Growth of high spatial frequency periodic ripple structures on SiC crystal surfaces irradiated with successive femtosecond laser pulses.

We present experimentally and theoretically the evolution of high spatial frequency periodic ripples (HSFL) fabricated on SiC crystal surfaces by irradiation with femtosecond laser pulses in a vacuum chamber. At early stages the seed defects are mainly induced by laser pulse irradiation, leading to the reduction in the ablation threshold fluence. By observing the evolution of these surface structures under illumination with successive laser pulses, the nanocraters are made by nanoablation at defects in the SiC surface. The Mie scattering by the nanoablated craters grows the periodic ripples. The number of HSFL is enhanced with increasing pulse number. At the edge of the laser spot the Mie scattering process is still dominant, causing the fabrication of HSFL. On the periphery of the spot SiC substrate remains a semiconductor state because the electron density in the SiC induced by laser irradiation is kept low. The HSFL observed is very deep in the SiC surface by irradiating with many laser pulses. These experimental results are well explained by 3D FDTD (three-dimensional finite-difference time-domain) simulation.

Low-level laser therapy for spinal cord injury in rats: effects of polarization.

The effects of laser polarization on the efficacy of near-infrared low-level laser therapy for spinal cord injury (SCI) are presented. Rat spinal cords were injured with a weight-drop device, and the lesion sites were directly irradiated with a linearly polarized 808-nm diode laser positioned either perpendicular or parallel to the spine immediately after the injury and daily for five consecutive days. Functional recovery was assessed daily by an open-field test. Regardless of the polarization direction, functional scores of SCI rats that were treated with the 808-nm laser irradiation were significantly higher than those of SCI alone group (Group 1) from day 5 after injury. The locomotive function of SCI rats irradiated parallel to the spinal column (Group 3) was significantly improved from day 10 after injury, compared to SCI rats treated with the linear polarization perpendicular to the spinal column (Group 2). There were no significant differences in ATP contents in the injured tissue among the three groups. We speculate that the higher efficacy with parallel irradiation is attributable to the deeper light penetration into tissue with anisotropic scattering.

Photomechanical wave-driven delivery of siRNAs targeting intermediate filament proteins promotes functional recovery after spinal cord injury in rats.

The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.

Propagation characteristics of photomechanical waves and their application to gene delivery into deep tissue.

Targeted gene transfection can be achieved by the use of photomechanical waves (PMWs) generated by irradiating a solid material with high-power nanosecond laser pulses. To examine the treatable tissue depth, we investigated propagation characteristics of PMWs and depth-dependent properties of gene transfection with different laser fluences and spot diameters. Pressure characteristics of PMWs were measured at different propagation distances using tissue phantoms and their propagation was imaged by shadowgraphing. Phantoms with various thicknesses were placed on rat dorsal skin that had been injected with plasmid DNA coding for a reporter gene and three pulses of PMWs were applied from the top of each phantom. Significant gene expression was observed in the skin even under a 15-mm-thick tissue phantom and the depth-dependent relationships between PMW parameters and gene expression level were revealed. The data obtained will be useful for determining appropriate laser parameters for PMW-based gene transfer into deep-located tissue.

Optical fiber-based photomechanical gene transfer system for in vivo application.

We developed an optical-fiber-based photomechanical gene transfer system for endoscopic or catheter-based application. A fiber tip with a laser-absorbing film covered with a transparent plastic disk for plasma confinement was attached to a quartz fiber; the film was irradiated with nanosecond laser pulses transmitted through the fiber to generate photomechanical waves (PMWs). Characteristics of PMWs emitted from the fiber tip were examined to confirm the necessary conditions for gene transfer. We then attempted to transfer reporter genes to the rat skin as a test tissue in vivo with the fiber system, and the results showed significantly high protein levels and spatially selective pinpoint gene expressions in the tissue.

Plasmonic and Mie scattering control of far-field interference for regular ripple formation on various material substrates.

We present experimental and theoretical results on plasmonic control of far-field interference for regular ripple formation on semiconductor and metal. Experimental observation of interference ripple pattern on Si substrate originating from the gold nanosphere irradiated by femtosecond laser is presented. Gold nanosphere is found to be an origin for ripple formation. Arbitrary intensity ripple patterns are theoretically controllable by depositing desired plasmonic and Mie scattering far-field pattern generators. The plasmonic far-field generation is demonstrated not only by metallic nanostructures but also by the controlled surface structures such as ridge and trench structures on various material substrates.

Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice.

BACKGROUND AND OBJECTIVE: Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI. STUDY DESIGN/MATERIALS AND METHODS: TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm(2) for 12-minutes giving a fluence of 36-J/cm(2). Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test. RESULTS: The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests. CONCLUSION: The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.

Site-specific gene transfer into the rat spinal cord by photomechanical waves.

Nonviral, site-specific gene delivery to deep tissue is required for gene therapy of a spinal cord injury. However, an efficient method satisfying these requirements has not been established. This study demonstrates efficient and targeted gene transfer into the spinal cord by using photomechanical waves (PMWs), which were generated by irradiating a black laser absorbing rubber with 532-nm nanosecond Nd:YAG laser pulses. After a solution of plasmid DNA coding for enhanced green fluorescent protein (EGFP) or luciferase was intraparenchymally injected into the spinal cord, PMWs were applied to the target site. In the PMW application group, we observed significant EGFP gene expression in the white matter and remarkably high luciferase activity only in the spinal cord segment exposed to the PMWs. We also assessed hind limb movements 24 h after the application of PMWs based on the Basso-Beattie-Bresnahan (BBB) score to evaluate the noninvasiveness of this method. Locomotor evaluation showed no significant decrease in BBB score under optimum laser irradiation conditions. These findings demonstrated that exogenous genes can be efficiently and site-selectively delivered into the spinal cord by applying PMWs without significant locomotive damage.

Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves.

We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

In vivo photoacoustic monitoring of photosensitizer distribution in burned skin for antibacterial photodynamic therapy.

For efficient antibacterial photodynamic therapy for wounds, information on the distribution of a photosensitizer in tissue is important, but conventional fluorescence measurement does not provide depth-resolved information. We previously proposed in vivo photoacoustic (PA) depth profiling of a photosensitizer, but the contrast of PA signals was not sufficiently high, mainly due to light absorption by blood in tissue. In this study, we performed dual-wavelength PA measurement; green light and red light were used to excite blood and photosensitzer, respectively, and the former signal was subtracted from the latter signal to compensate a blood-associated component. Methylene blue or porfimer sodium solution was injected into subcutaneous tissue in rats with deep dermal burn and two-dimensional PA measurement was performed. The signal subtraction diminished not only the signal originating from blood but also the signal originating from the stratum corneum and acoustic reflection noise, creating a high-contrast PA image. The distribution of PA signals was confirmed to coincide well with the distribution of photosensitizer-originating fluorescence measured for tissue biopsied after the PA measurement, demonstrating the validity of this method for in vivo photosensitizer dosimetry. On the basis of this method, temporal behaviors of two photosensitizers were compared.

Near-field interaction of two-dimensional high-permittivity spherical particle arrays on substrate in the Mie resonance scattering domain.

We describe theoretical and experimental results on near-field interaction of two-dimensionally (2D) arrayed, high-permittivity spherical particles on a substrate in the Mie resonance scattering domain for surface nano-patterning processing. When a touching particle pair of Mie resonance particles on the substrate is considered, an electromagnetic mode different from the single particle mode is excited inside the particles, resulting in an intensity enhancement in a gap between two hotspots at particle-substrate contact points. As for 2D hexagonal close-packed particle arrays on the substrate, the refractive index of particle exhibiting a maximal enhancement factor for the 2D particle arrays is found to be shifted from the Mie resonance conditions for the single particle system.

Accelerated adhesion of grafted skin by laser-induced stress wave-based gene transfer of hepatocyte growth factor.

Gene therapy using wound healing-associated growth factor gene has received much attention as a new strategy for improving the outcome of tissue transplantation. We delivered plasmid DNA coding for human hepatocyte growth factor (hHGF) to rat free skin grafts by the use of laser-induced stress waves (LISWs); autografting was performed with the grafts. Systematic analysis was conducted to evaluate the adhesion properties of the grafted tissue; angiogenesis, cell proliferation, and reepithelialization were assessed by immunohistochemistry, and reperfusion was measured by laser Doppler imaging as a function of time after grafting. Both the level of angiogenesis on day 3 after grafting and the increased ratio of blood flow on day 4 to that on day 3 were significantly higher than those in five control groups: grafting with hHGF gene injection alone, grafting with control plasmid vector injection alone, grafting with LISW application alone, grafting with LISW application after control plasmid vector injection, and normal grafting. Reepithelialization was almost completed on day 7 even at the center of the graft with LISW application after hHGF gene injection, while it was not for the grafts of the five control groups. These findings demonstrate the validity of our LISW-based HGF gene transfection to accelerate the adhesion of grafted skins.

Photoacoustic monitoring of burn healing process in rats.

We performed multiwavelength photoacoustic (PA) measurement for extensive deep dermal burns in rats to monitor the healing process of the wounds. The PA signal peak at 532 nm, an isosbestic point for oxyhemoglobin (HbO(2)) and deoxyhemoglobin (HHb), was found to shift to a shallower region of the injured skin tissue with the elapse of time. The results of histological analysis showed that the shift of the PA signal reflected angiogenesis in the wounds. Until 24 h postburn, PA signal amplitude generally increased at all wavelengths. We speculate that this increase in amplitude is associated with dilation of blood vessels within healthy tissue under the injured tissue layer and increased hematocrit value due to development of edema. From 24 to 48 h postburn, the PA signal showed wavelength-dependent behaviors; signal amplitudes at 532, 556, and 576 nm continued to increase, while amplitude at 600 nm, an HHb absorption-dominant wavelength, decreased. This seems to reflect change from shock phase to hyperdynamic state in the rat; in the hyperdynamic state, cardiac output and oxygen consumption increased considerably. These findings show that multiwavelength PA measurement would be useful for monitoring recovery of perfusion and change in local hemodynamics in the healing process of burns.

Enhanced angiogenesis in grafted skins by laser-induced stress wave-assisted gene transfer of hepatocyte growth factor.

Treatment to increase secretion of growth factors related to angiogenesis by gene transfection is a promising therapeutic solution for improving the outcome of tissue transplantation. We attempted to deliver a therapeutic vector construct carrying the human hepatocyte growth factor (hHGF) gene to skin grafts of rats using laser-induced stress waves (LISWs), with the objective of enhancing their adhesion. First we delivered the hHGF gene to rat native skin in vivo to determine the optimum gene transfer conditions. We then transferred the hHGF gene to excised rat skins, with which autografting was performed. We found that the density and uniformity of neovascularities were significantly enhanced in the grafted skins that were transfected using LISWs. These results suggest the efficacy of this method to improve the outcome of skin grafting. To our knowledge, this is the first experimental demonstration of a therapeutic efficacy based on LISW-mediated gene transfection. Since the present method can be applied not only to various types of tissues but also to bioengineered tissues, this technique has the potential to contribute to progress in transplantation medicine and future regenerative medicine.

Accelerated binocular rivalry with anxious personality.

When dissimilar figures are presented to the two eyes individually, perception alternates spontaneously between each monocular view. This phenomenon, designated binocular rivalry, has been used by many scientists as a tool for investigating visual awareness. Some recent studies have suggested involvement of serotonergic neural systems in this phenomenon. We explored the relationship between binocular rivalry and anxiety, a state thought to be associated with serotonergic neural activity. Perceptual alternation rate in dominance of binocular rivalry were compared between normal volunteers with high and low general anxiety as measured using the harm avoidance (HA) scale of the Tridimensional Personality Questionnaire. Perceptual alternation rate and rhythmicity in the high-HA group was significantly greater than that in the low-HA group. The perceptual alternation rate in binocular rivalry was accelerated in anxious subjects, suggesting that binocular rivalry and anxiety may share some common serotonergic neural substrates.

In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: effects of stress wave parameters, ambient temperature, and cell type.

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

Photoacoustic monitoring of neovascularities in grafted skin.

BACKGROUND AND OBJECTIVE: In skin grafting, evaluation of graft adhesion to the recipient site in the early postgrafting period is important. However, conventional diagnoses such as visual observation and thermography required about 1 week to obtain results and these methods cannot give quantitative information on the adhesion of a skin graft. We proposed a new method for monitoring adhesion of grafted skin that is based on measurement of photoacoustic signals. To investigate the validity of the method, we performed experiments using rat autografts models. STUDY DESIGN/MATERIALS AND METHODS: Grafted skin in a rat was irradiated with 200 microJ, 532-nm nanosecond laser pulses, and photoacoustic signals were detected with a piezoelectric transducer placed on the skin at various postgrafting time. Temporal profiles of the signals were converted to depth profiles using an assumed sound velocity of 1,500 m/second. Histological analysis was performed to observe neovascularities formed in the grafts. RESULTS: At 6 hours postgrafting, a photoacoustic signal peak appeared in the depth region corresponding to the graft. The results of histological analysis also showed formation of neovascularities in the graft after 6 hours postgrafting, indicating that photoacoustic signal peaks observed in the graft originated from the neovascularities, which are an indication of graft adhesion. For up to 24 hours postgrafting, no significant difference was observed between the results of visual observation and laser Doppler imaging of the same grafted skins. CONCLUSION: We have demonstrated that photoacoustic signals originating from neovascularities in grafts can be sensitively detected in the early postgrafting period, suggesting the validity of photoacoustic measurement for adhesion monitoring of skin grafts.

Evaluation of patello-femoral alignment using MRI.

In evaluating patello-femoral alignment, it is not enough to assess the conformity of the joint two-dimensionally, the direction of the extensor mechanism must be examined three-dimensionally. Using magnetic resonance imaging (MRI), we adopted an ideal extensor mechanism plane intersecting the patellar facet of the femur centrally and perpendicularly. We evaluated the alignment according to the extent to which the patella and/or the tibial tuberosity deviated from the plane. The results suggested that our method is useful for indicating proximal and distal realignment. Furthermore, two-dimensional finite element analysis in the patello-femoral joint showed that peak stress was significantly higher in the dislocation group than in the no-dislocation group. This indicates that osteoarthritic change in the future is a risk in the dislocation group.

In vivo scattering measurement of biological tissue by the use of a pyroelectric polymer transducer.

We propose a new method, to our knowledge, for noninvasive scattering measurements of tissues by the use of a pyroelectric polymer transducer, poly(vinylidene fluoride trifluoroethylene) film. In this method, samples are irradiated with nanosecond, low-energy light pulses delivered from an optical fiber, and the pyroelectric signal induced by the diffuse reflectance is measured with a transducer. The signal is then converted into diffuse reflectance by use of a calibration factor. The validity of this method was confirmed by our measuring the scattering coefficient of a white acrylic resin within an accuracy of +/- 15%. We attempted to apply this method to the estimation of scattering coefficients of normal and burned skins in rats in vivo.

Photoacoustic diagnosis of burns in rats.

BACKGROUND: Accurate burn depth assessment is important for determining the appropriate treatment plan for severe burn patients. However, conventional methods of diagnoses, such as visual observation and pinprick test, are often inaccurate. We previously proposed a new method for burn diagnosis in which photoacoustic signals originating from the blood in healthy tissue under the injured tissue are measured. In this study, we investigated the validity of this method by an experiment using rat scald burn models. METHODS: Superficial dermal burns (SDBs), deep dermal burns (DDBs), and deep burns (DBs) were made in the dorsal skin of rats by using a Walker-Mason template. Wounds were irradiated with low-energy, 550-nm, nanosecond pulsed light, and photoacoustic signals induced were measured with a piezoelectric film as a function of postburn time. Measurement in normal skin as a control was also performed. Temporal profiles of the photoacoustic signals were converted into depth profiles using sound velocity of tissue, and for each profile, a peak showing highest signal intensity was selected. For this peak, the depth at which the signal rose (signal rise depth) and the depth that gave a peak value (signal peak depth) were recorded. Statistical analysis was performed to clarify the difference in depth information of signals between burn groups. RESULTS: Depth profiles of photoacoustic signals showed unique features depending on the degree of burn; pronounced peaks shifted to deeper tissue as the burn severity increased. This indicates that the zone of stasis formed due to injuries can be monitored. There were significant differences in both the signal rise depth and the peak depth between the control and SDB groups, SDB and DDB groups, and DDB and DB groups (p < 0.001). CONCLUSION: SDBs, DDBs, and DBs can be differentiated by photoacoustic signals, suggesting that the method proposed is useful for diagnosing burn injuries.