral wall of the ear. Laser-manipulated embryos developed normally relative to the controls, with developmental patterning and morphology at 2 and 7 days indistinguishable from control larvae.

Near infrared laser tissue welding (LTW) is achieved by subjecting the closely approximated surgically incised tissues to a laser beam at a wavelength that is absorbed by water in the tissue. Full thickness welds are accomplished with optimum laser power and penetration depths appropriate for the thickness of welded tissues. No extrinsic cross-linking or bonding materials are used. The absorbed laser energy increases the entropy of collagen in the tissue. In LTW, tissue water temperatures transiently rises to approximately 60° C, causing partial denaturing of collagen and other structural proteins due to breaking of hydrogen bonds, electrostatic interactions and some interchain covalent bonds for a short duration of time. This is followed by cross linking of proteins on either side of weld line, with reformation of the above mentioned bonds as the tissue cools, resulting in the formation of water tight full thickness welds. In this study, a cw fiber laser emitting at 1455 nm, corresponding to absorption by a water vibrational overtone, is used for in vivo LTW of surgical incisions made in the skin of guinea pigs under general anesthesia. The tensile strength and healing rates of the welded incisions are compared to suturing of similar incisions. Laser parameters, including power, scanning rates, exposure area, and exposure duration, are optimized to reduce thermal damage while maintaining tensile strength.

A series of experiments were conducted in vivo on porcine skin to determine the ED50 damage thresholds for 1214 nm continuous wave laser irradiation. These results provide new information for refinement of Maximum Permissible Exposure (MPE). The study employed exposure durations of 1 sec, 3 sec, and 10 seconds with nominal spot diameters of 6 mm, 8 mm and 10 mm and as a function of laser power. The effect of each irradiation was evaluated acutely, one hour after exposure, and 24 hours post exposure. Probit analysis was conducted to estimate the dose for 50% probability of laser-induced damage (ED50); Damage was defined as persistent redness at the site of irradiation for the pig skin after 24 hours. The results indicated that Maximum Permissible Exposure (MPE) limits should be lowered for the laser beam diameters larger than 6 mm.

The Yucatan mini-pig (Sus scrofa) is one of the most widely used animal models for skin damage studies because it shares many of the same physical properties as human skin. While the Yucatan is ideal for laser exposure studies using a large spot size, its size and cost are excessive for projects using smaller beams. This experiment performed histological analysis of skin biopsies from pigmented Hairless Guinea Pigs (Cavia porcellus) for epidermal thickness and melanin concentration. That data was then compared to similar information on the Yucatan.

We studied the monitoring of heat-denature by autofluorescence spectrum from lung dissection plane during laser air leak sealing procedure. In order to seal the air leakage from lung in thoracotomy, we proposed novel laser sealing method with the combination of the diode laser (810nm wavelength) irradiation and indocyanine green staining (peak absorption wavelength: 805 nm). This sealing method is expected to preserve the postoperative ventilatory capacity and achieve minimally invasive surgery. We previously reported that this laser sealing only requires thin sealing margin (less than 300 μm in thickness) compared with that of the suturing or stapling. The most serious issue on the laser air leak sealing might be re-air-leakage due to rigid surface layer caused by excessive heat-denature, such as carbonization. We should achieve laser air leak sealing minimizing the degree of heat denature. Dissection planes of isolated porcine lung with /without the diode laser irradiation were prepared as samples. We measured the auto-fluorescence from these samples using a spectrometer. When the diode laser was irradiated with 400J/cm², the surface of diode laser irradiated lung was fully carbonized. The ration of auto-fluorescence emission of 450nm / 500 nm, with 280 nm excitation wavelength was decreased less tha 50 % of initial value. That of 600 nm / 500 nm was increased over 700 % of initial value. The decreasing of the 450 nm auto-fluorescence intensity might be attributed to the heat-denaturing of the interstitial collagen in lung. However, increasing of the 600 nm didn't specify the origins, we suppose it might be originated from heat-denature substance, like carbonization. We could establish the useful monitoring for lung heat-denaturing with simple methodology. We think the auto-fluorescence measurement can be helpful not only for understanding the sealing mechanism, but also for controlling the degree of heat-denaturing during the procedure.

Since lasers were first used in medicine and biomedical related research there have been a variety of documented effects following the irradiation of neural tissues. The first systematic studies to report the direct stimulatory effect of infrared light on neural tissues were performed by researchers at Vanderbilt University in the rat sciatic nerve. These initial studies demonstrated a set of associated advantages of standard stimulation methods, which lead to much excitement and anticipation from the neuroscience community and industry. The inception of this new field included a partnership between industry and academia to foster the development, not only of the applications but also a series of devices to support the research and ultimate commercialization of technology. Currently several institutions are actively utilizing this technique in various applications including in the cochlear and vestibular systems. As more researchers enter the field and new devices are developed we anticipate the number of applications will continue to grow. Some of the next steps will include the establishment of the safety and efficacy data to move this technique to clinical trials and human use.

Light can artificially stimulate nerve activity in vivo. A significant advantage of optical neural stimulation is the potential for higher spatial selectivity when compared with electrical stimulation. An increased spatial selectivity of stimulation could improve significantly the function of neuroprosthetics, such as cochlear implants. Cochlear implants restore a sense of hearing and communication to deaf individuals by directly electrically stimulating the remaining neural cells in the cochlea. However, performance is limited by overlapping electric fields from neighboring electrodes. Here, we report on experiments with a new laser, offering a previously unavailable wavelength, 1.94μm, and pulse durations down to 5μs, to stimulate cochlear neurons. Compound action potentials (CAP) were evoked from the gerbil cochlea with pulse durations as short as 1μs. Data show that water absorption of light is a significant factor in optical stimulation, as evidenced by the required distance between the optical fiber and the neurons during stimulation. CAP threshold measurements indicate that there is an optimal range of pulse durations over which to deposit the laser energy, less than ~100μs. The implications of these data could direct further research and design of an optical cochlear implant.

We studied the effect of infrared (IR) stimulation on rat sensory neurons. Primary sensory neurons were prepared by enzymatic dissociation of the inferior (or "nodose") ganglia from the vagus nerves of rats. The 1.85-μm output of a diode laser, delivered through a 200-μm silica fiber, was used for photostimulation. Nodose neurons express the vanilloid receptor, TRPV1, which is a non-selective cation channel that opens in response to significant temperature jumps above 37 C. Opening TRPV1 channels allows entry of cations, including calcium (Ca²⁺), into the cell to cause membrane depolarization. Therefore, to monitor TRPV1 activation consequent to photostimulation, we used fura-2, a fluorescent Ca²⁺ indicator, to monitor the rise in intracellular Ca²⁺ concentration ([Ca²⁺]i). Brief trains of 2-msec IR pulses activated TRPV1 rapidly and reversibly, as evidenced by transient rises in [Ca²⁺]i (referred to as Ca²⁺ transients). Consistent with the Ca²⁺ transients arising from influx of Ca²⁺, identical photostimulation failed to evoke Ca²⁺ responses in the absence of extracellular Ca²⁺. Furthermore, the photo-induced Ca²⁺ signals were abolished by capsazepine, a specific blocker of TRPV1, indicating that the responses were indeed mediated by TRPV1. We discuss the feasibility of using focal IR stimulation to probe neuronal circuit properties in intact neural tissue, and compare IR stimulation with another photostimulation technique-focal photolytic release of "caged" molecules.

One drawback with traditional cochlear implants, which use electrical currents to stimulate spiral ganglion cells, is the ability to stimulate spatially discrete cells without overlap and electric current spread. We have recently demonstrated that spatially selective stimulation of the cochlea is possible with optical stimulation. However, for light to be a useful stimulation paradigm for stimulation of neurons, including cochlear implants, the neurons must be stimulated at high stimulus repetition rates. In this paper we utilize single fiber recordings from the auditory nerve to demonstrate that stimulation is possible at high repetition rates of the light pulses. Results showed that action potentials occurred 2.5-4. ms after the laser pulse. Maximum rates of discharge were up to 300 Hz. The action potentials did not respond strictly after the light pulse with high stimulation rates, i.e. >300 pulses per second. The correlation between the action potentials and the laser pulses decreased drastically for laser pulse repetition rate larger than 300 pulses per second.

Many neural disorders are associated with aberrant activity in specific cell types or neural projection pathways embedded within the densely-wired, heterogeneous matter of the brain. An ideal therapy would permit correction of activity just in specific target neurons, while leaving other neurons unaltered. Recently our lab revealed that the naturally-occurring light-activated proteins channelrhodopsin-2 (ChR2) and halorhodopsin (Halo/NpHR) can, when genetically expressed in neurons, enable them to be safely, precisely, and reversibly activated and silenced by pulses of blue and yellow light, respectively. We here describe the ability to make specific neurons in the brain light-sensitive, using a viral approach. We also reveal the design and construction of a scalable, fully-implantable optical prosthetic capable of delivering light of appropriate intensity and wavelength to targeted neurons at arbitrary 3-D locations within the brain, enabling activation and silencing of specific neuron types at multiple locations. Finally, we demonstrate control of neural activity in the cortex of the non-human primate, a key step in the translation of such technology for human clinical use. Systems for optical targeting of specific neural circuit elements may enable a new generation of high-precision therapies for brain disorders.

While recording of compound actions potentials is a mature technique, it becomes problematic to use traditional electrical nerve stimulation on very short nerves due to the large electrical stimulus artifact and short propagation times. Novel methods of stimulation using focused infrared laser radiation may permit stimulation and measurement in closer proximity with much finer spatial resolution than conventional techniques. This project involved the development of a murine sciatic nerve model for action potential stimulus and measurement in vitro, including refinement of a dissection protocol and development of a small-scale stimulation and measurement dish specific to murine sciatic nerve. It demonstrated successful recording of compound nerve action potentials (CNAPs) using traditional current-mode electrical stimulus. Lastly, optical nerve stimulation was attempted on the same nerve prep using an infrared laser source of 1.86μm. In this preliminary study, the conventional stimulus was been successfully demonstrated, however the optical stimulus failed to produce measurable CNAPs, We later learned that other investigators have only succeeded with in vivo experiments in the mouse sciatic nerve, and in vitro experiments have not been demonstrated. More work is needed to achieve success in the in vitro model.

A combination of bioinformatics, biophysical, advanced laser studies and cell biology lead to the realization that laser-pulsed UV light stops cancer growth and induces apoptosis. We have previously shown that laser-pulsed UV (LP-UV) illumination of two different skin-derived cancer cell lines both over expressing the EGF receptor, lead to arrest of the EGFR signaling pathway. We have investigated the available sequence and experimental 3D structures available in the Protein Data Bank. The EGF receptor contains a Furin like cystein rich extracellular domain. The cystein content is highly unusual, 25 disulphide bridges supports the 621 amino acid extracellular protein domain scaffold (1mb6.pdb). In two cases a tryptophan is neighboring a cystein in the primary sequence, which in itself is a rare observation. Aromatic residues is observed to be spatially close to all observed 25 disulphide bridges. The EGF receptor is often overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV-light treatment. The discovery that UV light can be used to open disulphide bridges in proteins upon illumination of nearby aromatic amino acids was the first step that lead to the hypothesis that UV light could modulate the structure and therefore the function of these key receptor proteins. The observation that membrane receptors (EGFR) contained exactly the motifs that are sensitive to UV light lead to the prediction that UV light could modify these receptors permanently and stop cancer proliferation. We hereby show that the EGFR family of receptors has the necessary structural motifs that make this family of proteins highly sensitive to UV light.

We propose the application of early state photodynamic therapy (PDT) to treatment of atrial fibrillation, which is a kind of arrhythmia characterized by irregular rapid beating of heart. We had demonstrated that our PDT can block the propagation of electrical excitation in cardiac myocytes. However, the mechanism of the PDT-induced electrical blockade was not clear. In order to clarify this mechanism, changes in intracellular Ca²⁺ concentration during the PDT with Talaporfin sodium (water soluble photosensitizer) were measured by fluorescence Ca²⁺ indicator, fluo-4 AM. The PDT led to the rapid increase of intracellular Ca²⁺ concentration and the changes in cell shapes. These results indicated that extracellular Ca²⁺ flowed into the cells mediated by cell membrane. Moreover, we found bubble generation in the cells after the PDT. In conclusion, the PDT-induced electrical blockade in myocytes can be caused by cell death following the bubble generation, which is accompanied by the increase in intracellular Ca²⁺ concentration due to the cell membrane malfunction with the PDT.

The inactivation of viruses such as M13 bateriophages subject to excitations by a very low power visible femtosecond laser has been studied. Our experimental results show that for a visible femtosecond laser having λ = 425nm and a pulse width of 100 fs, the M13 bacteriophages are inactivated when the laser power density is greater than or equal to 50 MW / cm². The functionality of M13 bacteriophages has been shown to be critically dependent on the pulse width as well as power density of the excitation laser. Our work demonstrates that by using a very low power visible femtosecond laser, it is plausible to inactivate viruses such as the M13 bacteriophages through Impulsive Stimulated Raman Scattering (ISRS) process. These experimental findings lay down the foundation for a novel new avenue of selectively inactivating microorganisms while leaving the sensitive materials unharmed by manipulating and controlling with femtosecond laser systems.

The manipulation of cells by femtosecond (fs) laser pulses became a very important tool in cell biology. In terms of learning more about the function of the cell compartments and the cell dynamics, single cell organelles are manipulated by laser pulses. Meanwhile the cell reaction is observed by different microscopy methods. The parameters of the laser irradiation have to be chosen carefully to minimize unwanted side effects during the treatment and to prevent cell damage or cell death. In many applications, it is not known what happens due to the laser irradiation on the molecular level. The formation of reactive oxygen species (ROS) is an often predicted effect due to photo disruption in biologic tissue. In this paper, we present our study of the ROS formation during the irradiation of fs laser pulses for disruption of single cell organelles. The quantity of ROS formation depends strongly on the pulse energy of the laser. Therefore the creation of ROS was additionally studied while scanning the laser at low energy for multiphoton microscopy.

We investigated the correlation between the therapeutic effect by early irradiation Photodynamic Therapy (PDT) and vascular response. The early irradiation PDT has been proposed by our group. This PDT protocol is that pulse laser irradiates to tumors 1 h after intravenous injection of water-soluble photosensitizer. The intact layer appeared over the well treated layer, when the early irradiation PDT was performed at rat prostate subcutaneous tumors with high intensity pulse laser (over 1 MW/cm² in peak intensity) and Talaporfin sodium. In order to clarify the phenomenon mechanism, we monitored blood volume, surface temperature, photosensitizer amount, and oxygen saturation during the PDT. The rat prostate subcutaneous tumor was irradiated with excimer dye laser light at 1 h after the intravenous injection. The photosensitizer dose wa 2.0 mg/kg, and the pulse energy density was 2.5 mJ/cm² (low intensity) or 10 mJ/cm² (high intensity). Under the low intensity pulsed PDT, the fluorescence amount was decreasing gently during the irradiation, and the blood volume and oxygen saturation started decreasing just after the irradiation. Under the hgh intensity pulsed PDT, the fluorescence amount was decreaased rapidly for 20 s after the irradiation started. The blood volume and oxygen saturation were temporally decreased during the irradiation, and recovered at 48 hrs after the irradiation. According to these results, under the low intensity pulsed PDT, the blood vessel located near the surface started closing just after the irradiation. On the other hand, under the high intensity pulsed PDT the blood vessel was closing for 20 s after the irradiation started, moreover, the blood flow recovered at 48 hrs after the irradiation. We concluded that the vascular response depended on the pulse energy density, and then the therapeutic effect was attributed to the difference of the vascular response. In other words, the surface intact layer could be considered to be induced the temporal drug and oxygen depletion effect associated with the temporal vascular shutdown.

A brief introduction to time-resolved and steady-state Monte Carlo modeling is presented. The methods of time-resolved and steady-state Monte Carlo simulations of photon trajectories within a tissue are presented. An example simulation using Monte Carlo Multi-Layered (mcml) demonstrates the spatial distributions of power deposition and fluence rate within a layered tissue with different optical properties (absorption, reduced scattering) in each layer.

Time varying computer models of the interaction of laser light and tissue are very valuable in helping to understand the complexity of the human body and biological tissue. The electrical property of tissue known as permittivity is vital to accurately modeling the interaction of human tissue with lasers. Past laser and RF models have represented the permittivity of the tissue as constant or temperature dependent. This paper presents time dependent permittivity that changes as a result of tissue damage, temperature, blood flow, blood vessels, and tissue property. The models are compared to emphasize the importance of accounting for these different tissue properties. In particular, incorporating the time varying nature of the permittivity of human tissue into the model leads to a significant change seen in tissue damage. An important feature of the model is the feedback loop created between the permittivity, tissue damage, and temperature. The models focus on long time duration applications where the time dependent changes significantly alter the results of the model. The applications for these models lie mostly in the therapeutic area; however, the emphasized points are of consideration for any computer model of the interaction of lasers and tissue.

We have proposed a modified Monte Carlo approach to the solution of the radiative transport equation which has the unique feature of incorporating refractive index gradients within a multi-layer biological tissue model. In the approach, photon trajectories are computed using a solution of the Eikonal equation (ray-tracing methods) rather than linear trajectories. The method can be applied to the specific problem of incorporating thermal lensing and other non-linear effects in turbid media (biological tissues) by coupling the radiative transport solution into heat-transfer and damage models. In turn, the method can be applied in the establishment of laser exposure limits for tissue-penetrating wavelengths, as well as a number of additional applications in imaging, spectroscopy and vision science.

Contained herein is a spectral analysis of Pennes' Bio-heat equation in three dimensions. Each section focuses upon a necessary step in the total solving of the differential system. Complex analysis was found necessary in resolving the inversion of the inhomogeneous term, yielding an unexpected result, where the solution space apparently is divided into two main curvatures, elliptic and hyperbolic. Each space represents a fundamental difference in heat transfer modality; hyperbolic being a reduction in magnitude, elliptic being a transfer of heat. From differential geometry, elliptic curvature complements hyperbolic spaces and the former is considered to be nested within the later.

We derive a closed-form expression for the cell radius change induced by preferential absorption of continuous wave non-ionizing radiation. The result is an approximate solution to a nonlinear ordinary differential equation. The approximation is shown to be reasonable by numerical comparison.

Assessing the biological reaction to electromagnetic (EM) radiation of all frequencies and intensities is essential to the understanding of both the potential damage caused by the radiation and the inherent mechanisms within biology that respond, protect, or propagate damage to surrounding tissues. To understand this reaction, one may model the electromagnetic irradiation of tissue phantoms according to empirically measured or intelligently estimated dielectric properties. Of interest in this study is the terahertz region (0.2-2.0 THz), ranging from millimeter to infrared waves, which has been studied only recently due to lack of efficient sources. The specific interaction between this radiation and human tissue is greatly influenced by the significant EM absorption of water across this range, which induces a pronounced heating of the tissue surface. This study compares the Monte Carlo Multi-Layer (MCML) and Finite Difference Time Domain (FDTD) approaches for modeling the terahertz irradiation of human dermal tissue. Two congruent simulations were performed on a one-dimensional tissue model with unit power intensity profile. This works aims to verify the use of either technique for modeling terahertz-tissue interaction for minimally scattering tissues.

We experimentally demonstrate that the acoustic transients propagating as a result Free-Electron Laser (FEL) ablation in brain tissue exhibit a strong FEL wavelength dependence. These acoustic transients were measured with a time-resolved, polarization quadrature laser interferometer. The transients are multiphased, with displacements of tens of microns and durations of tens of milliseconds. We calculated the Fourier transforms, power spectra, and pressure transients based on these displacement data sets. For 3.0 μm irradiation, the bandwidth of the Fourier components extends to ~20 kHz, while for 6.45 μm irradiation the bandwidth of the Fourier components extend to ~8 kHz. For the 3.0 μm irradiation, the power spectra indicate acoustic energy propagates in the bandwidth up to ~12 kHz, with structure in the 1-4 kHz range. For the 6.45 μm radiation, the mechanical power spectra indicate the acoustic energy propagates in the bandwidth up to ~7 kHz, with structure throughout. The pressure transients resulting from 3.0 μm irradiation have a leading phase with a faster onset, shorter duration, and more than ten times the peak pressure compared to that observed in pressure transients resulting from 6.45 μm irradiation. For 3.0 μm irradiation, the observed pressure transients have peak pressures in the MPa range and durations of ~1 ms, while for 6.45 μm irradiation the pressure transients have peak pressures in the 0.1 MPa range and durations of about ~3 ms.

PC12 cells, which are derived from a rat pheochromocytoma, were independently patterned utilizing an impulsive force resulting in impulsive shockwave and cavitation bubble generation by focused femtosecond laser irradiation. Since the PC12 cells respond reversibly to nerve growth factor by induction of the neuronal phenotype, we can assess an influence that the impulsive force gives to the bioactivity in term of the cell differentiation. The patterned cells were accumulated on an intact dish and cultured for 3 days. The behavior of appearance and cell differentiation was observed by multipoint time-lapse system. On bases of these results, it was proved that the biological activity of the cell is unaffected by the femtosecond laser patterning.

Photothermal-based effects in and around gold nanoparticles under action of short (nano, pico- and femtosecond) laser pulses are analyzed with focus on photoacoustic effects due to the thermal expansion of nanoparticles and liquid around them, thermal protein denaturation, explosive liquid vaporization, melting and evaporation of nanoparticle, optical breakdown initiated by nanoparticles and accompanied to shock waves and explosion (fragmentation) of gold nanoparticles. Characteristic parameters for these processes such as the temperature and laser intensity thresholds are summarized to provide basis for comparison of different mechanisms of selective nanophotothermolysis of different targets (e.g., cancer cells, bacteria, viruses, fungi, and helminths).

Herein, we demonstrate the application of high-intensity femtosecond (fs) laser pulses for performing laser surgery on the embryonic cells of developing zebrafish (Danio rerio). When fs laser pulses were focused onto individual blastomeres, transient pores were formed exposing the extracellular space to the intracellular environment. Utilizing the transient pores as a pathway for delivery of exogenous material, both chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate (FITC)). Streptavidin-conjugated quantum dots or plasmid DNA (Simian-CMV-EGFP). Both FITC and quantum dots were found to disperse throughout the blastomere cells as the embryo developed. Gene expression was seen in 24 hour post-fertilized embryos, with fluorescence observed in the notochord, floor plates, somites and tails of the larvae. We also determined the survivability of laser-manipulated embryos by rearing zebrafish from early to mid cleavage stage (2-cell to 8/16-cell) to pec-fin stage. Survival rates of 89 and 100 % were found for dechorionated and chorionated embryos, respectively.

Injection and delivery of small amount reagent in aqueous solution for cell chip was performed utilizing regeneratively amplified femtosecond laser system. In our new trial, the reagent integrated on a solid strip are released and delivered to targeted cells with the femutosecond laser-induced impulsive-force. The reagent was fixed in poly(vinyl alcohol) or polystyrene film on a glass-substrate strip. When a single pulsed femtosecond laser was focused in the solution, the film near the focal point was fragmented and the reagent was dispersed in 45-μm φ area at 50 μm from the surface of the reagent strip. As examples cardiomyocyte beating cells of P19CL6 were bombed with epinephrine and acetylcholine, and as a result the beating ratio of the cells were quickly stimulated and suppressed, respectively. The results demonstrate that the present method is a promising key nano/micro technology for diagnosis and drug discovery.

Corneal organotypic cultures were generated as per existing methods, which included growth on polycarbonate inserts and air-lifting for one week. The corneal simulant cultures were exposed, with real-time IR imaging, to the 2-μm wavelength output of a thulium fiber laser with 4 mm beam diameter for 0.25 seconds in a thermally controlled environment and then assayed for damage. The in vitro threshold (ED₅₀ value of 12.5 W/cm²) and peak temperature (74.5 °C) at threshold irradiance are compared with rabbit corneal data in the literature.

Short pulse lasers are used for a variety of therapeutic applications in medicine. Recently ultra-short pulse lasers have gained prominence due to the reduction in collateral thermal damage to surrounding healthy tissue during tissue ablation. In this paper, ultra-short pulsed laser ablation of mouse skin tissue is analyzed by assessing the extent of damage produced due to focused laser beam irradiation. The laser used for this study is a fiber-based desktop laser (Raydiance, Inc.) having a wavelength of 1552 nm and a pulse width of 1.3 ps. The laser beam is focused on the sample surface to a spot size on the order of 10 microns, thus producing high peak intensity necessary for precise clean ablation. A parametric study is performed on in vitro mouse tissue specimens and live anaesthetized mice with mammary tumors through variation of laser parameters such as time-averaged laser power, repetition rate, laser scanning rate and irradiation time. Radial temperature distribution is measured using thermal camera to analyze the heat affected zone. Temperature measurements are performed to assess the peak temperature rise attained during ablation. A detailed histological study is performed using frozen section technique to observe the nature and extent of laser-induced damages.

The Er:YAG laser has been shown to be a effective and safe tool for middle ear surgery, due to its wavelength of 2.94 μm matching a peak in the absorption spectrum of tissue. The development of a compact laser provides similar optical properties with the additional advantage of being a smaller and more flexible system. The laser-tissue interaction of the laser with porcine otic capsule bone, including photoacoustic effects and ablation characteristics are presented here and compared to those of an Er:YAG laser, to show its suitable for middle ear surgery. Photoacoustic effects were recorded using a piezo-electric film. Ablation rates were determined by mass loss per pulse and etch depth per pulse.

Laser angioplasty, for example XeCl excimer laser angioplasty, has gained more attention in addition to conventional methods of surgical and interventional treatment of atherosclerotic diseases such as bypass operation and balloon dilatation. Low degrees of thermal damage after ablation of atherosclerotic lesions have been achieved by XeCl excimer laser at 308 nm. However, in most cases, laser ablation is not selective and normal arterial wall is also damaged. To avoid complications such as severe dissections or perforation of the arterial wall in an angioplasty, a laser light source with high ablation efficiency but low arterial wall injury is desirable. At atherosclerotic lesions, cholesterol accumulates on the tunica intima by establishing an ester bond with fatty acids such as oleic acid, and thus cholesterol ester is the main component of atherosclerotic plaques. Mid-infrared pulsed laser at 5.75 μm is selectively well absorbed in C=O stretching vibration mode of ester bonds. The purpose of this study is to determine the effectiveness of nanosecond pulsed laser at 5.75 μm irradiation of cholesterol ester in atherosclerotic plaques. In this study, we used a mid-infrared tunable solid-state laser which is operated by difference frequency generation method, with a wavelength of 5.75 μm, a pulse width of 5 nsec and a pulse duration of 10 Hz. It was confirmed that non-invasive interaction to normal thoracic aortas could be induce by the parameters, the wavelength of 5.75 μm, the average power densities of 35 W/cm² and the irradiation time under 10 sec. This study shows that nanosecond pulsed laser irradiations at 5.75 μm provide an alternative laser light source as an effectively cutting, less traumatic tool for removal of atherosclerotic plaque.

Laser alignment thermal sensitive paper can be used for laser applications such as spot size measurements, beam characteristics, and determining beam mode. Thermal sensitive paper interactions with a 1.54 micron, 35 ns, 3.75 J, Er-Glass laser produced spots that had three concentric zones of response. These spots interact with each other if a minimum distance between exposures is not maintained. The distance needed between spots is directly related to the energy density incident upon the paper. Although there was no lot designation for the box of thermal sensitive papers used in this research, we were able to determine that the response of different papers in the same box could vary under constant laser exposure parameters. Therefore, investigators need to be cautious when extrapolating experimental data from exposures using this type of laser alignment paper.

We report on a novel Electric field Monte Carlo (EMC) approach for directly simulating coherent backscattering (CBS) of coherent or partially coherent polarized light. The strong dependence of CBS on the polarization state of light is first demonstrated. The penetration depth of low coherence backscattering light is then investigated using the EMC approach. EMC simulations of linearly polarized light backscattering from a human epithelial tissue model show that the penetration depth of low coherence backscattering light is reduced to the level of one scattering length under illumination by a source of an extreme low spatial coherence. The penetration depth less than one scattering length has not been obtained even when the spatial coherence length is shortened to be one percent of the scattering mean free path. It is found that the penetration depth of low coherence backscattering light may remain orders of magnitude larger than the spatial coherence length of the partially coherent source when the coherence length is shortened in an attempt to shrink the probing depth of light. This calls for a careful interpretation of the penetration depth for low coherence backscattering techniques applied to tissue diagnostics.

Polarisation-sensitive optical coherence tomography (PSOCT) is a non destructive technique with great potential for tendinopathy diagnosis. Functional optical assessment can be used in operating theatres to delineate in depth the margin of the non-healthy area, and limit the amount of tissue to be removed. A clinical study of 21 patients has been undertaken to correlate the optical properties of tendons to their clinical conditions. Tendons were scanned ex vivo with a fibre based time domain PSOCT. The beam from a superluminescent diode with a bandwidth of 52nm is sent through a polarizer and a polarizer modulator, and split into a sample and reference arm. After passing through polarization beam splitter, the interferences fringes are detected with two balanced detectors, for horizontal and vertical polarization. Scattering, birefringence and in depth stokes vectors are extracted from the measurements. Direct microstructural variation and changes in scattering properties are correlated with different tendinopathy and presence of scar tissue, which is cross-validated by histology. Lack of tissue organization, detected as the disappearance of the bands of birefringence, is representative of tendon degeneration. Special attention is paid to the difference between crimp patterns of different patient's tendons. As in polarization microscopy, the crimp pattern appears as extinction bands, and is particularly important as its alteration is generally symptomatic and could be used as an early diagnosis. Its optical origin is investigated by varying polarization and scanning conditions.

Angular Domain Imaging (ADI) is a high resolution, ballistic imaging method that utilizes the angular spectrum of photons to filter multiply-scattered photons which have a wide distribution of angles from ballistic and quasi-ballistic photons which exit a scattering medium with a small distribution of angles around their original trajectory. Such spatial gating has been previously accomplished using a scanning array of collimating holes micromachined into a silicon wafer section. We now extend that work to include using a wide-beam, full-field, converging lens and pinhole aperture system to capture images in a single exposure. We have developed an analysis of resolution and sensitivity trade-offs of such a system using Fourier optics theory to show that the system resolution is primarily governed by collimation ability at larger aperture sizes and by spatiofrequency (Fourier space-gated) filtering at smaller aperture sizes. It is found that maximum sensitivity is achieved when spatiofrequency resolution and collimation resolution are equal. Planar, high contrast, phantom test objects are observed in 5 cm thick media with effective scattered to ballistic photon ratios >1.25×10⁷:1 using a wide-beam, full-field lens and aperture system. Comparisons are made between ballistic imaging with the lens and aperture system and with the scanning silicon micromachined collimating array. Monte-Carlo simulations with angular tracking validate the experimental results.

Imaging structures within a turbid medium using Angular Domain Imaging (ADI) employs angular filter array aligned to a laser source to separate ballistic and quasi-ballistic photons from the highly scattered light by means of angular filtration. The angular filter consists of a high aspect ratio linear array of silicon micromachined tunnels, 51 micron wide by 10mm long with a 0.29 degree acceptance angle. At heavy scattering ratios of >1E7 image detectability declines due to the non-uniform scattered background light fraction still within the acceptance angle. This scattered signal can be separated out by introducing a wedge prism to deviate the laser source where it enters the medium by an angle slightly larger than the acceptance angle. This creates a second image consisting of pure scattering photons with the filtration characteristics of the angular filter, and a pixel by pixel correspondence to the fully scattered illumination emitted from the medium. Experiments used an 808 nm laser diode, collimated to an 8×1 mm line of light, entering a 5cm thick medium with a scattering ratio of > 1E6, with a wedge prism creating a 0.44 degree deviation. Digitally subtracting the deviated scattered signal from the original image significantly reduced the scattered background and enhanced image contrast. We can have about images at least 40 times more of our previous scattering limits. Depending on test phantom object location, the contrast level can be increased from 4% of the total dynamic range to over 50% which results in higher definition and visibility of our micro-scale test structures in the turbid medium.

Optical changes in skin blood flow due to the presence of glycerol were measured from a two-dimensional map of blood flow in skin blood vessels with a dynamic imaging technique using laser speckle. In this study a dorsal skin-flap window was implanted on the hamster skin with and without a hyper-osmotic agent i.e. glycerol. The hyper-osmotic drug was delivered to the skin through the open dermal end of the window model. A two-dimensional map of blood flow in skin blood vessels were obtained with very high spatial and temporal resolution by imaging the speckle pattern with a CCD camera. Preliminary studies demonstrated that hyper-osmotic agents such as glycerol not only make tissue temporarily translucent, but also reduce blood flow. The blood perfusion was measured every 3 minutes up to 36-60 minutes after diffusion of anhydrous glycerol. Small capillaries blood flow reduced significantly within 3-9 minutes. Perfusion rate in lager blood vessels i.e. all arteries and some veins decreased (speckle contrasts increased from 0.0115 to 0.384) over time. However, the blood flow in some veins reduced significantly in 36 minutes. After 24 hours the blood perfusion further reduced in capillaries. However, the blood flow increased in larger blood vessels in 24 hours compared to an hour after application of glycerol. For further investigation the speckle contrast measurement were verified with color Doppler optical coherence tomography.

Neutral density (ND) filters are frequently used to vary radiant energy and irradiance in laser-tissue interactions. In the process of preparing an optical train for exposure of tissue to near infrared laser exposure, the absorbance of various ND filters was examined. ND filters were characterized for transmittance between 500 and 2000 nanometers using a CARY-500 spectrophotometer. By characterizing the optical transmittance of the ND filters we were able to accurately predict their effect on the near infrared laser beam. The ND filters were also characterized before and after laser exposure to see if any laser-induced changes in optical density occurred.

The aim of this study consisted in evaluating the influence of low power laser on the morfometry of white adipose cells in rats. The sample consisted of 20 adult female rats, from Wistar strain, randomized in four groups. All groups were submitted to tricotomy of the dorsal thoracic area and the first three groups were exposed to a InGaAlP laser (660 nm wavelength) with fluencies of 2, 8 and 16 J/cm² for groups L1, L2 and L3, respectively. L4 was the control. The groups were submitted to the protocols three times a week, for three weeks. The irradiated tissue was excised and submitted to histological treatment by HE for optical microscopy and the Kruskal-Wallis test was used for the statistical analysis. The average morphometry, in number of pixels, was: 3741 ± 704, 3762 ±947, 3737 ±1076 and 4619 ±781 for L1, L2, L3 and L4, respectively. There was no significant difference among groups L1 to L3 for the variable tested. From these results, it can be concluded that the application of low power laser - under the conditions proposed and for the fluency values chosen - influence the morphometry of white adipose cells in rats in the same manner, producing similar results.

Several kinds of manipulation of biological cells were performed utilizing regeneratively amplified femtosecond laser system. When single-shot pulse of an amplified Ti: Sapphire femtosecond laser pulse is focused on a culture medium, shockwave and cavitation bubble are generated with little heating. An impulsive force resulting in these phenomena was applied to pttern specific cells form a culture substrate. Furthermore, laser trapping of cells was realized using high-repetition rate pulses from the laser oscillator. Although the cell was trapped stably when the laser power was less than 100 mW, the cell was burst above the threshold laser power. The bursting would be due to heating inside cell, on which the laser was focused and multiphoton absorption was induced. On the bases of these results, we propose a new methodology to pattern biological cells, which is speedy and flexible when compared with previous micropatterning methods.

Efficient DNA delivery into single living cells would be a very powerful capability for cell biologists for elucidating basic cellular functions but also in other fields such as applied drug discovery and gene therapy. The ability to gently permeate the cell membrane and introduce foreign DNA with the assistance of lasers is a powerful methodology but requires exact focusing due to the required two-photon power density. Here, we demonstrate a laser-mediated delivery method of the red fluorescent protein DS-RED into Chinese hamster Ovary (CHO) cells. We used an elongated beam of light created by a Bessel beam (BB) which obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a threshold for transfection of 20%, the BB gives us transfection over twenty times the axial distance compared to the Gaussian beam of equivalent core diameter. In addition, by exploiting the BB property of reconstruction, we demonstrate successful transfection of CHO cells which involves the BB passing through an obstructive layer and re forming itself prior to reaching the cell membrane. In the light of this exciting result, one can envisage the possibility of achieving transfection through multiple cell monolayer planes and tissues using this novel light field, eliminating this way the stringent requirements for tight focusing.

In this article we propose an approach to improve the Monte Carlo simulation accuracy by implementing a full photon path integration simulation in a non-voxelized complex three-dimensional heterogeneous model. Mouse body shape, organs optical heterogeneities and fluorophore distribution are simulated by using boundary surface elements and basic analytical shapes. In addition, external and internal surface roughness and refractive index mismatch for complex angular objects are also considered and results are briefly compared with time sampled space voxelized Monte Carlo code, in order to illustrate the impact of these improvements on the simulation results.