Photonic Therapeutics and Diagnostics VII

Hemispherical Stokes polarimeter for early cancer diagnosis

Paul Lemaillet, Jessica C. Ramella-Roman

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Optimal treatment of skin cancer before it reaches metastasis depends critically on early diagnosis of the melanoma. Valuable information for this diagnosis can be obtained from the analysis of skin roughness. This information can aid in determining the necessity for skin removal. For this purpose, we developed a hemispherical imaging Stokes polarimeter designed to monitor skin cancer based on a roughness assessment of the epidermis. Our setup is composed of 16 out-of-plane polarized light illuminations tubes that contain a three color LED and a vertical polarizer, a Stokes polarimeter that contains 2 liquid crystal retarders, a reference vertical polarizer and a fast acquisition camera. The Stokes polarimeter was calibrated using a set of well-known input polarization states. Each illumination polarizer was positioned using a roughness gold standard and a facet model describing the principal angle of polarization of the analyzed light as a function of the angle of incidence. A set of phantoms mimicking the optical properties of skin at 633 nm as well as skin roughness was built using wax as the bulk material, titanium dioxide as the scatterer and a black dye as the absorber. Images of these phantoms are presented and they are analyzed using a facet model.

Comparison of various methods to enhance laser photon density in soft tissue: tissue temperature, laser pulse modulation, glycerol, and their combination

Changmin Yeo, Heesung Kang, Hunjeong Park, et al.

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Recently, tissue optical clearing (TOC) has been considered as a useful tool in low level laser therapy due to the enhancement of photon density in deep tissue layer. In this study, glycerol injection, tissue temperature, laser pulse modulation, and their combination methods were investigated and compared by analyzing 2D and 3D laser beam profile. A thermal plate was built to control tissue temperature from 40°C through 10°C at 10°C decrement. A continuous laser of 660 nm was modulated at the frequencies of 1, 10, 25, and 50 Hz. 95% glycerol was injected into a region of interest of sample where laser is irradiated and its effect was analyzed after 5 min. Finally, their combination method was evaluated. Analysis was performed with the diffusion images acquired by CCD and the optical properties measured by double integrating sphere. Results demonstrated that average peak intensity of laser beam profile was 1) 1.57-fold higher at 10°C than 40°C, 2) 1.79-fold higher at 10 Hz than continuous wave, 3) 1.65-fold higher with 95% glycerol injection than no glycerol application, and 4) 2.52-fold higher at the combination method than independent methods. Average total intensity at FWHM was 1) 1.44-fold higher with tissue cooling, 2) 1.71-fold higher at 10 Hz, 3) 1.61-fold higher with glycerol injection, and 4) 2.19-fold higher with the combination method. In conclusion, this study implies that tissuecooling, pulse modulation, glycerol injection, and their combination method can effectively deliver laser photon in LLLT by enhancing the photon density in soft tissue.

UV doses and skin effects during psoriasis climate therapy

Lise L. Randeberg, Julio Hernandez-Palacios, Mila Lilleeng, et al.

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Psoriasis is a common autoimmune disease with inflammatory symptoms affecting skin and joints. One way of dealing with psoriasis is by controlled solar UV exposure treatment. However, this treatment should be optimized to get the best possible treatment effect and to limit negative side effects such as erythema and an increased risk of skin cancer. In this study 24 patients at Valle Marina Treatment Center in Gran Canaria were monitored throughout a treatment period of three weeks starting at the beginning of November. The total UV dose to the location was monitored by UV-meters placed on the roof of the treatment centere, and the patients wore individual film dosimeters throughout the treatment period. Skin parameters were accessed by reflection spectroscopy (400-850nm). This paper presents preliminary findings from the skin measurements in the visible part of the spectrum, such as blood oxygenation, erythema and melanin indexes. Reflection spectroscopy was found to be a good tool for such treatment monitoring.

Monitoring human melanocytic cell responses to piperine using multispectral imaging

Ravikant Samatham, Kevin G. Phillips, Julia Sonka, et al.

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Vitiligo is a depigmentary disease characterized by melanocyte loss attributed most commonly to autoimmune mechanisms. Currently vitiligo has a high incidence (1% worldwide) but a poor set of treatment options. Piperine, a compound found in black pepper, is a potential treatment for the depigmentary skin disease vitiligo, due to its ability to stimulate mouse epidermal melanocyte proliferation in vitro and in vivo. The present study investigates the use of multispectral imaging and an image processing technique based on local contrast to quantify the stimulatory effects of piperine on human melanocyte proliferation in reconstructed epidermis. We demonstrate the ability of the imaging method to quantify increased pigmentation in response to piperine treatment. The quantization of melanocyte stimulation by the proposed imaging technique illustrates the potential use of this technology to quickly assess therapeutic responses of vitiligo tissue culture models to treatment non-invasively.

Fluorescence lifetime imaging of skin cancer

Rakesh Patalay, Clifford Talbot, Ian Munro, et al.

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Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Collateral damage-free debridement using 193nm ArF laser

James J. Wynne, Jerome M. Felsenstein, Robert Trzcinski, et al.

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Burn eschar and other necrotic areas of the skin and soft tissue are anhydrous compared to the underlying viable tissue. A 193 nm ArF excimer laser, emitting electromagnetic radiation at 6.4 eV at fluence exceeding the ablation threshold, will debride such necrotic areas. Because such radiation is strongly absorbed by aqueous chloride ions through the nonthermal process of electron photodetachment, debridement will cease when hydrated (with chloride ions) viable tissue is exposed, avoiding collateral damage to this tissue. Such tissue will be sterile and ready for further treatment, such as a wound dressing and/or a skin graft.

High-resolution multimodal clinical multiphoton tomography of skin

Karsten König

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This review focuses on multimodal multiphoton tomography based on near infrared femtosecond lasers. Clinical multiphoton tomographs for 3D high-resolution in vivo imaging have been placed into the market several years ago. The second generation of this Prism-Award winning High-Tech skin imaging tool (MPTflex) was introduced in 2010. The same year, the world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph. In particular, non-fluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen has been imaged with submicron resolution in patients suffering from psoriasis. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution wide-field systems such as ultrasound, optoacoustical, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer, optimization of treatment strategies, and cosmetic research including long-term testing of sunscreen nanoparticles as well as anti-aging products.

Comparison of skin responses from macroscopic and microscopic UV challenges

InSeok Seo, Paulo R. Bargo, Melissa Chu, et al.

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The minimal erythema dose induced by solar-simulated radiation is a useful measure of UV sensitivity of skin. Most skin phototests have been conducted by projecting a flat field of UV radiation onto the skin in an area greater than 15 cm × 15 cm with an increment of radiation doses. In this study, we investigated the responses of human skin to solar-simulated radiation of different field sizes. Twelve human subjects of skin phototype I-IV were exposed to solar-simulated radiation (SSR) on their upper inner arm or on their lower back with a series of doses in increments of 20% in order to determine the threshold dose to induce a minimal perceptible erythema response (MED). Each dose was delivered with a liquid light guide (8 mm diameter on the back or 6 mm on the upper inner arm) and with quartz optical fibers of 200 μm diameter. The resulting skin responses were evaluated visually and investigated with a reflectance confocal microscope and imaging. The erythema response to the microscopic challenge was always diffuse with no clear boundaries extending to several times the exposed site diameter at doses greater than 2 MED. The skin returned to normal appearance from the microscopic challenge after two weeks of exposure while change in appearance for the larger areas persisted for several weeks to months. This new modality of testing provides the possibility to study skin at the microscopic level with a rapid recovery following challenge.

Near-infrared laser treatment of complicated hemangiomas in children: ten-year clinical experience

Ivan A. Abushkin, Valeriy A. Privalov, Alexander V. Lappa

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Results of application of low invasive laser technology (developed by authors: Proc. SPIE 5863, 107-115 (2005), Russian Federation patent No.2290228 of.27.12.06) to treatment of hemangiomas in children are presented and analyzed in this work. From 2001 the technology was applied to about 1500 children with more than 2000 hemangiomas. Majority of them were complicated ones: belong to cavernous or combined types or (and) were localized on problem places: on face near eyes, nose, and lips, on auricles, on perineum near anus and genitals, in respiratory and gastrointestinal tracts. Diode laser with wavelength 920, 970, and 1060 nm at distant and interstitial irradiation were applied. In case of need there applied endoscopes. Excellent and good results have been achieved in 94% cases; there was a significant improvement in the rest cases.

Assessing topographic cutaneous autofluorescence variation using fluorescence UV and visible excitation emission matrix (EEM) spectroscopy

Jianhua Zhao, Soodabeh Zandi, Florina Feng, et al.

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Cutaneous autofluorescence properties were systematically studied using fluorescence excitation emission matrix spectroscopy. Twenty-six healthy subjects with a mean age of 34 (range 21-74) participated in this study. The fluorescence of major skin fluorophores such as tryptophan, collagen, elastin and NADH could be readily identified. On average, facial skin shows strong tryptophan and measurable porphyrin fluorescence; the palm and nail show strong tryptophan and keratin fluorescence. These results demonstrate that regional topographic variations exist not only in the amount of fluorescence but also in the relative distribution of fluorophores in normal skin. Moreover this provides a basis for future interpretation of autofluorescence in diseased skin.

Next generation Er:YAG fractional ablative laser

A. Heinrich, A. Vizhanyo, P. Krammer, et al.

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Pantec Biosolutions AG presents a portable fractional ablative laser system based on a miniaturized diode pumped Er:YAG laser. The system can operate at repetition rates up to 500 Hz and has an incorporated beam deflection unit. It is smaller, lighter and cost efficient compared to systems based on lamp pumped Er:YAG lasers and incorporates a skin layer detection to guarantee precise control of the microporation process. The pulse parameters enable a variety of applications in dermatology and in general medicine, as demonstrated by first results on transdermal drug delivery of FSH (follicle stimulating hormone).

Low-cost/high-efficiency lasers for medical applications in the 14XX-nm regime

J. J. Callahan, E. McIntyre, C. Rafferty, et al.

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Laser therapy is becoming an increasingly popular method of treating numerous dermatological conditions. The widespread use of these devices is often limited by the cost and size. Low cost, portable lasers would expand the laser market further into homes, general practitioners, dermatologists, plastic surgeons, and 3rd world countries. There are numerous light devices currently on the market for hair removal and growth, acne reduction, and wrinkles. In this paper, those efforts being made to develop manufacturing partners to lower the cost while increasing the production volume of long wavelength lasers will be discussed along with performance data and clinical results.

Motion correction in spatial frequency domain imaging; optical property determination in pigmented lesions

John Quan Nguyen, Rolf B. Saager, David J. Cuccia, et al.

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Background and Objective: Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field optical imaging technology currently being used to study the optical properties and chromophore concentrations of in-vivo malignant melanomas and benign pigmented lesions. Our objective is to develop a motion correction procedure in order to assess the concerns of subject-motion related variables during clinical measurements. Study Design/Materials and Methods: SFDI motion-correction is a two-part procedure which utilizes a fiduciary marker and canny-edge detection in order to reposition and align the frame-to-frame regions-of-interest (ROI). Motioninduced phase-shifts are subsequently sampled before the entire image-set is processed by a modified demodulation formula. By comparing the results of the adjusted processing method with data gathered from the current non-corrected method, we were able to systematically characterize the impact of motion variables on SFDI measurements. Results: Motion-corrected SFDI data from moving phantom measurements and clinical patient measurements showed up to 84.58% decrease in absorption (μa) variance and up to 92.63% decrease in reduced-scattering (μs') variance. Stationary phantom test-measurements showed almost no difference between motion corrected and standard processing. Conclusion: SFDI motion correction is necessary for obtaining high-fidelity in-vivo optical property measurements of pigmented lesions in a clinical setting.

In vivo analysis of human skin anisotropy by polarization-sensitive optical coherence tomography

Shingo Sakai, Masahiro Yamanari, Yiheng Lim, et al.

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Skin anisotropy is an important issue for plastic surgeons and cosmetics science. Cleavage lines, such as Langer's lines and relaxed skin tension lines (RSTLs), have been proposed as keys to understanding skin anisotropy. Collagen, a dominant dermal structural protein, forms a fibrous structure believed to play an important role in skin anisotropy. There have been few reports, however, on the relationship between the orientation of collagen fiber and the direction of the cleavage line. Collagen fiber has birefringence, a property analyzable in skin in three dimensions by high-speed polarization-sensitive optical coherence tomography (PS-OCT). Here we used PS-OCT for an in vivo analysis of anisotropic changes in the dermal birefringence of mechanically deformed human skin. The dermal birefringence of the forehead increased significantly when the skin was shrunk perpendicular to the RSTL and increased significantly when the skin was shrunk parallel to the RSTL. En-face images of dermal birefringence revealed that both shrinking perpendicular to and stretching in parallel to the RSTL promoted the formation of a macro rope-like collagen structure. Moreover, the birefringent change under shrinking conditions perpendicular to the RSTL showed negative correlation to Ra, a skin roughness parameter. These results suggest that PS-OCT enables the in vivo evaluation of skin anisotropy.

Intravital multiphoton tomography as an appropriate tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

Volker Huck, Christian Gorzelanny, Kai Thomas, et al.

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Increasing incidence of inflammatory skin diseases such as Atopic Dermatitis (AD) has been noted in the past years. According to recent estimations around 15% of newborn subjects are affected with a disease severity that requires medical treatment. Although its pathogenesis is multifactorial, recent reports indicate that an impaired physical skin barrier predispose for the development of AD. The major part of this barrier is formed by the stratum corneum (SC) wherein corneocytes are embedded in a complex matrix of proteins and lipids. Its components were synthesized in the stratum granulosum (SG) and secreted via lamellar bodies at the SC/SG interface. Within a clinical in vivo study we focused on the skin metabolism at the SC/SG interface in AD affected patients in comparison to healthy subjects. Measurement of fluorescence life-time of NADH provides access to the metabolic state of skin. Due to the application of a 5D intravital tomographic skin analysis we facilitate the non-invasive investigation of human epidermis in the longitudinal course of AD therapy. We could ascertain by blinded analysis of 40 skin areas of 20 patients in a three month follow-up that the metabolic status at the SC/SG interface was altered in AD compromised skin even in non-lesional, apparent healthy skin regions. This illustrates an impaired skin barrier formation even at non-affected skin of AD subjects appearing promotive for the development of acute skin inflammation. Therefore, our findings allow a deeper understanding of the individual disease development and the improved management of the therapeutic intervention in clinical application.

In vivo investigation of the evolution of skin barrier repair after mechanical injury

Steven Walston, Melissa Chu, Inseok Seo, et al.

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The stratum corneum (SC) serves a primary function of skin barrier and its maintenance is vital for the existence of terrestrial life. Few studies have been performed for evaluation of human SC repair in vivo, non-invasively. In the present study tape stripping was performed on the arms and legs of seven volunteers until all the SC was removed. The injured site and a control adjacent site were measured over a period of 10 days after the injury to assess functionality and repair. Transepidermal water loss (TEWL), tryptophan fluorescence and reflectance confocal microscopy were used to determine permeability of the skin barrier, cell turnover and epidermis morphology, respectively. The results show an exponential rate of recovery for the skin permeability (TEWL) which contrasted with a linear increase in the thickness of the SC as determined by confocal microscopy. Cell turnover increased rapidly immediately after the injury to 2.5 times the levels of the control site, attaining a maximum of 3.5-4 times greater levels after three days and slowly returned to baseline levels after the ten days. Correlation of the cell turnover to the thickness of the viable epidermis was observed and further studies are under way to interpret these results.

DNA-RNA, DNA-DNA, DNA-protein and protein-protein interactions in diagnosis of skin cancers by FT-IR microspectroscopy

Natalja Skrebova Eikje

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Skin tissue infrared (IR) microspectroscopy may work as an optical diagnostic method for common skin cancer detection, progression, and specific characterization of carcinogenesis in skin tumours. Spectral results from BCC, SCC and MM skin samples demonstrated significant levels of the multiplet at about 1055 cm^-1. Its activity level strongly correlated with the activity level of the most prominent peak in DNA/RNA triad in 5 BCC, in 3 SCC and only in 1 MM patient. DNA-RNA and DNA-DNA interactions as I₉₆₅ < I₁₀₅₅ < I_{max level DNA/RNA triad peak} were the most clearly observed in 3 BCC patients with high-leveled peaks of nucleic acids, that were presented as I₉₆₅ < I₁₀₅₅ > I_{max level DNA/RNA triad peak} in 3 SCC patients with the highest activity levels. Although mean values and the intensities of nucleic acids in the patients with MM showed strong correlation between each other, independent on the level of their activity, interactions differed individually. DNA-protein interactions were mostly expressed between intensities of DNA/RNA triad peaks and non-descriptive proteins, that were in agreement between BCC and SCC, but not in MM. Protein-protein interactions were similar among the patients, generally indicating the grade of activity in cells in tissues.

In vivo multiphoton imaging of collagen remodeling after microablative fractional rejuvenation

Riccardo Cicchi, Dimitrios Kapsokalyvas, Michela Troiano, et al.

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The potential of multiphoton microscopy in providing in-vivo early diagnosis of skin lesions has already been demonstrated, while its capability in therapy follow-up has not been deeply explored so far. Two-photon excited fluorescence and second-harmonic generation microscopy were used in combination to follow-up collagen remodeling after laser micro-ablative rejuvenation. Treated regions of volunteers were imaged with multiphoton microscopy before and after treatment, and we found a strong age-dependence of the treatment effectiveness. In particular, the photorejuvenating effect was negligible in young subjects (< 30 years), whereas a significant production of new collagen was observed in aged subjects (> 70 years). Quantification of the amount of newly produced collagen and its organization were performed by means of visual examination of two-photon images. The obtained results demonstrate the performance of laser fractional micro-ablative rejuvenation without the need of an invasive biopsy as well as the wide applicability range of applications for multiphoton microscopy in clinical dermatology.

Dual-effect laser handpiece for modification of tissue permeability

Kathleen McMillan

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A new approach for improving the availability of topically applied drugs by reducing the permeability of dermis has been evaluated. The premise of this work is that photothermal vascular injury will reduce vascular uptake of drug in the dermis. The dermal distribution of two topically applied drugs, 5-fluorouracil and mitomycin C, is calculated, considering molecular diffusion and vascular uptake according to a distributed model, in the presence and absence of vascular injury. Intradermal drug exposures obtained are compared to exposures known to be effective in killing tumor cells. Combining the reduction in dermal permeability with fractional photothermal epidermal ablation to increase epidermal permeability may allow higher drug concentrations to be achieved in the skin. A newly developed laser handpiece for implementing the technique is described.

In-vivo optical investigation of psoriasis

Dimitrios Kapsokalyvas, Riccardo Cicchi, Nicola Bruscino, et al.

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Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Cases of psoriasis were investigated in vivo with optical means in order to evaluate the potential of in vivo optical biopsy. A Polarization Multispectral Dermoscope was employed for the macroscopic observation. Features such as the 'dotted' blood vessels pattern was observed with high contrast. The average size of dot vessels in Psoriasis was measured to be 974 μm² which is much higher compared to healthy skin. High resolution image sections of the epidermis and the dermis were produced with a custom made Multiphoton Microscope. Imaging extended from the surface of the lesion down to the papillary dermis, at a depth of 200 μm. In the epidermis, a characteristic morphology of the stratum corneum found only in Psoriasis was revealed. Additionally, the cytoplasmic area of the cells in the stratum spinosum layer was found to be smaller than normal. In the dermis the morphological features were more pronounced, where the elongated dermal papillae dominated the papillary layer. Their length exceeds 100μm, which is a far greater value compared to that of healthy skin. These in vivo observations are consistent with the ex vivo histopathological observations, supporting both the applicability and potentiality of multispectral dermoscopy and multiphoton microscopy in the field of in vivo optical investigation and biopsy of skin.

Improvement of in vivo rat skin optical clearing with chemical penetration enhancers

Jing Wang, Xue Zhou, Shu Duan, et al.

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Optical method plays an important role in clinical diagnosis and treatment, but suffers from limited penetration depth of light in turbid tissue. The optical clearing technique can improve the light delivery significantly through immersion of tissues into Optical Clearing Agents (OCAs). However, the barrier function of stratum corneum makes it difficult for optical clearing of skin by topical application of OCAs. Addition of penetration enhancers to OCAs can improve the skin clearing efficacy, but most investigations were performed on in vitro skin. Here, to evaluate the efficacy of this method on in vivo skin, direct observation and measurement of diffuse reflectance spectra were performed after topical application of different mixtures. One OCA, PEG-400, and three penetration enhancers (PEs), Thiazone, Azone and Propylene Glycol (PG), were used. The results indicated that the addition of penetration enhancers could improve the optical clearing efficacy of rat skin in vivo significantly, the dermal blood vessels could be observed directly with PEs. Among the three penetration enhancers, Thiazone induced the largest enhancement of clearing efficacy, and the enhancement induced by PG is the least. This study is very helpful for in vivo application of OCAs to enhance skin optical clearing non- invasively.

Raman spectra and optical coherent tomography images of skin

A. E. Villanueva-Luna, J. Castro-Ramos, S. Vazquez-Montiel, et al.

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The optical coherence tomography images are useful to see the internal profile and the structure of material samples. In this work, OCT images were recorded in 10 volunteers with different skin tone which were related to Raman spectra. The areas where we obtained OCT images and Raman spectra were a) index finger nail, b) between index finger and middle finger, c) middle finger tip, d) half of middle finger, e) the thumb finger tip and f) between index finger and thumb, areas measured were for the purpose of finding extracellular fluids with contain triglycerides, cholesterol and glucose that are reported in the literature. The excitation wavelength used for this work was 785 nm, a spectrometer of 6 cm^-1 resolution. The spectral region used ranges from 300 to 1800 cm^-1. We use an OCT with 930 nm of Central Wavelength, 1.6 mm of Image Depth, 6 mm of image width and 6.2 μm of axial resolution.

Spectral characteristics of two-photon autofluorescence and second harmonic generation from human skin in vivo

Hans Georg Breunig, Karsten König

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We performed multiphoton imaging of human skin and recorded in combination the complete spectral content of the signals in vivo. The spectra represent the integration of multiphoton signals over the investigated regions of the epidermis and dermis. They are used to study depth-resolved in vivo emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, collagen and elastin. The identification of the specific fluorophores is supported by analysis of additional in vivo fluorescence lifetime imaging. Furthermore, as a potential application of spectrally selective imaging the possibility to investigate the penetration of nanoparticles from sunscreen lotion into skin in vivo is discussed.

Multi-spectral imaging analysis of pigmented and vascular skin lesions: results of a clinical trial

Ilona Kuzmina, Ilze Diebele, Lauma Valeine, et al.

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A clinical trial comprising 266 pigmented lesions and 49 vascular lesions has been performed in three Riga clinics by means of multi-spectral imaging analysis. The imaging system Nuance 2.4 (CRI) and self-developed software for mapping of the main skin chromophores were used. The obtained results confirm clinical potential of this technology for non-contact quantitative assessment of skin pathologies.

Fourier domain versus time domain optical coherence tomography of the prostate nerves

Shahab Chitchian, Gwen Lagoda, Arthur Burnett, et al.

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Theoretical comparisons of detection performance for Fourier domain (FD) and time domain (TD) optical coherence tomography (OCT) have been previously reported. In this study, we compare several image quality metrics including signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and equivalent number of looks (ENL) for TD-OCT and FD-OCT images taken of the rat prostate, in vivo. The results show that TD-OCT has inferior CNR, but superior SNR compared to FD-OCT, and that TD-OCT is better for deep imaging of opaque tissues, including the prostate gland.

Holmium:YAG (wavelength=2120 nm) vs. Thulium fiber laser (wavelength=1908 nm) ablation of kidney stones: thresholds, rates, and retropulsion

Richard L. Blackmon, Pierce B. Irby, Nathaniel M. Fried

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The Holmium:YAG (Ho:YAG) laser lithotriptor is capable of operating at high pulse energies, but its efficient operation is limited to relatively low pulse rates (~10 Hz) during lithotripsy. On the contrary, the Thulium Fiber Laser (TFL) is limited to low pulse energies, but can operate at very high pulse rates (up to 1000 Hz). This study compares stone ablation threshold, ablation rate, and retropulsion effects for different Ho:YAG and TFL operation modes. The TFL (λ=1908 nm) was operated with pulse energies of 5-35 mJ, 500-μs pulse duration, and pulse rates of 10-400 Hz. The Ho:YAG laser (λ=2120 nm) was operated with pulse energies of 30-550 mJ, 350-μs pulse duration, and pulse rate of 10 Hz. Laser energy was delivered through small-core (200-270-μm) optical fibers in contact mode with human calcium oxalate monohydrate (COM) stones for ablation studies and plaster-of-Paris stone phantoms for retropulsion studies. The COM stone ablation threshold for Ho:YAG and TFL measured 82.6 J/cm²and 20.8 J/cm², respectively. Stone retropulsion with Ho:YAG laser increased linearly with pulse energy. Retropulsion with TFL was minimal at pulse rates < 150 Hz, then rapidly increased at higher pulse rates. For minimal stone retropulsion, Ho:YAG operation at pulse energies < 175 mJ at 10 Hz, and TFL operation at 35 mJ at 100 Hz is recommended, with both lasers producing comparable ablation rates. Further development of a TFL operating with both high pulse energies (e.g. 100-200 mJ) and high pulse rates (100-150 Hz) may also provide higher ablation rates, when retropulsion is not the primary concern.

Continuous-wave vs. pulsed infrared laser stimulation of the rat prostate cavernous nerves

Serhat Tozburun, Christopher M. Cilip, Gwen A. Lagoda, et al.

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Optical nerve stimulation has recently been developed as an alternative to electrical nerve stimulation. However, recent studies have focused primarily on pulsed delivery of the laser radiation and at relatively low pulse rates. The objective of this study is to demonstrate faster optical stimulation of the prostate cavernous nerves using continuouswave (CW) infrared laser radiation, for potential diagnostic applications. A Thulium fiber laser (λ = 1870 nm) was used for non-contact optical stimulation of the rat prostate cavernous nerves, in vivo. Optical nerve stimulation, as measured by an intracavernous pressure (ICP) response in the penis, was achieved with the laser operating in either CW mode, or with a 5-ms pulse duration at 10, 20, 30, 40, 50, and 100 Hz. Successful optical stimulation was observed to be primarily dependent on a threshold nerve temperature (42-45 °C), not an incident fluence, as previously reported. CW optical nerve stimulation provides a significantly faster ICP response time using a laser with lower power output than pulsed stimulation. CW optical nerve stimulation may therefore represent an alternative mode of stimulation for intra-operative diagnostic applications where a rapid response is critical, such as identification of the cavernous nerves during prostate cancer surgery.

Comparison of 808, 980, and 1075nm lasers for noninvasive thermal coagulation of the canine vas deferens, ex vivo

Christopher M. Cilip, Gino R. Schweinsberger, Nathaniel M. Fried

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Successful noninvasive laser coagulation of the canine vas deferens, in vivo, has been previously reported. However, the therapeutic window for treatment is relatively narrow. This study determines the dependence of vas thermal coagulation on laser wavelength for development of a noninvasive laser vasectomy procedure. Noninvasive laser coagulation of canine vas tissue, ex vivo, was performed using three commonly available near-infrared laser wavelengths: 808, 980, and 1075 nm. Each laser delivered an average power of 9.2 W, 500-ms pulse duration, pulse rate of 1.0-Hz, and 3.2-mm diameter laser spot, synchronized with cryogen spray cooling of the scrotal skin surface for a total treatment time of 60 s. Vas burst pressures were measured to determine strength of vas closure and compared to previously reported ejaculation pressures. Gross inspection of vas and scrotal skin was also performed immediately after the procedure as an indicator of thermal coagulation and skin burns. The 1075 nm laser produced the highest vas burst pressures (288 ± 28 mmHg), significantly greater than previously reported ejaculation pressures (136 ± 29 mmHg). The 808 nm wavelength produced insufficient vas burst pressures of 141 ± 61 mmHg, and minor scrotal skin burns were observed in at least one case. The 980 nm wavelength was unable to produce thermal coagulation of the vas, with low burst pressures (89 ± 58 mmHg) and severe scrotal skin burns. The 1075 nm wavelength was the only near-IR wavelength that consistently thermally coagulated the vas with a strong degree of closure and without any scrotal skin burns.

Optical and thermal simulations of noninvasive laser coagulation of the human vas deferens

Gino R. Schweinsberger, Christopher M. Cilip, Susan R. Trammell, et al.

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Successful noninvasive laser coagulation of the canine vas deferens, in vivo, has been previously reported. However, there is a significant difference between the optical properties of canine and human skin. In this study, Monte Carlo simulations of light transport through tissue and heat transfer simulations are performed to determine the feasibility of noninvasive laser vasectomy in humans. A laser wavelength of 1064 nm was chosen for deep optical penetration in tissue. Monte Carlo simulations determined the spatial distribution of absorbed photons inside the tissue layers (epidermis, dermis, and vas). The results were convolved with a 3-mm-diameter laser beam, and then used as the spatial heat source for the heat transfer model. A laser pulse duration of 500 ms and pulse rate of 1 Hz, and cryogen spray cooling were incident on the tissue for 60 s. Average laser power (5-9 W), cryogen pulse duration (60-100 ms), cryogen cooling rate (0.5-1.0 Hz), and increase in optical transmission due to optical clearing (0-50 %), were studied. After application of an optical clearing agent to increase skin transmission by 50%, an average laser power of 6 W, cryogen pulse duration of 60 ms, and cryogen cooling rate of 1 Hz resulted in vas temperatures of ~ 60°C, sufficient for thermal coagulation, while 1 mm of the skin surface (epidermis and dermis) remained at a safe temperature of ~ 45 °C. Monte Carlo and heat transfer simulations indicate that it is possible to noninvasively thermally coagulate the human vas without adverse effects (e.g. scrotal skin burns), if an optical clearing agent is applied to the skin prior to the procedure.

MoXy fiber with active cooling cap for bovine prostate vaporization with high power 200W 532 nm laser

Steven Yihlih Peng, Hyun Wook Kang, Homa Pirzadeh, et al.

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A novel MoXy^TM fiber delivery device with Active Cooling Cap (ACC^TM) is designed to transmit up to 180W of 532 nm laser light to treat benign prostatic hyperplasia (BPH). Under such high power tissue ablation, effective cooling is key to maintaining fiber power transmission and ensuring the reliability of the fiber delivery device To handle high power and reduce fiber degradation, the MoXy fiber features a larger core size (750 micrometer) and an internal fluid channel to ensure better cooling of the fiber tip to prevent the cap from burning, detaching, or shattering during the BPH treatment. The internal cooling channel was created with a metal cap and tubing that surrounds the optical fiber. In this study MoXy fibers were used to investigate the effect of power levels of 120 and 200 W on in-vitro bovine prostate ablation using a 532 nm XPS^TM laser system. For procedures requiring more than 100 kJ, the MoXy fiber at 200W removed tissue at twice the rate of the current HPS fiber at 120W. The fiber maintained a constant tissue vaporization rate during the entire tissue ablation process. The coagulation at 200W was about 20% thicker than at 120W. In conclusion, the new fibers at 200W doubled the tissue removal rate, maintained vaporization efficiency throughout delivery of 400kJ energy, and induced similar coagulation to the existing HPS fiber at 120W.

Interaction between high power 532nm laser and prostatic tissue: in vitro evaluation for laser prostatectomy

Hyun Wook Kang, Yihlih Steven Peng, Douglas Stinson

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Photoselective vaporization of the prostate (PVP) has been developed for effective treatment of obstructive benign prostatic hyperplasia. To maximize tissue ablation for large prostate gland, identifying the optimal power level for PVP is still necessary. We investigated the effect of various power levels on in vitro bovine prostate ablation with a 532-nm laser system. A custom-made 532-nm laser was employed to provide various power levels, delivered through a newly designed 750-μm side-firing fiber. Tissue ablation efficiency was evaluated in terms of power (P; 120~200W), treatment speed of fiber (TS; 2~8 mm/s), and working distance between fiber and tissue surface (WD; 1~5 mm). Coagulation depth was also estimated macroscopically and histologically (H&E) at various Ps. Both 180 and 200W yielded comparable ablated volume (104.3±24.7 vs. 104.1±23.9 mm³ at TS=4 mm/s and WD=2 mm; p=0.99); thus, 180W was identified as the optimal power to maximize tissue ablation, by removing tissue up to 80% faster than 120W (41.7±9.9 vs. 23.2±3.4 mm³/s at TS=4 mm/s and WD=2 mm; p<0.005). Tissue ablation was maximized at TS=4 mm/s and ablated equally efficiently at up to 3 mm WD (104.5±16.7 mm³ for WD=1 mm vs. 93.4±7.4 mm³ for WD=3 mm at 180W; p=0.33). The mean thickness of coagulation zone for 180W was 20% thicker than that for 120W (1.31±0.17 vs. 1.09±0.16 mm; p<0.005). The current in vitro study demonstrated that 180W was the optimal power to maximize tissue ablation efficiency with enhanced coagulation characteristics.

Interaction between high power 532nm laser and prostatic tissue: in vivo evaluation for laser prostatectomy

Reza Malek, Hyun Wook Kang, Steven Yihlih Peng, et al.

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A previous in vitro study demonstrated that 180W was the optimal power to reduce photoselective vaporization of the prostate (PVP) time for larger prostate glands. In this study, we investigated anatomic and histologic outcomes and ablation parameters of 180W laser performed with a new 750-μm side-firing fiber in a survival study of living canines. Eight male canines underwent anterograde PVP with the 180W 532-nm laser. Four each animals were euthanized 3 hours or 8 weeks postoperatively. Prostates were measured and histologically analyzed after hematoxylin and eosin (H&E), triphenyltetrazolium chloride (TTC), or Gomori trichrome (GT) staining. Compared to the previous 120W laser, PVP with the 180W laser bloodlessly created a 76% larger cavity (mean 11.8 vs. 6.7 cm³; p=0.014) and ablated tissue at a 77% higher rate (mean 2.3 vs. 1.3 cm³/min; p=0.03) while H&E- and TTC-staining demonstrated its 33% thicker mean coagulation zone (2.0±0.4 vs. 1.5±0.3 mm). H&E-stained cross-sectional prostatic tissue specimens from the 3-hour (acute) group showed histologic evolution of concentric non-viable coagulation zone, partially viable hyperemic transition zone of repair, and viable non-treated zone. H&E- and GT-stained specimens from the 8-week (chronic) group revealed healed circumferentially epithelialized, non-edematous, prostatic urethral channels with no increase in collagen in the subjacent prostatic tissue vis-á-vis the normal control. Our canine study demonstrates that 180W 532-nm laser PVP with its new fiber has a significantly higher ablation rate with a more hemostatic coagulation zone, but equally favorable tissue interaction and healing, compared with our previous 120W canine study.

Laser laparoscopic partial nephrectomy in clinical cases (N=17)

Oleg Teodorovich, Natalia Zabrodina, Eduard Galljamov, et al.

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A pulsed Nd:YAG laser approved for clinical use in Russian Federation was used for laparoscopic partial nephrectomy(LPN). Patients with T₁N₀M₀ (N=17) cancer underwent laser LPN during 2006-2009 for removal tumor sized from 2.0 to 3.9 cm. Successful laser LPN was performed without ischemia in all cases. Bleeding during laser LPN was substantially reduced due to laser coagulation of tissue. Currently all patients are under medical supervision with no recurrence of tumor. A pulsed Nd:YAG laser showed safety and efficacy of LPN in humans.

Ablative efficiency of lithium triborate laser vaporization and conventional transurethral resection of the prostate: a comparison using transrectal three-dimensional ultrasound volumetry

Oliver Gross, Tullio Sulser, Lukas J. Hefermehl, et al.

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Introduction and objectives: It is unknown if tissue ablation following 120W lithium triborate (LBO) laser vaporization (LV) of the prostate is comparable to that following transurethral resection of the prostate (TURP). Therefore, transrectal 3D-ultrasound volumetry of the prostate was performed to compare the efficiency of tissue ablation between LBO-LV and TURP. Methods: Between 03/2008 and 03/2010 110 patients underwent routine LBO-LV (n=61) or TURP (n=49). Transrectal 3D-ultrasound with planimetric volumetry of the prostate was performed pre-operatively, after catheter removal, 6 weeks and 6 months. Results: Median prostate volume was 52.5ml in the LV group and 46.9ml in the TURP group. After catheter removal, median absolute volume reduction (LV: 7.05ml, TURP: 15.8ml) and relative volume reduction (15.9% vs. 34.2%) were significantly lower in the LV group (p<0.001). After 6 weeks/ 6 months, the relative volume reduction but not the absolute remained significantly lower in the LV group. Conclusions: LBO-LV is an efficient procedure evidenced by an absolute tissue ablation not significantly different to that after TURP. However, TURP seems to be superior due to a higher relative tissue ablation. The differences in tissue ablation had no impact on the early clinical outcome. Delayed volume reduction indicates that prostatic swelling occurs early after LV and then decreases subsequently.

In vitro assessment of fiber sweeping angle during Q-switched 532-nm laser tissue ablation

Danop Rajabhandharaks, Hyun Wook Kang, Woo Jin Ko, et al.

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Photoselective vaporization of the prostate (PVP) has been widely used to treat benign prostatic hyperplasia (BPH). It is well regarded as a safe and minimally invasive procedure and an alternative to the gold standard transurethral resection of the prostate (TURP). Despite of its greatness, as well aware of, the operative procedure time during the PVP is still prolonged. Such attempts have been tried out in order to shorten the operative time and increase its efficacy. However, scientific study to investigate techniques used during the PVP is still lacking. The objective of this study is to investigate how sweeping angle might affect the PVP performance. Porcine kidneys acquired from a local grocery store were used (N=140). A Q-switched 532-nm GreenLight XPS^TM (American Medical Systems, Inc., MN, USA), together with 750- μm core MoXy^TM fiber, was set to have power levels of 120 W and 180 W. Treatment speed and sweeping speed were fixed at 2 mm/s and 0.5 sweep/s, respectively. Sweeping angles were varied from 0 (no sweeping motion) to 120 degree. Ablation rate, depth, and coagulation zone were measured and quantified. Tissue ablation rate was peaked at 15 and 30 degree for both 120- and 180-W power levels and dramatically decreased beyond 60 degree. At 180 W, ablation rate increased 20% at 30 degree compared to 0 degree. This study demonstrated that ablation rate could be maximized and was contingent upon sweeping angle.

In vitro assessment of fiber sweeping speed during Q-switched 532-nm laser tissue ablation

Danop Rajabhandharaks, Hyun Wook Kang, Woo Jin Ko, et al.

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Photoselective vaporization of the prostate (PVP) is considered a minimally invasive procedure to treat benign prostatic hyperplasia (BPH). During the PVP, the prostate gland is irradiated by the 532-nm laser and the fiber is swept and dragged along the urethra. In this study the speed of sweeping fiber during the PVP is being investigated. In vitro porcine kidney model was used (N=100) throughout the experiment. A Q-switched 532-nm laser, equipped with sidefiring 750-Um fiber, was employed and set to power levels of 120 and 180 W. The speed of fiber sweeping was the only variable in this study and varied at 0 (i.e. no sweeping), 0.5, 1.0, 1.5, and 2.0 sweep/s. Ablation rate, depth, and coagulation thickness were quantified. Based on the current settings, ablation rate decreased as sweeping speed increased and was maximized between 0 to 1.0 sweep/s for 120-W power level and between 0 to 0.5 sweep/s for 180-W power level. Ablation rate at 180 W was higher than that at 120 W, regardless of sweeping speed. Ablation depth at both 120 and 180 W was maximized at 0 sweep/s and decreased 35% at 0.5 sweep/s. The overall coagulation thickness was less than 1.5 mm and comparable from 0 to 1.5 sweep/s (0.8~0.9 mm) and increased at 2.0 sweep/s (~1.1 mm). This study demonstrated that tissue ablation performance was contingent upon sweeping speed and maximized at slow sweeping speed due to longer laser-tissue interaction time and larger area coverage by the 532-nm light.

Spatio-temporal processing of massive glottic images from high-speed videoendoscopy

Yuling Yan, Krzysztof Izdebski, Emma Marriott

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We present here development and application of new approaches for quantitative spatio-temporal analyses of vocal fold (VF) vibrations derived from high-speed digital imaging (HSDI) data of the glottis. We develop image processing methods to track the motion of the VF and target the analysis of HSDI-derived glottal area waveform (GAW), glottal width function (GWF) and displacements of the VF tissues for the characterization of the VF dynamic properties. In particular, a combined threshold and region growing method is used for the glottis segmentation, and an analytic signal approach and the Nyquist plot and associated parameters are used to represent and to characterize the VF vibratory behaviors in normal and specific pathologic voice productions.

OCT in the field of laryngology: further perspectives

T. Just, H. W. Pau, E. Lankenau, et al.

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Early detection of cancerous lesions of the larynx may be the best method of improving patient quality of life and survival rates. New in-vivo technologies may be of great clinical relevance in improving the accuracy of sampling during microlaryngeal surgery. Optical coherence tomography (OCT) is an optical imaging technique that clearly identifies basement membrane violation caused by laryngeal cancer. With a microscope-based spectral domain OCT (SD-OCT) we reached in vivo a fairly accurate assessment of benign and dysplastic laryngeal lesions. Recent improvements in OCT technology have led to the development of high-speed OCT systems displaying millions of pixels per second. These systems allow non-contact real-time imaging of large sections of laryngeal tissue. Polarization contrast OCT (PS-OCT) may provide additional information about the lamina propria of the true vocal cord because of the birefringence of connective tissue. We present microscope-based high-speed SD-OCT images with and without polarization contrast and 3D volumes of selected laryngeal pathologies in order to demonstrate our current concepts for the intended intraoperative application. High-speed SD-OCT and polarization contrast can also be complemented by our recently developed rigid confocal endoscopic system to obtain cellular and sub-cellular information about the tissue. Further perspectives will be presented.

Comparison of lasers used in stapedotomy using specialized visualization techniques for mechanical and thermal effects in an inner ear model

Digna M. A. Kamalski, Rudolf M. Verdaasdonk, Tjeerd de Boorder, et al.

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The outcome of stapedotomy depends on several surgical steps. Using laser light, the ossicular chain can be handled and the oval window can be punctured with a non-touch method. Various lasers are being used or considered, however, it is not clear which settings and characteristics will contribute to optimal or adverse effects (vestibule damage and loss hearing frequencies). Using a unique high speed thermal imaging setup based on Schlieren techniques, the mechanical and thermal effects during laser stapedotomy were studied in an inner ear model consisting of human, fresh frozen stapes positioned on a liquid filled cavity in a gel cast. The cw KTP (532 nm), cw CO₂ (10.6 μm), cw Thulium (2.0 μm), pulsed Er,Cr;YSGG (2.78 μm) coupled to special fiber delivery systems were applied at typical clinical settings for comparison. The imaging techniques provided a good insight in the extent of heat conduction beneath the footplate and (explosive) vapour formation on both sides. For the pulsed laser modes, explosive vapour expansion can to be controlled with optimized pulse energies while for continuous wave lasers the thermal effects can be controlled with the pulse length and repetition rate. The fluence at the tip of the delivery system and the distance to the footplate has a major impact on the ablation effect. The pulsed IR lasers with fiber delivery show to be promising for a controlled stapedotomy.

Cochlea hair cell rescue after a noise-induced hearing loss using a low level laser therapy (LLLT)

Chung-Ku Rhee, Chan Woong Bahk, Jae Yun Jung, et al.

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Aim: To see the effect of LLLT on noise-induced hearing loss. Methods: Eleven rats were exposed to noise (120 dB, 16 kHz, 6 h) and left ears were irradiated at 60J/cm², 830 nm laser for 12 days. Right ears were control. Hearing levels were measured at frequencies of 4, 8, 12, 16, 32 kHz before noise exposure and after 12th irradiations. Results: The initial hearing levels were 26.5±4.7, 24.5±5.0, 24.0±5.2, 24.0±3.2, 24.5±5.5 dB SPL. After noise exposure, thresholds were 63.5±15.1, 64±16.8, 71.5±11.3, 73.5±15.6, 67.5±14.4 dB SPL in 4, 8, 12, 16, 32 kHz. After 12th irradiation, thresholds of treated ears recovered significantly 21±4.2, 20±3.5, 24±11.9, 24±12.9, 21±2.2 dB SPL and that of the untreated right ears measured 36.3±22.9, 45±15.8, 66.3±22.9, 50±16.8, 43.8±21.4 dB SPL. Conclusion: LLLT may promote recovery of hearing after noiseinduced hearing loss.

Phase contrast imaging of cochlear soft tissue

Stephanie Shintani Smith, Margaret Hwang, Christoph Rau, et al.

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A noninvasive technique to image soft tissue could expedite diagnosis and disease management in the auditory system. We propose inline phase contrast imaging with hard X-rays as a novel method that overcomes the limitations of conventional absorption radiography for imaging soft tissue. In this study, phase contrast imaging of mouse cochleae was performed at the Argonne National Laboratory Advanced Photon Source. The phase contrast tomographic reconstructions show soft tissue structures of the cochlea, including the inner pillar cells, the inner spiral sulcus, the tectorial membrane, the basilar membrane, and the Reissner's membrane. The results suggest that phase contrast X-ray imaging and tomographic techniques hold promise to noninvasively image cochlear structures at an unprecedented cellular level.

Diode laser (980nm) cartilage reshaping

A. El Kharbotly, T. El Tayeb, Y. Mostafa, et al.

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Loss of facial or ear cartilage due to trauma or surgery is a major challenge to the otolaryngologists and plastic surgeons as the complicated geometric contours are difficult to be animated. Diode laser (980 nm) has been proven effective in reshaping and maintaining the new geometric shape achieved by laser. This study focused on determining the optimum laser parameters needed for cartilage reshaping with a controlled water cooling system. Harvested animal cartilages were angulated with different degrees and irradiated with different diode laser powers (980nm, 4x8mm spot size). The cartilage specimens were maintained in a deformation angle for two hours after irradiation then released for another two hours. They were serially measured and photographed. High-power Diode laser irradiation with water cooling is a cheep and effective method for reshaping the cartilage needed for reconstruction of difficult situations in otorhinolaryngologic surgery. Key words: cartilage,diode laser (980nm), reshaping.

Enhanced transfection of brain tumor suppressor genes by photochemical internalization

Chih H. Chou, Chung-Ho Sun, Yi-Hong Zhou, et al.

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One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

Oral cancer screening approach based on labeling exfoliated oral cells with molecularly-targeted optical contrast agents

Veronica Leautaud, Charles R. Horres, Vijayashree S. Bhattar, et al.

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Early detection is a potential key to improving the survival rates of oral cancer patients and reducing the morbidity associated with treatment. We seek to improve upon methods of detecting of early malignancies with oral brush biopsies by using immunofluorescence-based assessment of the expression of multiple well-described markers commonly overexpressed in oral cancers, such as Epidermal Growth Factor Receptor (EGFR) and Cytokeratin 8 (CK8). Furthermore, since abnormal cells are often scarce in brush biopsy samples, we seek to use magnetic microparticles targeted to these markers as a means of enriching the concentration of abnormal cells. Finally, we plan to conduct a small pilot study using these methods with brush biopsies from patients of the M. D. Anderson Cancer Center Head and Neck Clinic.

Autofluorescence guided diagnostic evaluation of suspicious oral mucosal lesions: opportunities, limitations, and pitfalls

Nadarajah Vigneswaran

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Wide-filed autofluorescence examination is currently considered as a standard of care for screening and diagnostic evaluation of early neoplastic changes of the skin, cervix, lung, bladder, gastrointestinal tract and oral cavity. Naturally occurring fluorophores within the tissue absorb UV and visible light and can re-emit some of this light at longer wavelengths in the form of fluorescence. This non-invasive tissue autofluorescence imaging is used in optical diagnostics, especially in the early detection of cancer. Usually, malignant transformation is associated with thickening of the epithelium, enhanced cellular density due to increased nuclear cytoplasmic ratio which may attenuate the excitation leading to a decrease in collagen autofluorescence. Hence, dysplastic and cancerous tissues often exhibit decreased blue-green autofluorescence and appear darker compared to uninvolved mucosa. Currently, there are three commercially available devices to examine tissue autofluorescence in the oral cavity. In this study we used the oral cancer screening device Identafi^TM 3000 to examine the tissue reflectance and autofluorescence of PML and confounding lesions of the oral cavity. Wide-field autofluorescence imaging enables rapid inspection of large mucosal surfaces, to aid in recognition of suspicious lesions and may also help in discriminate the PML (class 1) from some of the confounding lesions (class II). However, the presence of inflammation or pigments is also associated with loss of stromal autofluorescence, and may give rise to false-positive results with widefield fluorescence imaging. Clinicians who use these autofluorescence based oral cancer screening devices should be aware about the benign oral mucosal lesions that may give false positivity so that unnecessary patient's anxiety and the need for scalpel biopsy can be eliminated.

Real-time spectroscopic evaluation of oral lesions and comparisons with histopathology

Richard A. Schwarz, Wen Gao, Jennifer Nguyen, et al.

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Optical techniques including widefield autofluorescence and reflectance imaging, depth-sensitive optical spectroscopy, and high-resolution imaging can be used to noninvasively detect dysplastic and cancerous alterations in oral tissue. The diagnostic performance of depth-sensitive optical spectroscopy with respect to histopathology is examined. A compact, portable spectroscopy device for clinical use is described. Practical considerations for the comparison of optical measurements to histopathologic diagnoses are outlined. Important considerations for comparison to histopathology include the physical correspondence of the measured region to the biopsy or specimen; data collection and processing procedures; and data analysis procedures. Multimodal combinations of widefield imaging, point spectroscopy, and highresolution imaging may enhance the ability of clinicians to accurately assess the margins of neoplastic oral lesions in vivo.

Laryngeal imaging with polarization-sensitive optical coherence tomography

James A. Burns, Ki Hean Kim, R. Rox Anderson

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Objectives/Hypothesis: Optical coherence tomography (OCT), an imaging technology that provides crosssectional subsurface tissue structure images using backscattered light, is a promising noninvasive, imaging modality for in-vivo assessment of vocal fold layered microstructure. Polarization-sensitive OCT (PS-OCT) augments conventional OCT by detecting changes in the polarization state of reflected light. This study imaged various benign laryngeal pathologies in patients undergoing direct laryngoscopy under general anesthesia to determine whether PS-OCT would provide useful additional information about vocal fold microstructure and glottic surface pathology. Study Design:Prospective clinical trial. Methods: Eighteen patients who were undergoing microlaryngoscopy under general anesthesia for benign glottic disease were imaged bilaterally with OCT and PS-OCT (N=34 vocal folds). Intraoperative microphotography guided placement of the imaging probe. Normalappearing glottic tissue was also imaged if present. When clinically indicated, biopsy or complete removal of the lesion established histologic confirmation. Results: PS-OCT provided high quality, vertical, cross-sectional images up to 1.2mm deep that complemented microlaryngoscopy, and conventional OCT for vocal fold pathologies. Scar tissue was visualized by PS-OCT, characterized by a birefringence pattern more intense than that of normal glottic tissue. Conclusions: Combining PS-OCT with OCT during human vocal cord imaging provides useful information in characterizing vocal cord lesions, particularly scar tissue.

pH-dependent mechanisms of electromechanical cartilage reshaping

Edward C. Wu, Cyrus T. Manuel, Dmitriy E. Protsenko, et al.

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Electromechanical reshaping of cartilage is a novel modality that has significant clinical applications in otolaryngology and plastic surgery. Although EMR dosimetry has been extensively studied, little is known about the mechanisms of EMR, of which local tissue pH changes is believed to play a role. In this study, rabbit nasal septal cartilage is subject to a number of experiments aimed at elucidating pH-related changes using phenol red. The lateral extent and magnitude of pH change as well as factors that impact pH change are studied. Increasing voltage and application appear to increase the area and intensity of color change. With parameters known to produce thermal tissue injury, a transitional zone likely representing a confluence of acid-base products is noted in the region around the bend axis. Furthermore, rehydration and pH indicator application time do not appear to play a role in the quality of pH change. These simple experiments may provide insight into the role of pH changes in EMR that may allow correlation of dosimetry to tissue damage, further optimizing the clinical potential of EMR.

Fluorescence-guided surgical resection of oral cancer reduces recurrence

Pierre Lane, Catherine F. Poh, J. Scott Durham, et al.

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Approximately 36,000 people in the US will be newly diagnosed with oral cancer in 2010 and it will cause 8,000 new deaths. The death rate is unacceptably high because oral cancer is usually discovered late in its development and is often difficult to treat or remove completely. Data collected over the last 5 years at the BC Cancer Agency suggest that the surgical resection of oral lesions guided by the visualization of the alteration of endogenous tissue fluorescence can dramatically reduce the rate of cancer recurrence. Four years into a study which compares conventional versus fluorescence-guided surgical resection, we reported a recurrence rate of 25% (7 of 28 patients) for the control group compared to a recurrence rate of 0% (none of the 32 patients) for the fluorescence-guided group. Here we present resent results from this ongoing study in which patients undergo either conventional surgical resection of oral cancer under white light illumination or using tools that enable the visualization of naturally occurring tissue fluorescence.

Selective sinoatrial node optical mapping to investigate the mechanism of sinus rate acceleration

Shien-Fong Lin, Tetsuji Shinohara, Boyoung Joung M.D., et al.

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Studies using isolated sinoatrial node (SAN) cells indicate that rhythmic spontaneous sarcoplasmic reticulum Ca release (Ca clock) plays an important role in SAN automaticity. However, it is difficult to translate these findings into intact SAN because the SAN is embedded in the right atrium (RA). Cross contamination of the optical signals between SAN and RA prevented the definitive testing of Ca clock hypothesis in intact SAN. We use a novel approach to selectively map intact SAN to examine the Ca clock function in intact RA. We simultaneously mapped intracellular Ca (Cai) and membrane potential (Vm) in 7 isolated, Langendorff perfused normal canine RA. Electrical conduction from the SAN to RA was inhibited with high potassium (10 mmol/L) Tyrode's solution, allowing selective optical mapping of Vm and Cai of the SAN. Isoproterenol (ISO, 0.03 μmol/L) decreased cycle length of the sinus beats from 586±17 ms at baseline to 366±32 ms, and shifted the leading pacemaker site from the middle or inferior SAN to the superior SAN in all RAs. The Cai upstroke preceded the Vm in the leading pacemaker site by up to 18±2 ms. ISO-induced changes to SAN were inhibited by ryanodine (3 μmol/L), but not ZD7288 (3 μmol/L), a selective If blocker. We conclude that a high extracellular potassium concentration results in intermittent SAN-RA conduction block, allowing selective optical mapping of the intact SAN. Acceleration of Ca cycling in the superior SAN underlies the mechanism of sinus tachycardia during sympathetic stimulation.

Fluorescence imaging of macrophages in atherosclerotic plaques using plasmonic gold nanorose

Tianyi Wang, Veronika Sapozhnikova, J. Jacob Mancuso, et al.

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Macrophages are one of the most important cell types involved in the progression of atherosclerosis which can lead to myocardial infarction. To detect macrophages in atherosclerotic plaques, plasmonic gold nanorose is introduced as a nontoxic contrast agent for fluorescence imaging. We report macrophage cell culture and ex vivo tissue studies to visualize macrophages targeted by nanorose using scanning confocal microscopy. Atherosclerotic lesions were created in the aorta of a New Zealand white rabbit model subjected to a high cholesterol diet and double balloon injury. The rabbit was injected with nanoroses coated with dextran. A HeNe laser at 633 nm was used as an excitation light source and a acousto-optical beam splitter was utilized to collect fluorescence emission in 650-760 nm spectral range. Results of scanning confocal microscopy of macrophage cell culture and ex vivo tissue showed that nanoroses produce a strong fluorescence signal. The presence of nanorose in ex vivo tissue was further confirmed by photothermal wave imaging. These results suggest that scanning confocal microscopy can identify the presence and location of nanorose-loaded macrophages in atherosclerotic plaques.

Design, construction, and validation of a multimodal intravascular diagnostic catheter combining IVUS and fluorescence lifetime spectroscopy detection channels

Julien Bec, Hongtao Xie, Diego Yankelevich, et al.

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We report the development and validation of an intravascular rotary catheter that enables bi-modal interrogation of arterial pathologies based on fast-frame time-resolved fluorescence spectroscopy (TRFS) and intravascular ultrasound (IVUS). The catheter is based on a parallel design that allows for independent rotation of the ultrasonic and optical channels within an 8 Fr outer diameter catheter sheath and integrates a low volume flushing channel for blood removal in the optical pathways. In current configuration, the two channels consist of a) a standard 8 Fr IVUS catheter with single element transducer (15 MHz) and b) a side-viewing UV-grade silica/silica fiber optic (400 μm core). The catheter is terminated by a small (0.82 mm internal diameter) polyimide tube to keep the fiber stable within the sheath. To clear the field of view from blood, a saline solution can be flushed in a sheath channel, concentric with the fiber optic, through the tube and in a radial opening aligned with the fiber's optical beam. The flushing function was optimized with a computational fluid dynamics (CFD) model pursued in a parallel study. The ability of the catheter to operate in intraluminal setting in blood flow, the effect of probe-to-tissue distance on optical signal and ability to generate co-registered TRFS and IVUS data were demonstrated in blood vessel phantoms. Current results demonstrate the feasibility of the described catheter for parallel interrogation of vessel walls based on TRFS and IVUS and to generate robust TRFS data. These results facilitate further development of a bi-modal TRFS-IVUS technique for intravascular diagnosis of atherosclerotic cardiovascular diseases including vulnerable plaques.

Tools for experimental characterization of the non-uniform rotational distortion in intravascular OCT probes

Marc L. Dufour, Charles-Etienne Bisaillon, Guy Lamouche, et al.

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The Industrial Material Institute (IMI) together with the Institute for Biodiagnostic (IBD) has developed its own optical catheters for cardiovascular imaging applications. Those catheters have been used experimentally in the in vitro coronary artery model of the Langendorff beating heart and in a percutaneous coronary intervention procedure in a porcine model. For some catheter designs, non-uniform rotational distortion (NURD) can be observed as expected from past experience with intra-vascular ultrasound (IVUS) catheters. A two-dimensional (2D) coronary artery test bench that simulates the path into the coronary arteries has been developed. The presence or absence of NURD can be assessed with the test bench using a custom-built cardiovascular Optical Coherence Tomography (OCT) imaging system. A square geometry instead of the circular shape of an artery is used to simulate the coronary arteries. Thereby, it is easier to visualize NURD when it is present. The accumulated torsion induced by the friction on the catheter is measured along the artery path. NURD is induced by the varying friction force that is balanced by the accumulated torsion force. The pullback force is measured and correlated with NURD observed in the 2D test bench. Finally, a model is presented to help understanding the mechanical constraint that leads to the friction force variations.

In vivo swine myocardial tissue characterization and monitoring during open chest surgery by time-resolved diffuse near-infrared spectroscopy

Lorenzo Spinelli, Davide Contini, Andrea Farina, et al.

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Cardiovascular diseases are the main cause of death in industrialized countries. Worldwide, a large number of patients suffering from cardiac diseases are treated by surgery. Despite the advances achieved in the last decades with myocardial protection, surgical failure can still occur. This is due at least in part to the imperfect control of the metabolic status of the heart in the various phases of surgical intervention. At present, this is indirectly controlled by the electrocardiogram and the echographic monitoring of cardiac mechanics as direct measurements are lacking. Diffuse optical technologies have recently emerged as promising tools for the characterization of biological tissues like breast, muscles and bone, and for the monitoring of important metabolic parameters such as blood oxygenation, volume and flow. As a matter of fact, their utility has been demonstrated in a variety of applications for functional imaging of the brain, optical mammography and monitoring of muscle metabolism. However, due to technological and practical difficulties, their potential for cardiac monitoring has not yet been exploited. In this work we show the feasibility of the in-vivo determination of absorption and scattering spectra of the cardiac muscle in the 600-1100 nm range, and of monitoring myocardial tissue hemodynamics by time domain near-infrared spectroscopy at 690 nm and 830 nm. Both measurements have been performed on the exposed beating heart during open chest surgery in pigs, an experimental model closely mimicking the clinical cardio-surgical setting.

Towards development of an intravascular diagnostic catheter based on fluorescence lifetime spectroscopy: study of an optimized blood flushing system

Narugopal Ghata, Ralph C. Aldredge, Julien Bec, et al.

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Fluorescence lifetime spectroscopy has demonstrated potential for characterization and diagnosis of arterial vessels pathologies. However, the intravascular application of such technique is hampered by the presence of blood hemoglobin that affects both the delivery of the excitation light to and the collection of the fluorescence light from the vessel wall. We report here a computational fluid dynamics model that allows for the optimization of blood flushing parameters in a manner that minimizes the amount of saline needed to clear the optical field of view. A 3D turbulence (k - ∈) model was employed to simulate the flow inside and around a side-viewing fiber-optic catheter. The influence of various infusion rates, blood flow rates and vessel diameters on the flow around the catheter tip and its effects on the wall shear stress (WSS) are studied. Current results suggest that low flushing rates in smaller-diameter vessels (e.g., stenotic vessels) can produce better flushing efficiency by removing the blood cells in the path of the fluorescence light and reducing wall shear stress. The comparison of the results for blood vessels with equal diameter but different flow rates suggests that the effect of systolic and diastolic conditions on the maximum wall shear stress is not substantial. The results from this study can be utilized in determining the optimal flushing rate depending on the diameter of the vessels, blood flow rate, and the maximum wall shear stress that vessel wall can sustain, which can be estimated from the feedback of the fluorescent light from the wall.

Non-thermal myocardial electrical conduction block by photosensitization reaction with catheterization in right atrium isthmus of porcine heart in vivo

Arisa Ito, Takuro Kajihara, Tsukasa Suenari, et al.

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We have studied a new type of myocardial catheter ablation with photosensitization reaction to realize non-thermal therapy for atrial arrhythmia, such as atrial fibrillation. Photochemically-generated reactive oxygen species may induce myocardial electrophysiological damage without heat generation. In this study, to demonstrate photosensitization reaction-induced myocardial electrical conduction block, the inferior vena cava to tricuspid annulus (IVC-TA) isthmus linear ablation was conducted with photosensitization reaction in porcine heart in vivo, using a newly developed laser catheter (7 Fr.). The end point of the procedure was the production of IVC-TA isthmus block under the electrophysiological analysis by diagnostic catheter with 10-bipole electrodes placed in right atrium along the isthmus. Talaporfin sodium (NPe6) as a photosensitizer was injected intravenously to pigs at 2.5-5.0 mg/kg. About 15 min after the injection, the laser light at the wavelength of 663 nm with a catheter output power density of 40-60 W/cm² in about 1.4 mm spot size was irradiated through the laser catheter point by point in line crossing the isthmus under the fluoroscopic guidance. Before the photosensitization procedure, pacing signal from the distal electrodes of the diagnostic catheter, propagated through the isthmus in order. During the irradiation, electrical potential at the irradiated area was diminished. After the completion of the irradiation line, the bidirectional conduction block on the IVC-TA isthmus was validated by pacing from the distal and proximal bipole. These results indicated that photosensitization reaction could achieve the electrical conduction block of myocardial tissue immediately after the irradiation. We think that photosensitization reaction could become a novel therapy for atrial arrhythmia.

Basic study of effects on the smooth muscle cells' proliferation with novel short-term thermal angioplasty in vitro and in vivo

M. Kunio, N. Shimazaki, A. Ito, et al.

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We investigated the effect on smooth muscle cells' proliferation with stretch-fixing in both in vitro and in vivo porcine study to determine the optimum heat condition of novel short-term thermal angioplasty, Photo-thermo Dynamic Balloon Angioplasty (PTDBA). With PTDBA, we have obtained the sufficient arterial dilatation by short-term heating (< 15 s, < 70 °C) and low dilatation pressure (< 0.4 MPa) without excessive neo-intimal hyperplasia on chronic phase. The smooth muscle cells were found to be fixed with stretched shape in vascular wall after PTDBA in vivo. The deformation rate of smooth muscle cells' nuclei was 1.6 ± 0.1 after PTDBA (15 s, 65 °C, 0.35 MPa). The smooth muscle cells, which were extracted from porcine arteries, were cultured on the specially designed equipment to give stretch-fixing stimulus in vitro. The cell proliferation was inhibited at 20 % stretching compared to 15 % stretching significantly (p < 0.05). The immunostaining specimens of basic Fibroblast Growth Factor (bFGF) and its receptor FGFR-1 were made from the porcine arteries in vivo. We found that the expressions of bFGF and FGFR-1 in the media were not observed after PTDBA. We think that these results suggested the possibility for the inhibition of the excessive cell proliferation after PTDBA.

In vivo experimental study on laser welded ICG-loaded chitosan patches for vessel repair

Francesca Rossi, Paolo Matteini, Giuseppe Esposito, et al.

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Laser welding of microvessels provides several advantages over conventional suturing techniques: surgical times reduction, vascular healing process improvement, tissue damage reduction. We present the first application of biopolymeric patches in an in vivo laser assisted procedure for vessel repair. The study was performed in 20 New Zealand rabbits. After anesthesia, a 3-cm segment of the right common carotid artery was exposed and clamped proximally and distally. A linear lesion 3 mm in length was carried out. We used a diode laser emitting at 810 nm and equipped with a 300 μm diameter optical fiber. To close the cut, ICG-loaded chitosan films were prepared: chitosan is characterized by biodegradability, biocompatibility, antimicrobial, haemostatic and wound healing-promoting activity. ICG is an organic chromophore commonly used in the laser welding procedures to mediate the photothermal conversion at the basis of the welding effect. The membranes were used to wrap the whole length of the cut, and then they were welded in the correct position by delivering single laser spots to induce local patch/tissue adhesion. The result is an immediate closure of the wound, with no bleeding at clamps release. The animals were observed during follow-up and sacrificed after 2, 7, 30 and 90 days. All the repaired vessels were patent, no bleeding signs were documented. The carotid samples underwent histological examinations. The advantages of the proposed technique are: simplification of the surgical procedure and shortening of the operative time; good strength of the vessel repair; decreased foreign-body reaction, reduced inflammatory response and improved vascular healing process.

The usefulness of optical analyses for detecting vulnerable plaques using rabbit models

Kanji Nakai, Miya Ishihara, Satoko Kawauchi, et al.

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Purpose: Carotid artery stenting (CAS) has become a widely used option for treatment of carotid stenosis. Although technical improvements have led to a decrease in complications related to CAS, distal embolism continues to be a problem. The purpose of this research was to investigate the usefulness of optical methods (Time-Resolved Laser- Induced Fluorescence Spectroscopy [TR-LIFS] and reflection spectroscopy [RS] as diagnostic tools for assessment of vulnerable atherosclerotic lesions, using rabbit models of vulnerable plaque. Materials & Methods: Male Japanese white rabbits were divided into a high cholesterol diet group and a normal diet group. In addition, we used a Watanabe heritable hyperlipidemic (WHHL) rabbit, because we confirmed the reliability of our animal model for this study. Experiment 1: TR-LIFS. Fluorescence was induced using the third harmonic wave of a Q switch Nd:YAG laser. The TR-LIFS was performed using a photonic multi-channel analyzer with ICCD (wavelength range, 200 - 860 nm). Experiment 2: RS. Refection spectra in the wavelength range of 900 to 1700 nm were acquired using a spectrometer. Results: In the TR-LIFS, the wavelength at the peak was longer by plaque formation. The TR-LIFS method revealed a difference in peak levels between a normal aorta and a lipid-rich aorta. The RS method showed increased absorption from 1450 to 1500 nm for lipid-rich plaques. We observed absorption around 1200 nm due to lipid only in the WHHL group. Conclusion: These methods using optical analysis might be useful for diagnosis of vulnerable plaques. Keywords: Carotid artery stenting, vulnerable plaque, Time-Resolved Laser-Induced Fluorescence

ALA-induced PpIX spectroscopy for brain tumor image-guided surgery

Pablo A. Valdes, Frederic Leblond, Anthony Kim, et al.

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Maximizing the extent of brain tumor resection correlates with improved survival and quality of life outcomes in patients. Optimal surgical resection requires accurate discrimination between normal and abnormal, cancerous tissue. We present our recent experience using quantitative optical spectroscopy in 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence-guided resection. Exogenous administration of ALA leads to preferential accumulation in tumor tissue of the fluorescent compound, PpIX, which can be used for in vivo surgical guidance. Using the state of the art approach with a fluorescence surgical microscope, we have been able to visualize a subset of brain tumors, but the sensitivity and accuracy of fluorescence detection for tumor tissue with this system are low. To take full advantage of the biological selectivity of PpIX accumulation in brain tumors, we used a quantitative optical spectroscopy system for in vivo measurements of PpIX tissue concentrations. We have shown that, using our quantitative approach for determination of biomarker concentrations, ALA-induced PpIX fluorescence-guidance can achieve accuracies of greater than 90% for most tumor histologies. Here we show multivariate analysis of fluorescence and diffuse reflectance signals in brain tumors with comparable diagnostic performance to our previously reported quantitative approach. These results are promising, since they show that technological improvements in current fluorescence-guided surgical technologies and more biologically relevant approaches are required to take full advantage of fluorescent biomarkers, achieve better tumor identification, increase extent of resection, and subsequently, lead to improve survival and quality of life in patients.

Blood interference in fiber-optical based fluorescence guided resection of glioma using 5-aminolevulinic acid

Neda Haj-Hosseini, Shannely Lowndes, Göran Salerud, et al.

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Fluorescence guidance in brain tumor resection is performed intra-operatively where bleeding is included. When using fiber-optical probes, the transmission of light to and from the tissue is totally or partially blocked if a small amount of blood appears in front of the probe. Sometimes even after rinsing with saline, the remnant blood cells on the optical probe head, disturb the measurements. In such a case, the corresponding spectrum cannot be reliably quantified and is therefore discarded. The optimal case would be to calculate and take out the blood effect systematically from the collected signals. However, the first step is to study the pattern of blood interference in the fluorescence spectrum. In this study, a fiber-optical based fluorescence spectroscopy system with a laser excitation light of 405 nm (1.4 J/cm²) was used during fluorescence guided brain tumor resection using 5-aminolevulinic acid (5-ALA). The blood interference pattern in the fluorescence spectrum collected from the brain was studied in two patients. The operation situation was modeled in the laboratory by placing blood drops from the finger tip on the skin of forearm and the data was compared to the brain in vivo measurements. Additionally, a theoretical model was developed to simulate the blood interference pattern on the skin autofluorescence. The blood affects the collected fluorescence intensity and leaves traces of oxy and deoxy-hemoglobin absorption peaks. According to the developed theoretical model, the autofluorescence signal is considered to be totally blocked by an approximately 500 μm thick blood layer.

ALA-induced PpIX fluorescence in epileptogenic tissue

Jonathan K. Kleen, Pablo A. Valdes, Brent T. Harris, et al.

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Astrogliotic tissue displays markedly increased levels of ALA-induced PpIX fluorescence, making it useful for fluorescence-guided resection in glioma surgery. In patients with temporal lobe epilepsy (TLE) and corresponding animal models, there are areas of astrogliosis that often co-localize with the epileptic focus, which can be resected to eliminate seizures in the majority of treated patients. If this epileptogenic tissue can exhibit PpIX fluorescence that is sufficiently localized, it could potentially help identify margins in epilepsy surgery. We tested the hypothesis that ALA-induced PpIX fluorescence could visually accentuate epileptogenic tissue, using an established animal model of chronic TLE. An acute dose of pilocarpine was used to induce chronic seizure activity in a rat. This rat and a normal control were given ALA, euthanized, and brains examined post-mortem for PpIX fluorescence and neuropathology. Preliminary evidence indicates increased PpIX fluorescence in areas associated with chronic epileptic changes and seizure generation in TLE, including the hippocampus and parahippocampal areas. In addition, strong PpIX fluorescence was clearly observed in layer II of the piriform cortex, a region known for epileptic reorganization and involvement in the generation of seizures in animal studies. We are further investigating whether ALA-induced PpIX fluorescence can consistently identify epileptogenic zones, which could warrant the extension of this technique to clinical studies for use as an adjuvant guidance technology in the resection of epileptic tissue.

Photothermal ablation of malignant brain tumors by nanoparticle loaded macrophages

Henry Hirschberg, Seung-Kuk Baek, Young Jik Kwon, et al.

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Nanoshells are a new class of optically tunable nanoparticles composed of a dielectric core (silica) coated with an ultrathin metallic layer (gold). Since nanoshells are roughly one million times more efficient at converting NIR light into heat than conventional dyes when exposed to NIR light, they can generate sufficient heat to induce cell death. Macrophages are frequently found in and around glioblastomas in both experimental animals and patient biopsies. Inflammatory cells loaded with nanoparticles could therefore be used to target tumors.

Nonlinear optical imaging: toward chemical imaging during neurosurgery

Tobias Meyer, Benjamin Dietzek, Christoph Krafft, et al.

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Tumor recognition and precise tumor margin detection presents a central challenge during neurosurgery. In this contribution we present our recent all-optical approach to tackle this problem. We introduce various nonlinear optical techniques, such as coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG) and two-photon fluorescence (TPEF), to study the morphology and chemical composition of (ex vivo) brain tissue. As the experimental techniques presented are contact-free all-optical techniques, which do not rely on the administration of external (fluorescence) labels, we anticipate that their implementation into surgical microscopes will provide significant advantages of intraoperative tumor diagnosis. In this contribution an introduction to the different optical spectroscopic methods will be presented and their implementation into a multimodal microscopic setup will be discussed. Furthermore, we will exemplify their application to brain tissue, i.e. both pig brain as a model for healthy brain tissue and human brain samples taken from surgical procedures. The data to be discussed show the capability of a joint CARS/SHG/TPEF multimodal imaging approach in highlighting various aspects of tissue morphochemistry. The consequences of this microspectroscopic potential, when combined with the existing technology of surgical microscopes, will be discussed.

Real-time intraoperative full-range complex FD-OCT guided cerebral blood vessel identification and brain tumor resection in neurosurgery

Kang Zhang, Yong Huang, Gustavo Pradilla, et al.

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This work utilized an ultra-high-speed full-range complex-conjugate-free optical coherence tomography (FD-OCT) system to perform real-time intraoperative imaging to guide two common neurosurgical procedures: the cerebral blood vessel identification and the brain tumor resection. The cerebral blood vessel identification experiment is conducted ex vivo on human cadaver specimen. Specific cerebral arteries and veins in different positions of the specimen are visualized and the spatial relations between adjacent vessels are indentified through real-time 3D visualization. The brain tumor resection experiment is conducted in vivo on 9L gliomas established in rat brains. The normal brain-tumor margin can be clearly identified in depth of the tissue from sagittal, coronal and axial slices of the intraoperatively acquired 3D data set. The real-time full-range FD-OCT guided in vivo rat flank tumor resection is also conducted.

Fluorescence and reflectance spectroscopy for protoporphyrin IX quantification in tissue-like media

Gesa Palte, Ann Johansson, Georg Hennig, et al.

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PpIX induced by administration of ALA is being successfully employed for tissue diagnosis and photodynamic therapy (PDT) of, for example, brain malignancies. To guide tissue biopsy by fluorescence during stereotaxy, correct quantification of the PpIX accumulation is required. However, the detected fluorescence intensity and spectral shape are influenced and distorted by the varying optical properties of tissue. Quantitative PpIX measurements thus need to disentangle these effects in order to provide the undistorted, intrinsic fluorescence. Numerous methods for obtaining the intrinsic fluorescence have been developed and optimized for certain fluorochromes. PpIX poses a particular case where excitation and fluorescence are spectrally well separated. Furthermore, the fluorescence appears within the red wavelength region where absorption in tissue is relatively weak. Here, three experimental approaches towards assessing the intrinsic fluorescence for PpIX in homogeneous phantom materials for four subsets at tissue-like conditions, were tested and compared; 1) single-fiber with diameters 200-800 μm, 2) a two-fiber probe with evaluation based on an empirical ratio between fluorescence and reflectance signals or 3) a multi-fiber probe for combined fluorescence and reflectance measurements with evaluation based on a theoretical model of light propagation. All methods could be realized with an outer probe diameter of less than 1.5 mm, thus applicable during stereotaxy. Method 3 could quantify the PpIX concentration best, regarding all four subsets and thus covering a broad, physiologically relevant range of optical parameters. With accuracies between ± 3.2 % and ± 24.8 % for different subsets it was overall a great improvement to the accuracies resulting from calculations based on a plane wave geometry, which vary from ± 4.6 % to ± 84.3 %.

Neuro-endovascular optical coherence tomography imaging: clinical feasibility and applications

Marlon S. Mathews, Jianping Su, Esmaeil Heidari, et al.

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The authors report on the feasibility of clinical neuroendovascular optical coherence tomography (OCT) imaging as well as its efficacy and safety by comparing findings with histology in animal, cadaveric and clinical studies. Catheter-based in vivo endovascular OCT imaging was carried out intracranially in four patients, three in the anterior circulation and one in the posterior circulation (vertebral artery). The neuroendovascular OCT device was delivered to the desired location using groin access and standard endovascular procedures. In vivo findings were reproduced using ex vivo OCT imaging in corresponding animal and human (cadaveric) harvested tissue segments with findings matched by histology. OCT images correlated well with the images obtained after histologic sectioning, and visualized in vivo the laminar vascular structure. Satisfactory imaging findings were obtained with no complications. Neuroendovascular OCT imaging is thus feasible for clinical use and can detect with high resolution the structure of arterial segments. Understanding OCT imaging in non-diseased arteries is important in establishing baseline findings necessary for interpreting pathologic processes. This allows neuroendovascular optical biopsies of vascular tissue to be obtained without the need for excision and processing, and potentially allows prophylactic interventions against stroke and other cerebrovascular disease before they become symptomatic.

Cortical blood flow imaging of mouse stroke model by high-speed Spectral OCT

Ireneusz Grulkowski, Grzegorz Wilczynski, Danuta Bukowska, et al.

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We have developed and applied a high-speed Spectral OCT system to image small animal brains. OCT imaging with high spatial resolution and application of multi-parameter approach enabled cortical blood flow visualization. We imaged the brain vascular network of an anesthetized mouse stroke model. We demonstrated the impact of induced stroke on the brain vasculature. The preliminary studies have revealed local ischemia in the areas of the stroke.

Joint attention studies in normal and autistic children using NIRS

Ujwal Chaudhary, Michael Hall, Anibal Gutierrez, et al.

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Autism is a socio-communication brain development disorder. It is marked by degeneration in the ability to respond to joint attention skill task, from as early as 12 to 18 months of age. This trait is used to distinguish autistic from nonautistic. In this study Near infrared spectroscopy (NIRS) is being applied for the first time to study the difference in activation and connectivity in the frontal cortex of typically developing (TD) and autistic children between 4-8 years of age in response to joint attention task. The optical measurements are acquired in real time from frontal cortex using Imagent (ISS Inc.) - a frequency domain based NIRS system in response to video clips which engenders a feeling of joint attention experience in the subjects. A block design consisting of 5 blocks of following sequence 30 sec joint attention clip (J), 30 sec non-joint attention clip (NJ) and 30 sec rest condition is used. Preliminary results from TD child shows difference in brain activation (in terms of oxy-hemoglobin, HbO) during joint attention interaction compared to the nonjoint interaction and rest. Similar activation study did not reveal significant differences in HbO across the stimuli in, unlike in an autistic child. Extensive studies are carried out to validate the initial observations from both brain activation as well as connectivity analysis. The result has significant implication for research in neural pathways associated with autism that can be mapped using NIRS.

Synthetic reconstruction of dynamic blood flow in cortical arteries using optical coherence tomography for the evaluation of vessel compliance

Edward Baraghis, Virginie Bolduc, Marc-Antoine Gillis, et al.

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Optical Coherence Tomography (OCT) has recently been used to produce 3D angiography of microvasculature in the rodent brain in-vivo and blood flow maps of large vessels. Key enabling developments were novel algorithms for detecting Doppler shifts produced by moving scatterers and new scanning protocols tailored to increase sensitivity to small flow speeds. These progresses were pushed by the need for a non invasive imaging modality to monitor quantitative blood flow at a higher resolution and a greater depth than could be achieved by other means. The rationale for this work originates from new hypotheses regarding the role of blood regulation in neurodegenerative diseases and from current investigations of animal models of vascular degeneration. In this work we demonstrate the synthetic reconstruction of dynamic blood flow in mice over the course of a single cardiac cycle in an 800μm wide by ~ 3mm deep B-Frame slice with a lateral resolution of 10μm and a depth resolution of 7μm. Images were taken using a cranial window over the exposed parietal bone of mice skull. Electrocardiography (ECG) recordings were co registered with the OCT A lines at high temporal resolution. QRS peak detection was then used to locate the time value of each A-line in the cardiac cycle and to reconstruct a synthetic temporal frame over one cardiac cycle. Doppler speed in this cardiac cycle was used to measure temporal variations of flow inside arteries and of their area. Three dimensional volume scans yielded measurements of quantitative blood flow on the same arteries. Using these informations a measure of compliance could be established. Comparing measures between atherosclerotic (ATX) and wild type (WT) mice revealed higher blood flow in WT mice, suggested lower systemic compliance in the ATX group but higher compliance of cerebral vasculature on these mice. These results are consistent with expectations showing that OCT is a potential tool for in-vivo arterial compliance evaluation.

Identification of prefrontal cortex (BA10) activation while performing Stroop test using diffuse optical tomography

Sabin Khadka, Srujan R. Chityala, Fenghua Tian, et al.

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Stroop test is commonly used as a behavior-testing tool for psychological examinations that are related to attention and cognitive control of the human brain. Studies have shown activations in Broadmann area 10 (BA10) of prefrontal cortex (PFC) during attention and cognitive process. The use of diffuse optical tomography (DOT) for human brain mapping is becoming more prevalent. In this study we expect to find neural correlates between the performed cognitive tasks and hemodynamic signals detected by a DOT system. Our initial observation showed activation of oxy-hemoglobin concentration in BA 10, which is consistent with some results seen by positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). Our study demonstrates the possibility of combining DOT with Stroop test to quantitatively investigate cognitive functions of the human brain at the prefrontal cortex.

Temporal mapping and connectivity using NIRS for language-related tasks

Michael Hall, Ujwal Chaudhary, Gustavo Rey, et al.

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Near Infrared Spectroscopy (NIRS) offers an invaluable tool to monitor the functionality of the brain. NIRS with its high temporal resolution and good spatial resolution has been applied towards various area of brain research in order to map the cortical regions of the brain. The present study is aimed at using NIRS to understand the functionality of the temporal cortex in response to language-related tasks. A 32-channel NIRS system (Imagent ISS Inc.) is used to perform experimental studies on 15 normal adults. A block-design based Word Expression and Word Reception tasks were independently presented to the participants during the imaging study. Unlike past research where only the brain activation was determined for language tasks, in the current study the activation, connectivity, and lateralization in the temporal cortex are correlated. In the future, the work is focused to target the pediatric epileptic populations, where understanding the temporal brain functionality in response to language is essential in pre-surgical clinical environment.

Steroid-induced osteoporosis monitored by Raman spectroscopy

Jason R. Maher, Masahiko Takahata, Hani A. Awad, et al.

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Glucocorticoids are frequently used to treat inflammatory disorders such as rheumatoid arthritis. Unfortunately, extended exposure to this steroid is the leading cause of physician-induced osteoporosis, leaving patients susceptible to fractures at rates of 30-50%. In this presentation, we report correlations between Raman spectra and biomechanical strength tests on bones of glucocorticoid- and placebo- treated mice. Both wild-type mice and a transgenic model of rheumatoid arthritis have been studied. A two-way ANOVA model reveals statistically significant spectral differences as influenced by glucocorticoid treatment and mouse type.

Transcutaneous Raman spectroscopy for assessing progress of bone-graft incorporation in bone reconstruction and repair

Paul I. Okagbare, Francis W. L. Esmonde-White, Steven A. Goldstein, et al.

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Allografts and other bone-grafts are frequently used for a variety of reconstructive approaches in orthopaedic surgery. However, successful allograft incorporation remains uncertain. Consequently, there is significant need for methods to monitor the fate of these constructs. Only few noninvasive methods can fully assess the progress of graft incorporation and to provide information on the metabolic status of the graft, such as the mineral and matrix composition of the regenerated-tissue that may provide early indications of graft success or failure. For example, Computed-tomography and MRI provide information on the morphology of the graft/host interface. Limited information is also available from DXA. To address this challenge, we present here the implementation of a noninvasive Raman spectroscopy technique for in-vivo assessment of allograft incorporation in animal-model. In an animal use committee approved osseointegration experiment, a 3mm defect is created in rat's tibia. The defect is reconstructed using auto or allograft and Raman spectra are collected at several time-points during healing using an array of optical-fibers in contact with the skin of the rat over the tibia while the rat is anaesthetized. The array allows excitation and collection of Raman spectra through the skin at various positions around the tibia. Raman parameters such as mineral/matrix, carbonate/phosphate and cross-linking are recovered and monitored. The system is calibrated against locally-constructed phantoms that mimic the morphology, optics and spectroscopy of the rat. This new technology provides a non-invasive method for in-vivo assessment of bone-graft incorporation in animal-models and can be adapted for similar study in human subjects.

Space simulations of thermal fields generated in bone tissue for application to nanophotohyperthermia and nanophotothermolysis

Renat R. Letfullin, Colin E. W. Rice, Thomas F. George

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The use of nanoparticles in medical applications has been gaining momentum as antibody-conjugated nanoparticles are becoming more and more feasible as a means of targeted delivery of various therapies. Irradiating nanoparticles with light of strongly-absorbed wavelengths allows them to act as heat generation sites. Two therapies utilize these nanoparticle heat sources to kill the target cells: nanophotohyperthermia, which heats the particles just enough to disrupt cell function and trigger cell death; and nanophotothermolysis, which heats the particles to such extremes as to destroy the cell membrane. The use of optical wavelengths in the range of 750-1100 nm has been to capitalize on the "optical transparency window" of biotissues between the absorption peaks of hemoglobin in the visible end and water in the near-IR. However, further research has shown that a plasmon resonance can greatly affect the absorption characteristics of nanoparticles at the plasmon resonant frequency, allowing for increased absorption characteristics at desirable wavelengths. Thus, other transparency windows may find use in a similar manner, such as nanoparticle heating by RF waves. This paper presents the modeling of 3D thermal fields around nanoparticle absorbers in bone tissue for various frequencies. A comparison of the heating effectiveness across multiple wavelengths is discussed for application to nanophotothermolysis and nanophotohyperthermia treatments in or near biological hard tissue.

Quantitative evaluation of simulated human enamel caries kinetics using photothermal radiometry and modulated luminescence

Adam Hellen, Andreas Mandelis, Yoav Finer, et al.

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Photothermal radiometry and modulated luminescence (PTR-LUM) is a non-destructive methodology applied toward the detection, monitoring and quantification of dental caries. The purpose of this study was to evaluate the efficacy of PTRLUM to detect incipient caries lesions and quantify opto-thermophysical properties as a function of treatment time. Extracted human molars (n=15) were exposed to an acid demineralization gel (pH 4.5) for 10 or 40 days in order to simulate incipient caries lesions. PTR-LUM frequency scans (1 Hz - 1 kHz) were performed prior to and during demineralization. Transverse Micro-Radiography (TMR) analysis followed at treatment conclusion. A coupled diffusephoton- density-wave and thermal-wave theoretical model was applied to PTR experimental amplitude and phase data across the frequency range of 4 Hz - 354 Hz, to quantitatively evaluate changes in thermal and optical properties of sound and demineralized enamel. Excellent fits with small residuals were observed experimental and theoretical data illustrating the robustness of the computational algorithm. Increased scattering coefficients and poorer thermophysical properties were characteristic of demineralized lesion bodies. Enhanced optical scattering coefficients of demineralized lesions resulted in poorer luminescence yield due to scattering of both incident and converted luminescent photons. Differences in the rate of lesion progression for the 10-day and 40-day samples points to a continuum of surface and diffusion controlled mechanism of lesion formation. PTR-LUM sensitivity to changes in tooth mineralization coupled with opto-thermophysical property extraction illustrates the technique's potential for non-destructive quantification of enamel caries.

Image-guided photoacoustic spectroscopy in diagnosis of osteoarthritis in hands: an initial study

Zhen Yuan, J. Xiao, Yao Sun, et al.

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We present for the first time in vivo experimental evidence that multispectral quantitative photoacoustic tomography (qPAT) has the potential to detect osteoarthritis (OA) in the finger joints. In this pilot study, 2 OA patients and 4 healthy volunteers were enrolled, and their distal interphalangeal (DIP) joints were examined photoacoustically by a multispectral PAT scanner. Images of tissue physiological/functional parameters including oxy-hemoglobin, deoxyhemoglobin, oxygen saturation and water content along with tissue acoustic velocity of all the examined joints were simultaneously recovered using a finite element reconstruction algorithm for multispectral photoacoustic measurements. The recovered multispectral photoacoustic images show that the OA joints have significantly elevated water content, decreased oxygen saturation, and increased acoustic velocity compared to the normal joints.

3D imaging of dental hard tissues with Fourier domain optical coherence tomography

Yueli L. Chen, Yi Yang, Jing Ma, et al.

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A fiber optical coherence tomography (OCT) probe is used for three dimensional dental imaging. The probe has a lightweight miniaturized design with a size of a pen to facilitate clinic in vivo diagnostics. The probe is interfaced with a swept-source / Fourier domain optical coherence tomography at 20K axial scanning rate. The tooth samples were scanned from occlusal, buccal, lingual, mesial, and distal orientations. Three dimensional imaging covers tooth surface area up to 10 mm x 10 mm with a depth about 5 mm, where a majority of caries affection occurs. OCT image provides better resolution and contrast compared to gold standard dental radiography (X-ray). In particular, the technology is well suited for occlusal caries detection. This is complementary to X-ray as occlusal caries affection is difficult to be detected due to the X-ray projectile scan geometry. The 3D topology of occlusal surface as well as the dentin-enamel junction (DEJ) surface inside the tooth can be visualized. The lesion area appears with much stronger back scattering signal intensity.

Detection of chemical changes in bone after irradiation with Er,Cr:YSGG laser

Carolina Benetti, Moises O. Santos, Jose S. Rabelo, et al.

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The use of laser for bone cutting can be more advantageous than the use of drill. However, for a safe clinical application, it is necessary to know the effects of laser irradiation on bone tissues. In this study, the Fourier Transform Infrared spectroscopy (FTIR) was used to verify the molecular and compositional changes promoted by laser irradiation on bone tissue. Bone slabs were obtained from rabbit's tibia and analyzed using ATR-FTIR. After the initial analysis, the samples were irradiated using a pulsed Er,Cr:YSGG laser (2780nm), and analyzed one more time. In order to verify changes due to laser irradiation, the area under phosphate (1300-900cm^-1), amides (1680-1200cm^-1), water (3600-2400cm^-1), and carbonate (around 870cm^-1 and between 1600-1300cm^-1) bands were calculated, and normalized by phosphate band area (1300-900cm^-1). It was observed that Er,Cr:YSGG irradiation promoted a significant decrease in the content of water and amides I and III at irradiated bone, evidencing that laser procedure caused an evaporation of the organic content and changed the collagen structure, suggesting that these changes may interfere with the healing process. In this way, these changes should be considered in a clinical application of laser irradiation in surgeries.

Diffuse reflectance study of the effects of bleaching agents in damaged dental pieces

J. Bante-Guerra, R. Trejo-Tzab, J. D. Macias, et al.

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One of the most important subjects of interest in dentistry and teeth preservation is related to the effects of bleaching agents on the integrity of the dental pieces. This is especially crucial when teeth surface has received some damage, generated by chemical, biological and mechanical agents or weathering in the case of dental pieces recovered from burial sites. In this work the time evolution of the effects of bleaching agents on the surface of dental pieces is monitored using diffuse reflectance in the visible spectrum is reported. The effects were monitored in teeth previously subject to chemical agents. Bleaching was induced using commercial whitening products. It is shown that the time evolution of the reflectance depends strongly on the condition of the surface as well as on the thickness of enamel. Additionally the colorimetric analysis of the samples during the bleaching is presented. This is especially useful in for comparing with previous studies. In order to complement our studies, the effects of the bleaching on the surface of the teeth were monitored by scanning electron microscopy.

Time and frequency-domain biomedical photoacoustic imaging: a comparative study

Bahman Lashkari, Andreas Mandelis

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In this study, we compare the time-domain (pulsed laser) and frequency-domain (FD) photoacoustic (PA) imaging techniques with respect to their signal-to-noise ratio (SNR), contrast and axial resolution. Experiments are performed using a dual-mode PA system and under the condition of maximum permissible exposure (MPE) for both methods. An analytical model of photoacoustic effect and a Krimholtz-Leedom-Matthaei (KLM) model for employed transducers are developed and used to analyze the experimental results. Experiments reveal that the contrast of the pulsed method suffers from the oscillating baseline and the resolution of the FD-PA is limited by the finite bandwidth as well as combining the in-phase and quadrature signals to generate the envelope signal; both are the requirements to maximize the SNR. It is shown that by increasing the laser power and decreasing the chirp duration within the safety limits, the SNR of the FD-PA method can be enhanced. Also it is demonstrated that the axial resolution of the FD method can be improved by combining its two channels; amplitude and phase. The improved resolution competes with the high resolution generated by pulsed technique.

Efficient delivery of small interfering RNA into injured spinal cords in rats by photomechanical waves

Takahiro Ando, Shunichi Sato, Terushige Toyooka, et al.

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In the central nervous system, lack of axonal regeneration leads to permanent functional disabilities. In spinal cord injury (SCI), the over-expressions of intermediate filament (IF) proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are mainly involved in glial scar formation; these proteins work as both physical and biochemical barriers to axonal regeneration. Thus, silencing of these IF proteins would be an attractive strategy to treat SCI. In this study, we first attempted to deliver fluorescent probe-labeled siRNAs into injured spinal cords in rats by applying photomechanical waves (PMWs) to examine the capability of PMWs as a tool for siRNA delivery. Intense fluorescence from siRNAs was observed in much broader regions in the spinal cords with PMW application when compared with those with siRNA injection alone. Based on this result, we delivered siRNAs for GFAP and vimentin into injured spinal tissues in rats by applying PMWs. The treatment resulted in efficient silencing of the proteins at five days after SCI and a decrease of the cavity area in the injured tissue at three weeks after SCI when compared with those with siRNA injection alone. These results demonstrate the capability of PMWs for efficient delivery of siRNAs into injured spinal cords and treatment of SCIs.

Simultaneous imaging of light-evoked activities in retinal photoreceptors and inner neurons

Xin-Cheng Yao, Yi-Chao Li, Christianne Strang, et al.

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Simultaneous imaging of different retinal neurons functioning together can benefit better understanding of visual signal processing mechanisms. In this paper we demonstrate that intrinsic optical signal (IOS) imaging allows simultaneous monitoring of stimulus-evoked responses propagating from the photoreceptors to inner retinal neurons. High resolution imaging revealed robust IOSs at the photoreceptor, inner plexiform and ganglion cell layers. While IOSs of the photoreceptor layer were mainly confined to the area directly stimulated by the visible light; IOSs of inner retinal layers spread from the stimulus site into relatively large areas.

Functional near infrared brain imaging with a brush-fiber optode to improve optical contact on subjects with dense hair

Bilal Khan, Chester Wildey, Robert Francis, et al.

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A major obstacle in functional near infrared spectroscopy (fNIRS) and imaging is poor optical contact of the optodes with the scalp due to obstruction by hair. To overcome this problem a novel brush-fiber extension was made as an attachment to existing commercial flat-face fiber bundle optodes. The goal was that the brush-fiber extension would thread through hair and improve optical contact on subjects with dense hair. Simulations and experiments were performed to assess the magnitude of these improvements. Firstly, a Monte Carlo (MC) simulation of photon propagation through the scalp with hair and hair follicles was performed showing a drop in light transmission for different hair densities in the range of 3 to 9 dB. Similar levels of signal loss at individual optodes were determined experimentally in preliminary measurements on a human subject, when comparisons were made for finger tapping activation measurements with and without the brush-fiber extensions. The effects of such losses on reconstructed images were also simulated by using neutral density filters during tissue phantom measurements. As brush-fibers attenuated the signal by approximately 2.5 dB due to imperfect optical contact with the flat-face optode tips we explored the idea of using larger diameter brush-fibers to overcome that loss.

Infrared laser stimulation of retinal and vestibular neurons

Fabrice Bardin, Jean-Michel Bec, Emmanuelle S. Albert, et al.

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The study of laser-neuron interaction has gained interest over the last few years not only for understanding of fundamental mechanisms but also for medical applications such as prosthesis because of the non-invasive characteristic of the laser stimulation. Several authors have shown that near infrared lasers are able to stimulate neurons. It is suggested that a thermal gradient induced by the absorption of the laser radiation on cells is the primary effect but the exact mechanism remains unclear. We show in this work that infrared laser radiations provide a possible way for stimulating retinal and vestibular ganglion cells. We describe relevant physical characteristics allowing safe and reproducible neuron stimulations by single infrared pulses. Calcium fluorescence imaging and electrophysiological recordings have been used to measure ionic exchanges at the neuron membrane. The stimulation system is based on a pulsed laser diode beam of a few mW. Effects of three different wavelengths (from 1470 to 1875 nm) and stimulation durations have been investigated. Variations of the stimulation energy thresholds suggest that the main physical parameter is the water optical absorption. Measurements of the temperature at the cell membrane show that a constant temperature rise is required to stimulate neurons, suggesting a photothermal process.

Development of VCSELs for optical nerve stimulation

Matthew Dummer, Klein Johnson, Mary Hibbs-Brenner, et al.

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Neural stimulation using infrared optical pulses has numerous potential advantages over traditional electrical stimulation, including improved spatial precision and no stimulation artifact. However, realization of optical stimulation in neural prostheses will require a compact and efficient optical source. One attractive candidate is the vertical cavity surface emitting laser. This paper presents the first report of VCSELs developed specifically for neurostimulation applications. The target emission wavelength is 1860 nm, a favorable wavelength for stimulating neural tissues. Continuous wave operation is achieved at room temperature, with maximum output power of 2.9 mW. The maximum lasing temperature observed is 60° C. Further development is underway to achieve power levels necessary to trigger activation thresholds.

Continuous-wave optical stimulation of the rat prostate nerves using an all-single-mode 1455 nm diode laser and fiber system

Serhat Tozburun, Gwen A. Lagoda, Arthur L. Burnett M.D., et al.

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Optical nerve stimulation (ONS) has recently been reported as a potential alternative to electrical nerve stimulation. Continuous-wave (CW) laser stimulation of the prostate cavernous nerves (CN) in a rat model, in vivo, has also been demonstrated in our previous studies. The objective of this study is to present a new all-single-mode-fiber configuration for ONS with the laser operating in CW mode for potential diagnostic applications. An infrared pigtailed single-mode diode laser (λ = 1455 nm) was used in this study for noncontact ONS. This new all-fiber approach introduces several advantages including: (1) a less expensive and more compact ONS system, (2) elimination of alignment of optical components, and (3) an improved spatial beam profile. Successful optical stimulation of the rat CN using this new design was observed after the CN reached a threshold temperature of ~ 41 °C with response times as short as 3 s. Upon further study, this configuration may be useful for identification and preservation of the cavernous nerves during prostate cancer surgery.

Nerve fiber recruitment in the context of hybrid neural stimulation

Austin R. Duke, Hui Lu, Michael W. Jenkins, et al.

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Recently, hybrid neural stimulation combining electrical and optical techniques was demonstrated. By applying a subthreshold electrical stimulus with infrared neural stimulation (INS), hybrid stimulation was shown to reduce INS thresholds as much as 3-fold while maintaining spatial selectivity, thus overcoming the risk of thermally-induced tissue damage associated with INS and the fundamental lack of spatial specificity associated with electrical stimulation. While the potential of hybrid stimulation is evident, a better fundamental understanding of the interaction between tissue, light, thermal gradients and current is necessary before this new stimulation paradigm can be further refined and optimized for clinical implementation. A key element of this understanding is the spatial superposition of the electrical and optical stimuli. A successful hybrid stimulation paradigm requires accurate recruitment of the same neurons by each modality. If the same neurons are not targeted by both electrical and optical stimulation, then hybrid stimulation will suffer from lack of repeatability and consistency. Here we present evidence as to how light and current interact spatially within neural tissue. There exists a finite spatial region that is excitable by hybrid stimulation. This region is shown to change in size and location by altering the optical and electrical components of the hybrid stimulus. By taking advantage of these results, we are now able to achieve greater control of hybrid stimulation and can better apply this promising technology.

Nanopore formation in neuroblastoma cells following ultrashort electric pulse exposure

Caleb C Roth, Jason A Payne, Gerald J. Wilmink, et al.

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Ultrashort or nanosecond electrical pulses (USEP) cause repairable damage to the plasma membranes of cells through formation of nanopores. These nanopores are able to pass small ions such as sodium, calcium, and potassium, but remain impermeable to larger molecules like trypan blue and propidium iodide. What remains uncertain is whether generation of nanopores by ultrashort electrical pulses can inhibit action potentials in excitable cells. In this paper, we explored the sensitivity of excitable cells to USEP using Calcium Green AM 1 ester fluorescence to measure calcium uptake indicative of nanopore formation in the plasma membrane. We determined the threshold for nanopore formation in neuroblastoma cells for three pulse parameters (amplitude, pulse width, and pulse number). Measurement of such thresholds will guide future studies to determine if USEP can inhibit action potentials without causing irreversible membrane damage.

Non-linear stimulation of excitable cells with and without optogenetic sensitization

Shivaranjani Shivalingaiah, Ling Gu, Samarendra K. Mohanty

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Here, we report development of an integrated system for co-registration of patch-clamp measurements with calcium imaging during two-photon stimulation (TPS) of excitable cells sensitized with optogenetic probe, chanelrhodopsin-2 (ChR2). Comparison of calcium changes due to focused two-photon micro-irradiation of excitable cells with and without optogenetic sensitization, revealed wavelength-insensitive injection of extra-cellular calcium via pore formation at high laser beam powers. However, use of defocused/weakly-focused beam allowed sub-threshold stimulation of the excitable cells, revealed by both calcium imaging and whole-cell patch-clamping. Irregular calcium spiking was observed for continuous two-photon defocused micro-irradiation. Even at high extra-cellular calcium conditions, since presence of alltrans- retinal (ATR) was necessary even for detectable calcium increase (and inward current) under defocused twophoton irradiation, role of ChR2 was confirmed as opposed to optoporation, for defocused condition. In the subthreshold stimulation regime, while peak-photocurrents variation with TPS wavelength followed ChR2 two-photon activation spectrum, the power dependence of the current was highly non-linear. Though defocused two-photon beam may cause minimal photo damage while stimulating the cells, the threshold average power required for generating action potential in the ChR2-sensitized cells is higher than that used for routine two-photon imaging.

Millimeter wave-induced changes in membrane properties of leech Retzius neurons

Victor Pikov, Peter H. Siegel

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This study evaluated a novel method for modulation of neuronal excitability using non-invasive delivery of millimeter waves. Millimeter waves at 60 GHz and incident power density of 100-600 μW/cm² were applied to three intact segmental ganglia of the adult leach, and intracellular neuronal activity was recorded from the Retzius neurons using intracellular glass electrode. Transient dosedependent increase in the plasma membrane permeability was observed. In addition, in one of the examined neurons, a decrease in the neuronal firing rate was also evident. The results provide strong evidence for the feasibility of modulating neuronal excitability using non-invasive delivery of millimeter waves, and will be explored further for applications in basic neuroscience and treatment of neurological disorders.

Optical path of infrared neural stimulation in the guinea pig and cat cochlea

Suhrud M. Rajguru, Margaret Hwang, Laura E. Moreno, et al.

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It has been demonstrated previously that infrared neural stimulation (INS) can be used to stimulate spiral ganglion cells in the cochlea. With INS, neural stimulation can be achieved without direct contact of the radiation source and the tissue and is spatially well resolved. The presence of fluids or bone between the target structure and the radiation source may lead to absorption or scattering of the radiation and limit the efficacy of INS. To develop INS based cochlear implants, it is critical to determine the beam path of the radiation in the cochlea. In the present study, we utilized noninvasive X-ray microtomography (microCT) to visualize the orientation and location of the optical fiber within the guinea pig and cat cochlea. Overall, the results indicated that the optical fiber was directed towards the spiral ganglion cells in the cochlea and not the nerve fibers in the center of the modiolus. The fiber was approximately 300 μm away from the target structures. In future studies, results from the microCT will be correlated with physiology obtained from recordings in the midbrain.

Optical stimulation of the auditory nerve: effects of pulse shape

Renee M. Banakis, Agnella I. Matic, Suhrud M. Rajguru, et al.

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It has been demonstrated that spiral ganglion cells in the cochlea can be stimulated with infrared radiation (IR). The potential benefits of infrared neural stimulation (INS) include the possibility of stimulating the neurons without direct physical contact between the stimulation source and the neural tissue and the improved spatial selectivity. In order to determine how INS can be best incorporated in both research and in neural interfaces, it is critical to identify the optimal stimulation parameters for the laser. This study focuses on direct comparison of amplitudes of neural responses evoked by various IR pulse shapes and durations. With the present experiments, the results indicate that the peak power is an important variable for stimulating auditory neurons. While the radiant energy has little effect on amplitudes of compound potentials (CAP) evoked by infrared pulses shorter than 70 μs, it impacts the amplitudes for pulses 80 μs and longer. In addition, we show that the shape of the infrared pulse is important and varying the shape may allow an expansion of response dynamic range while stimulating neurons. The results indicate that square pulses were the most effective pulse shape to evoke CAPs.

Photonic Therapeutics and Diagnostics VII

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