Diffuse optical spectroscopy (DOS) and laser-induced fluorescence (LIF) techniques have widely been used as noninvasive tools for early cancer detection in several organs including the cervix, oral cavity and gastrointestinal tract. Using a combined DOS/LIF approach, one can simultaneously measure the morphology and biochemical composition of tissue and use these features to diagnose malignancy. We report for the first time to our knowledge both the optical properties and native fluorophore characteristics of non-melanoma skin cancer in the UV-visible range. We collected in vivo diffuse reflectance and intrinsic fluorescence measurements from 44 skin lesions on 37 patients. The skin sites were further categorized into three groups of non-melanoma skin cancer according to histopathology: 1) pre-cancerous actinic keratosis 2) malignant squamous cell carcinoma (SCC) and 3) basal cell carcinoma (BCC). We used a custom-built probe-based clinical system that collects both white light reflectance and laser-induced fluorescence in the wavelength range of 350-700 nm. We extracted the blood volume fraction, oxygen saturation, blood vessel size, tissue microarchitecture and melanin content from diffuse reflectance measurements. In addition, we determined the native fluorophore contributions of NADH, collagen and FAD from laser-induced fluorescence for all groups. The scattering from tissue decreased with progression from clinically normal to precancerous actinic keratosis to malignant SCC. A similar trend was observed for clinically normal skin and malignant BCC. Statistically significant differences were observed in the collagen contributions, which were lower in malignant SCC and BCC as compared to normal skin. Our data demonstrates that the mean optical properties and fluorophore contributions of normal, benign and malignant nonmelanoma cancers are significantly different from each other and can potentially be used as biomarkers for the early detection of skin cancer.

The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

As applied to microscopy, the confocal technique is a powerful method that can non-invasively provide depth-resolved information on tissue because of its optical sectioning capability. Raman spectroscopy is a non-invasive optical technique that is very sensitive to the structure and conformation of biochemical constitutes. Combining these two distinct techniques can non-invasively provide depth-resolved biochemical information of the skin in vivo. In this paper, we present a novel confocal Raman spectrometer and preliminary results on in vivo Raman spectral measurements of mouse tumor model. A total of 255 Raman spectra were taken from 15 mice in vivo. We found that Raman spectra of different sub-layers in mouse skin differ significantly. Obvious spectral changes for the dermis were also observed between normal skin and skin immediately overlying subcutaneous tumor.

The light does not penetrate deeply into the skin tissue because of tissue turbidity. Light penetration depth in skin tissue can be increased by using optical clearing agents such as glycerol, glucose and dimethyl sulfoxide(DMSO). The stratum corneum prevents most optical skin clearing agent from diffusing into the tissue. Previous studies demonstrated the optical tissue clearing effect using optical clearing agents and presented several physical methods to improve transdermal delivery of optical clearing agents. In previous study, we introduced a micro-needling method to enhance optical clearing efficacy against skin barrier and suggested quantitative analysis method to evaluate the optical tissue clearing efficacy. In this study, we present a new physical micro-needling method combined with sonophoresis to further enhance the optical tissue clearing efficacy. The optical tissue clearing effect was quantitatively evaluated with a modulation transfer function target placed under ex-vivo porcine skin samples. To improve transdermal delivery of glycerol, 70% glycerol solution as optimal concentration was topically applied. In conclusion, the samples treated with micro-needling method and sonophoresis resulted in noticeable optical tissue clearing effect.

The appearance and color distribution of skin are important characteristics that affect the human perception of health and vitality. Dermatologists and other skin researchers often use color and appearance to diagnose skin conditions and monitor the efficacy of procedures and treatments. Historically, most skin color and chromophore measurements have been performed using reflectance spectrometers and colorimeters. These devices acquire a single measurement over an integrated area defined by an aperture, and are therefore poorly suited to measure the color of pigmented lesions or other blemishes. Measurements of spots smaller than the aperture will be washed out with background, and spots that are larger may not be adequately sampled unless the blemish is homogenous. Recently, multispectral imaging devices have become available for skin imaging. These devices are designed to image regions of skin and provide information about the levels of endogenous chromophores present in the image field of view. This data is presented as four images at each measurement site including RGB color, melanin, collagen, and blood images. We developed a robust segmentation technique that can segment skin blemishes in these images and provide more precise values of melanin, blood, and collagen by only analyzing the segmented region of interest. Results from hundreds of skin images show this to be a robust automated segmentation technique over a range of skin tones and shades.

Hyperspectral imaging is a modality which combines spatial resolution and spectroscopy in one technique. Analysis of hyperspectral data from biological samples is a demanding task due to the large amount of data, and due to the complex optical properties of biological tissue. In this study it was investigated if depth information could be revealed from hyperspectral images using a combination of image analysis and analytic simulations of skin reflectance. It was also investigated if hyperspectral imaging could be utilized to monitor changes in the distribution of hemoglobin species after smoking. Hyperspectral data in the wavelength range 400-1000nm were collected from the forearm of 15 non-smokers and 5 smokers. The hyperspectral images were analyzed with respect to the distribution of hemoglobin species and vascular structures. Changes in the vascular system due to smoking were also evaluated. Principal component analysis (PCA), Spectral angle mapping (SAM), and Mixture tuned matched filtering (MTMF) were used to enhance vascular structures. Emphasis was put on identifying apparent and true absorption spectra for the present chromophores by combining image analysis and an analytical photon transport model. The results show that the depth resolution of hyperspectral images can be better understood using analytical simulations. Modulation of the chromophore spectra by the optical properties of overlying tissue was found to be an important mechanism causing the depth resolution in hyperspectral images. It was also found that hyperspectral imaging and image analysis can be successfully applied to quantify skin changes following smoking.

High-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspect™ has been performed on hundreds of patients and volunteers in Australia, Asia, and Europe. The system enables the in vivo detection of the elastin and the collagen network as well as the imaging of melanin clusters in aging spots. The epidermis-dermis junction can be detected with submicron resolution. One major applications of this novel HighTech imaging tool is the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In particular, the stimulated biosynthesis of collagen can be investigated over long periods of time. The system with its sub-500 nm lateral resolution is able to image age-related modifications of the extracellular matrix on the level of a single elastin fiber.

Background: The intermixing of light reflected from tissue surface and scattered from tissue volume complicates skin surface roughness assessment by laser speckle technique, a non-invasive optical method based on the analysis of the contrast of a speckle pattern. Objective: In this study we investigated optical discrimination methods to separate the two contributions in a speckle pattern. Methods: Three discrimination methods, spatial, polarization and spectral filtering, were implemented to suppress light from skin internal volume in a laser speckle device. In order to determine the effectiveness of the discrimination methods, speckle patterns were obtained from healthy volunteers, and polychromatic speckle contrast was computed before and after each filtering procedure. Results: Speckle contrast increased after discrimination filtering. A simple formula was derived to calculate the speckle contrast associated with light scattered from the skin surface. This corrected speckle contrast was proposed to be used for skin roughness assessment.

Nowadays, documenting the face appearance through imaging is prevalent in skin research, therefore detection and quantitative assessment of the degree of facial wrinkling is a useful tool for establishing an objective baseline and for communicating benefits to facial appearance due to cosmetic procedures or product applications. In this work, an algorithm for automatic detection of facial wrinkles is developed, based on estimating the orientation and the frequency of elongated features apparent on faces. By over-filtering the skin texture image with finely tuned oriented Gabor filters, an enhanced skin image is created. The wrinkles are detected by adaptively thresholding the enhanced image, and the degree of wrinkling is estimated based on the magnitude of the filter responses. The algorithm is tested against a clinically scored set of images of periorbital lines of different severity and we find that the proposed computational assessment correlates well with the corresponding clinical scores.

In our previous study, we delivered plasmid DNA coding for human hepatocyto growth factor (hHGF) to rat skin grafts based on laser-induced stress wave (LISW), by which production of CD31-positive cells in the grafted skins was found to be enhanced, suggesting improved angiogenesis. In this study, we validated the efficacy of this method to accelerate adhesion of grafted skins; reperfusion and reepithelialization in the grafted skins were examined. As a graft, dorsal skin of a rat was exsected and its subcutaneous fat was removed. Plasmid DNA expression vector for hHGF was injected into the graft; on its back surface a laser target with a transparent sheet for plasma confinement was placed, and irradiated with three nanosecond laser pulses at a laser fluence of 1.2 J/cm2 (532 nm; spot diameter, 3 mm) to generate LISWs. After the application of LISWs, the graft was transplanted onto its donor site. We evaluated blood flow by laser Doppler imaging and analyzed reepithelialization based on immunohistochemistry as a function of postgrafting time. It was found that both reperfusion and reepithelialization were significantly enhanced for the grafts with gene transfection than for normal grafts; reepithelialization was completed within 7 days after transplantation with the transfected grafts. These findings demonstrate that adhesion of grafted skins can be accelerated by delivering HGF gene to the grafts based on LISWs.

An infrared pulsed laser was used to irradiate toenails with visible signs of bacterial/fungal infections. Seventeen subjects with both great toes involved were recruited in a podiatric private practice. Toes were randomized to receive either a single treatment or no treatment. The treatment was tolerated by all subjects without anesthesia and there were no occurrences of serious adverse effects. Eleven out of 14 (79%) treated toes improved. Improvement ranged from 2.1 to 6.1 mm over 90 days following a single treatment. There was a highly significant difference (p<0.001) between treated and untreated toes for nail bed clearing.

The great selectivity and the lack of side effects of Photodynamic Therapy make it more advantageous than radiotherapy or chemotherapy. The application of PDT to skin diseases is particularly appropriate, due to the accessibility of this tissue. Common disorders like nonmelanoma skin cancer, that includes basocelullar or squamous cell carcinomas, can be treated with PDT. Conventional procedures, like surgery or radiotherapy, are not so efficient and do not, in general, obtain the same favourable results. PDT in dermatology medical praxis uses fixed protocols depending on the photosensitizer and the optical source used. These protocols are usually provided by the photosensitizer laboratory, and every lesion is treated with the same parameters. In this work we present a photo-chemical model of PDT applied to skin disorders treated with topical photosensitizers. Optical propagation inside the tissue is calculated by means of a 3D diffusion equation, solved via a finite difference numerical method. The photosensitizer degradation or photobleaching is taken into account, as the drug looses efficiency with the irradiation time. With these data the necrosis area is estimated, so this model could be used as a predictive tool to adjust the optical power and exposition time for the particular disease under treatment.

Noninvasive low-level laser devices have been introduced for therapeutic purpose in medicine. However, low-level laser cannot deliver enough photon density to expect positive therapeutic results in deep tissue layer due to light scattering property in tissue. In order to overcome the limitation, this study was aimed to develop a negative pressure applied low-level laser probe to enhance laser transmission and, therefore, photon density in soft tissue. In order to evaluate clinical feasibility of developed laser probe, ex-vivo experiments were performed with porcine skin samples and laser transmissions were quantitatively measured as a function of tissue compression. The laser probe has an air suction hole to apply negative pressure to skin, a transparent plastic body to observe tissue deformation, and a small metallic optical fiber guide to support the optical fiber when negative pressure was applied. By applying negative pressure to the laser probe, the porcine skin under the fiber guide is compressed down and, low-level laser is emitted into the skin. Diffusion images of laser in the skin samples were acquired with a CCD camera and analyzed. Compared to the intensity without compression, the peak intensity of laser beam profiles increased about 2~2.5 times and FWHM (Full Width at Half Maximum) decreased about 1.67~2.85 times. In addition, the peak intensity was linearly increased as a function of compression. In conclusion, we verified the enhancement of laser transmission and therefore, photon density in tissue by applying negative pressure to the developed low-level laser probe and its potential for clinical usefulness.

A clinical trial was conducted at the National Rehabilitation Hospital on 15 individuals to assess whether Rheparan Skin, a bio-engineered component of the extracellular matrix of the skin, is effective at promoting healing of a variety of wounds. Along with standard clinical outcome measures, a spectroscopic camera was used to assess the efficacy of Rheparan skin. Gauzes soaked with Rheparan skin were placed on volunteers wounds for 5 minutes twice weekly for four weeks. Images of the wounds were taken using a multi spectral camera and a digital camera at baseline and weekly thereafter. Spectral images collected at different wavelengths were used combined with optical skin models to quantify parameters of interest such as oxygen saturation (SO2), water content, and melanin concentration. A digital wound measurement system (VERG) was also used to measure the size of the wound. 9 of the 15 measured subjects showed a definitive improvement post treatment in the form of a decrease in wound area. 7 of these 9 individuals also showed an increase in oxygen saturation in the ulcerated area during the trial. A similar trend was seen in other metrics. Spectral imaging of skin wound can be a valuable tool to establish wound-healing trends and to clarify healing mechanisms.

A multi-spectral dermatoscope was used to investigate the effect of laser hair removal. Ten volunteers underwent three laser treatments, 6 weeks apart. In a subsequent trial, three volunteers received one laser treatment after which the skin region was imaged at short intervals. Practical solutions were developed to re-locate the investigated skin area. After exact matching using rigid and elastic registration software, the images showed acute and delayed effects on the hairs, pigment and vasculature after laser hair removal and subsequent healing response. The multi-spectral dermatoscope provides a perfect tool to study the efficacy and side effects of laser hair removal procedures and can be used to optimize the treatment plan.

During laser therapy of port wine stain (PWS) birthmarks, regions of persistent perfusion may exist. We hypothesize that such regions, which are not readily visible, exist even during laser surgery performed by highly experienced clinicians. The objective of this study was to use objective feedback to assess the acute vascular response to laser therapy. We have developed a clinic-friendly laser speckle imaging (LSI) instrument to provide the clinician with real-time images of blood flow during laser therapy. We acquired images from patients undergoing laser therapy of PWS birthmarks at Scripps Clinic and Beckman Laser Institute and Medical Clinic. We extracted blood flow maps from the acquired imaging data. Collectively, we have observed two regimes of patient response to therapy: 1) an immediate increase in perfusion within minutes after laser therapy; and 2) an overall decrease in blood perfusion approximately one hour after laser therapy, with distinct regions of persistent perfusion apparent in the majority of post-treatment blood-flow images. A comparison of blood flow in PWS and adjacent normal skin demonstrated that PWS blood flow can be greater than or sometimes equivalent to that of normal skin. Regions of persistent perfusion frequently exist immediately after laser therapy of PWS birthmarks. Existence of these regions may be correlated to the need for multiple treatment sessions to improve substantially PWS skin appearance. With the use of intraoperative LSI, immediate retreatment of these regions may improve the outcome of each session.

Currently, most investigations of wound healing rely on invasive biopsy followed by histology and immunohistochemistry staining. There is a great need to develop non-invasive techniques for in vivo diagnostic, clinical and scientific evaluation. Here, we performed a comprehensive investigation on the dynamic wound healing process as a response to laser-induced microinjuries using non-invasive imaging techniques such as reflectance laser-scanning confocal microscopy and video microscopy. Eight healthy subjects ranging from Fitzpatrick skin type II-VI with age from 27 to 57 years were recruited. The volar forearm of each subject was treated with a laser device that generates an array of microbeams with an infrared wavelength. The microscopic changes of epidermal cells and collagen during the wound healing process were assessed non-invasively using confocal microscopy. We also developed a quantitative method to evaluate the dynamic wound healing process at the microscopic level in three areas of interest: (1) treated micro-wounding zone, (2) surrounding collateral damage zone and (3) normal area. The depth-dependent intensity profile derived from reflectance confocal microscope images clearly distinguishes the three areas of interest and quantitatively measures the cellular structure-associated changes. A progressive change in depth-dependent intensity profiles in subjects with different ages parallels the clinical observation of wound healing rate. The quantitative analysis developed in this study may find broad applications in assessing the skin response to treatment at a microscopic level.

Nonablative skin remodeling is a new light treatment approach for photodamaged skin. Compared to ablative CO₂ or Er:YAG laser resurfacing, dermabrasion, and chemical peels, the clinical objective of nonablative skin remodeling is to maximize thermal damage to upper dermis while minimizing injury to the epidermis and surrounding tissue, consequently decreasing potential complications and shortening long recuperation periods. Histological analysis of preoperative and postoperative biopsies using H&E or special stains has indicated the dermal thermal injury, which resulting in collagen denaturation, is the most important mechanism of nonablative skin remodeling for improving skin situation. And the extent of improvement of skin situation corresponded to the formation of a new band of dense, compact collagen bundles in the papillary dermis. The diversity of individual skin condition influences the choice of pulsed light treatment parameters, and further influences the degree of dermal thermal damage, thus the efficacy of nonablative skin remodeling remains unstable. Recently, multiphoton microscopy has show a promising application for monitoring skin thermal damage, because collagen could produce strong second harmonic generation (SHG). And SHG intensity is presumably proportional to the percentage of collagen in dermis. In this paper, the auto-fluorescence (AF) intensity and SHG intensity of mice skin irradiated by pulsed Nd:YAG laser were measured and imaged with multiphoton microscope, and the results show the ratio of SHG to AF decreases with the increase of irradiation exposure dose, and could be a quantitative technique to assess dermal thermal damage, and could further benefit the choice of light treatment parameters.

We have demonstrated that the 1.3 µm Swept-Source Optical Coherence Tomography (SS-OCT) may be used in vivo in a non invasive manner to analyze the characteristics of microablative injuries produced by fractional laser treatments. We have found that the depth of the ablative columns depended approximately linearly on the pulse energy ranging from 180±20 µm for 5 mJ laser pulses to 420±60 µm for 20 mJ pulses.

We investigated the validity of photoacoustic (PA) measurement for monitoring granulation tissue formed in the grafted artificial dermis (AD) in rats. We found that the depths of granulation tissue or neovascularities in the grafted AD can be monitored by PA measurement. There was a significant correlation between the PA signal amplitude and the density of neovascularities in the granulation tissue. These results suggest the usefulness of PA measurement for monitoring the adhesion of grafted ADs.

With low risk of complications and no down-time, the non-ablative photorejuvenation is playing an increasing role in the therapy of the photodamaged skin. The light dose is one of the key factors that affect the performance of the photorejuvenation. Monitoring the tissue response during the procedure of laser irradiation would help to determine whether the light dose is appropriate. In this study, we developed a new approach to monitor the instant response of tissue irradiated by laser device by measuring the change of the total attenuation coefficient of the tissue with optical coherence tomography. The total attenuation coefficient was deduced from the raw data obtained by OCT with single scattering mode. Spatial and temporal equalizations were employed to improve the signal-to-noise ratio. We measured in vivo the total attenuation coefficients of the mouse back skin before and after laser irradiation. The total attenuation coefficients of the tissue reduced approximately 60% immediately after laser irradiation and then kept constant in a short time. The reduction of the attenuation coefficient depended on the light dose. These results demonstrated that the new approach could be a potential tool for monitoring in clinic in the future.

The aim of our work was to determine a therapeutic effect of photodynamic therapy (PDT). Twenty five patients with the Bowen's disease, actinic keratosis and basal cell carcinoma (superficial, nodular) were examined. They were treated with photosensitizer - aminolevulinic acid (metabolized in protoporphyrin IX), and the new red light source built of high-power diodes. A new method, based on numerical analysis of fluorescence imaging of tissues, was proposed as a way for controlling therapy.

The performance of wavelet shrinkage algorithms for image-denoising can be improved significantly by considering the statistical dependencies among wavelet coefficients as demonstrated by several algorithms presented in the literature. In this paper, a locally adaptive denoising algorithm using a bivariate shrinkage function is applied to reduce speckle noise in time-domain (TD) optical coherence tomography (OCT) images of the prostate. The algorithm is illustrated using the dual-tree complex wavelet transform. The cavernous nerve and prostate gland can be separated from discontinuities due to noise, and image quality metrics improvements with signal-to-noise ratio (SNR) increase of 14 dB are attained with a sharpness reduction of only 3%.

The cavernous nerves are responsible for erectile function and course along the prostate surface, varying in size and location among patients, making preservation of sexual function challenging after prostate cancer surgery. Electrical stimulation has proven inconsistent and unreliable in identifying these nerves and evaluating nerve function. Optical stimulation of the rat cavernous nerves has recently been reported as a alternative to electrical stimulation, with potential advantages including noncontact stimulation and improved spatial selectivity. This study describes the design of a compact laparoscopic probe for future clinical use in optical nerve stimulation. The 10-Fr (3.4-mm-OD) prototype laparoscopic probe includes an aspheric lens for collimation of the laser beam with a 0.8- mm-diameter spot, coupled with a 200-μm-core optical fiber. A 45° gold-coated rod mirror in the probe tip provides side-firing delivery of the laser radiation. The probe handle houses a miniature linear motorized stage for lateral scanning of the probe tip over a 25-mm line along the prostate surface. A 5.5-W Thulium fiber laser with tunable wavelength range of 1850-1880 nm was tested with the probe. The probe fits through a standard 5-mm-ID laparoscopic port and is capable of delivering pulse energies up to 8 mJ (1.6 J/cm²) at a 2.5-ms pulse duration, well above the threshold (~ 0.35 J/cm²) for optical stimulation of the cavernous nerves.

Raman spectroscopy is an optical technique that can be used to obtain specific molecular information of biological tissues. It has been used successfully to differentiate normal and pre-malignant tissue in many organs. The goal of this study is to determine the possibility to distinguish normal tissue from bladder cancer using this system. The endoscopic Raman system consists of a 6 Fr endoscopic probe connected to a 785nm diode laser and a spectral recording system. A total of 107 tissue samples were obtained from 54 patients with known bladder cancer during transurethral tumor resection. Immediately after surgical removal the samples were placed under the Raman probe and spectra were collected and stored for further analysis. The collected spectra were analyzed using multivariate statistical methods. In total 2949 Raman spectra were recorded ex vivo from cold cup biopsy samples with 2 seconds integration time. A multivariate algorithm allowed differentiation of normal and malignant tissue with a sensitivity and specificity of 78,5% and 78,9% respectively. The results show the possibility of discerning normal from malignant bladder tissue by means of Raman spectroscopy using a small fiber based system. Despite the low number of samples the results indicate that it might be possible to use this technique to grade identified bladder wall lesions during endoscopy.

Human tissues intrinsically contain many fluorophores, as such NADH, elastin, collagen, and flavins, that can be excited and imaged using multiphoton microscopy, up to 150 μm depth. In this work we have used combined two-photon excited fluorescence (TPE), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses have allowed to characterize both healthy and pathological tissue samples. In particular, we have examined tissue samples from healthy bladder mucosa, and bladder carcinoma in-situ (CIS), finding both morphological and spectroscopic differences. From the morphological point of view, cancer cells appeared more elongated with respect to corresponding normal cells; they also exhibited a different nucleus to cytoplasm ratio. From the spectroscopic point of view, we have found differences between the two tissue types in both spectral emission and fluorescence lifetime distribution. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or cystoscope to be used in in-vivo imaging applications.

At the time of diagnosis, approximately 75% of bladder cancers are non-muscle invasive. Appropriate diagnosis and surgical resection at this stage improves prognosis dramatically. However, these lesions, being small and/or flat, are often missed by conventional white-light cystoscopes. Furthermore, it is difficult to assess the surgical margin for negativity using conventional cystoscopes. Resultantly, the recurrence rates in patients with early bladder cancer are very high. This is currently addressed by repeat cystoscopies and biopsies, which can last throughout the life of a patient, increasing cost and patient morbidity. Multiphoton endoscopes offer a potential solution, allowing real time, noninvasive biopsies of the human bladder, as well as an up-close assessment of the resection margin. While miniaturization of the Multiphoton microscope into an endoscopic format is currently in progress, we present results here indicating that Multiphoton imaging (using a bench-top Multiphoton microscope) can indeed identify cancers in fresh, unfixed human bladder biopsies. Multiphoton images are acquired in two channels: (1) broadband autofluorescence from cells, and (2) second harmonic generation (SHG), mostly by tissue collagen. These images are then compared with gold standard hematoxylin/eosin (H&E) stained histopathology slides from the same specimen. Based on a "training set" and a very small "blinded set" of samples, we have found excellent correlation between the Multiphoton and histopathological diagnoses. A larger blinded analysis by two independent uropathologists is currently in progress. We expect that the conclusion of this phase will provide us with diagnostic accuracy estimates, as well as the degree of inter-observer heterogeneity.

Fluorescence detection of early superficial bladder cancer has been well established over the last years. This technique exploits the selective production and accumulation within cancerous tissues of photoactive porphyrins (PaP), mainly protoporphyrin IX (PpIX), after the instillation of hexaminolevulinic acid (Hexvix®) in the bladder. Although the selective production of PpIX and the sensitivity of this procedure are outstanding, its specificity can be improved due to false positive (FP) lesions. Therefore, our current research focuses on the Characterization of positive sites by high magnification cystoscopy. Cancerization process often combines with changes in vascular architecture. It is likely that the visualization of these modifications should allow us to differentiate false and true positive (TP). New methods, using high magnification (HM) endoscopy, are being investigated by our group, and hopefully resulting in a reduced number of biopsies. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during "macroscopic" white light and fluorescence observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. This is realized by an optical setup directly integrated in the cystoscope. We describe here an offclinics calibration procedure that will allow us to quantify the vessel structure and size once we use this optics to observe the bladder mucosa.

Laparoscopic radical prostatectomy (LRP) has revolutionized the surgical treatment of prostate cancer. This procedure permits complete removal of the prostate and seminal vesicles while minimizing pain and recovery time. However, the laparoscopic approach greatly limits the surgeon's tactile sensation during the procedure. This is particularly true with robot-assisted LRP where no tactile feedback is available forcing the surgeon to rely solely on visual cues. The surgeon and pathologist perform intraoperative frozen section pathologic analysis of a few select tissue fragments, but this is time consuming and costly. Concrete conclusions based on such samples are unreliable as they do not reflect the entire surgical margin status. In this case a conservative approach might dictate removal of more marginal material than necessary, thereby compromising the important nerve-sparing aspects of the procedure. In this study, we demonstrate the feasibility of using multi-modal time-gated optical imaging, i.e. time-resolved light reflectance and auto-fluorescence life-time imaging performed by an ICCD (Intensified Charge-Coupled Device) imaging system to enable clinicians to detect positive tumor margins with high sensitivity and specificity over the prostate. Results from animal experiments present the potential of identifying differences in optical signals between prostate cancer and control tissues. Results also show that the use of classification algorithms can identify cancerous regions with high sensitivity and specificity.

Urolithiasis is a common, disturbing disease with high recurrent rate (60% in five years). Accurate diagnosis of urinary stone composition is important in preventing stone recurrence. With the improvement in minimal invasive urological surgery, such as ureteroscopic lithotripsy, and extracorporeal shock wave lithotripsy, stone management becomes not so suffering and effective than before. However, the new problem arises in that less and less stone fragments could be collected because of tiny expelled stone powder after MIUS. The goal of this study is to use Raman spectroscopy (RS) to analyze small stone fragments collected from urine of patients with urolithiasis after MIUS. First, data from five main urinary stones [Calcium oxalate monohydrate (COM), Dicalcium phosphate dehydrate(DCPD), Calcium phosphate hydroxide(hydroxyl apatite, or HAP), Calcium oxalate dehydrate(COD), and uric acid] were established in RS database. Second, we used RS and clinical Fourier Transform Infrared Spectroscopy (FTIR) to analyze stone fragments collected from patients with urolithiasis. Seventeen patients were enrolled in the study and all had comparable results between RS detection and clinical analysis by FTIR. RS approach has successfully detected tiny stone powders with or without fluorescence photobleaching. We successfully measured COM, DCPD, HAP, COD, and uric acid stones. This study demonstrated the feasibility of using RS for conducting the clinical stone analysis from the tiny urinary stone sample. It provided satisfying results and could be applied on clinical practice.

The technique of nephron sparing surgery has matured significantly over the past decade and is emerging as an oncologically sound procedure for the management of renal tumors. Methods of tumor excision as well as parenchymal reconstruction in a hemostaticallly controlled field have evolved to make this procedure safer. In an attempt to find an improoved hemostatic cutting instrument we developed a 1.94 micrometer Laser-Scalpel system in a porcine model. We evaluated data for partial porcine kidney resection performed by a 1.94 micrometer Laser-Scalpel and compared the data to those of a standard HF- (High- Frequency) dissection device. In 12 pigs general anesthesia and a median laparotomy was performed to expose both kidneys. In each pig one kidney was partially resected with the Laser-Scalpel and the other side with the HF-dissection device. The first 6 pigs were euthanized immediately after the procedure. The following 6 pigs were allowed to recover and underwent 2-3 weeks later euthanasia. The final evaluation data included total resection time, blood loss, mass of dissected tissue, total ischemic time and histological examination. Mean resected kidney tissue mass was 4.75 g with the laser system and 5.57 g for the HF-dissector, respectively. Mean estimated blood loss was 22 ml for the Laser- Scalpel and 78.2 ml for the HF-dissection device. Resection time was 9.45 min for the Laser-scalpel compared to 10.16 min. No complications, specifically no postoperative bleeding, occured in any of the animals. Histological evaluation with H&E staining showed a carbonized zone of about 0.57 mm directly at the dissected edge followed by a thermal damaged zone of about 1.25 mm in width. Thereafter healthy tissue was found in all histological samples. Partial kidney resection was easily and fast performed by the use of a 1.94 micrometer Laser-Scalpel system. Hemostasis was highly sufficient, so blood loss was minimal compared to conventional HF-dissection device. Therefore the 1.94 micrometer Laser-Scalpel system is a very promising dissection device for urological surgery.

Complications during laser lithotripsy include optical fiber bending failure resulting in endoscope damage and low irrigation rates leading to poor visibility. Both problems are related to fiber diameter and limited by the Holmium:YAG laser (λ = 2120 nm) multimode beam profile. This study exploits the Thulium fiber laser (λ = 1908 nm) beam profile for higher power transmission through smaller fibers. Thulium fiber laser radiation with 1-ms pulse duration, pulse rates of 10-30 Hz, and 70-μm-diameter spot was coupled into silica fibers with 100, 150, and 200 μm core diameters. Fiber transmission, bending, and endoscope irrigation tests were performed. Damage thresholds for 100, 150, 200 μm fibers averaged 40 W, 60 W, and > 80 W. Irrigation rates measured 35, 26, and 15 ml/min for no fiber, 100, and 200 μm fibers. Thulium fiber laser energy of 70-mJ delivered at 20 Hz through a 100 μm fiber resulted in vaporization and fragmentation rates of 10 and 60 mg/min for uric acid stones. The Thulium fiber laser beam profile provides higher laser power through smaller fibers than the Ho:YAG laser, potentially reducing fiber failure and endoscope damage and allowing greater irrigation rates for improved visibility.

The holmium:yttrium aluminum garnet (YAG) laser is the gold standard laser for intracorporeal lithotripsy. Optical fibers are utilized to transmit laser energy to the surface of a stone for fragmentation. During lithotripsy, fiber tip degradation (burn back) can occur. The exact mechanism for tip degradation and related factors are not completely understood, and have not been investigated. This characteristic is important because fiber burn back may affect diminish fragmentation efficiency, increase operative time, and increase cost due to the need for fiber replacement. We hypothesize that fiber tip degradation (burn back) varies amongst different commercially available holmium:YAG laser fibers.

The contemporary medicine heads towards the minimally-invasive diagnosis and treatment methods. Laser-assisted lithotripsy is a minimally-invasive method for destroying or disruption of human urinary stones. The basic principle is the delivery of laser light to the place of urinary stones followed by the absorprion of laser radiation by the urinary stones material which resulted in ablation or plasma vaporization, and finished by fragmentation of urinary stones and spontaneous draining. For the purpose of minimally-invasive laser light delivery the optical fibres or sealed and flexible hollow waveguides are used. In this study we have compared the ablation effect of Ho:YAG laser (with the generated wavelength 2100 nm) and Er:YAG laser (with the generated wavelength 2940 nm) on the artificial samples (special compressed plaster) and human urinary stones in vitro. The reason for the investigating of Er:YAG laser radiation is the fact that the generated wavelength 2940 nm matches the local absorption of water and therefore it is potentially applicable in various medical branches. Both lasers operated in pulsed free-running regime. The Ho:YAG laser was clinically used laser system with low-OH fibre delivery. The Er:YAG laser was laboratory laser system and for the radiation delivery the special COP/Ag hollow glass waveguide sealed with fused silica cap. The ablation or perforation rates were measured and compared for both laser systems, various pulse energy levels and various interaction samples thickness. The interaction environment was water with temperature 25°C. Finally both lasers were tested for human urinary stones lithotripsy in vitro.

It has recently been shown that laser fibre deterioration leads to a significant decrease of power output during 80 W potassium titanyl phosphate (KTP) laser vaporisation (LV) of the prostate. This decrease results in inefficient vaporisation especially towards the end of the procedure. For the new 120 W lithium-triborate (LBO) High Performance System (HPS) laser not only higher power but also changes in beam characteristics and improved fibre quality have been advertised. However, high laser power has been identified as a risk factor for laser fibre degradation. Between July and September 2008 25 laser fibres were investigated during routine 120 W LBO-LV in 20 consecutive patients. Laser beam power was measured at baseline and after the application of every 25 kJ during the LV procedure. Postoperatively, the surgeon subjectively rated the performance of the respective fibre on a scale from 1 to 4 (1 indicating perfect and 4 insufficient performance). Additionally, microscopic examination of the fibre tip was performed. Median energy applied was 212 kJ. Changes of power output were similar for all fibres. Typically, a steep decrease of power output within the first 50 kJ was followed by a continuous mild decrease until the end of the procedure. After the application of 50 kJ the median power output was 63% (58-73% interquartile range) of the baseline value. The median power output at the end of the 275 kJ-lifespan of the fibres was 42% (40-47%). The median surgeons' rating of the overall performance of the laser fibres was 2 and the median estimated final decrease of power output 60%. Some degree of degradation at the emission window was microscopically detectable in all cases after the procedure. However, even after the application of 275 kJ, these structural changes were only moderate. Minor degradation of the laser fibre was associated with a significant decrease of power output during 120 W LBO-LV. However, following an early, steep decrease, power output remained relatively constant on a medium level for the rest of the fibre's lifespan. The subjective rating by the surgeons is in accordance with these findings. Improved properties of the LBO laser and enhanced fibre quality resulted in an only moderate decrease of power output which allowed for a consistently effective performance.

Preservation of continence and potency after Robotic Assisted Laparoscopic Radical Prostatectomy (RALP) are two key outcome measures that patients consider when comparing different treatment options for localized prostate cancer. Ensuring that positive surgical margins are as low as possible provides oncologic control. Various techniques to optimize these outcomes have been employed. This study presents the early outcomes for Hemostatic Hydrodissection of the Neurovascular Bundles during 86 consecutive RALPs. Positive margin rates were 12.5% overall (9% for pT2 and 28.6% for pT3); continence at 6 months was 100%, at 3 months 90% and at 1 month 66%. In patients with no preoperative erectile dysfunction (preoperative SHIM of 25), 79% had return of erections sufficient for intercourse by 6 months. 2 of these patients were able to have intercourse 2 weeks after surgery. These preliminary findings appear promising.

Our goal was to develop a robotic approach for vasoepididymostomy (RAVE) and vasovasostomy (RAVV) using a 4 arm High Definition Platform (Intuitive Surgical, CA) and present our human in-vivo results. All 3 RAVV procedures were successful and one patient had 102 million motile sperm/ml of ejaculate at 1 month post-op. The RAVE procedure patient only had a few non-motile sperm at 6 month follow up in his ejaculate. The use of robotics seems to offer advantages in terms of ergonomics and suture control. Further evaluation is needed to assess the clinical potential of robotics in vasectomy reversal.

Histotripsy is an extracorporeal ablative technology that utilizes microsecond pulses of intense ultrasound (< 1% duty cycle) to produce nonthermal, mechanical fractionation of targeted tissue. We have previously demonstrated the feasibility of histotripsy prostate ablation. In this study we sought to assess the chronic tissue response, tolerability and safety of histotripsy in a chronic in vivo canine model. Five acute and thirteen chronic canine subjects were anesthetized and treated with histotripsy targeting the prostate. Pulses consisted of 3 cycle bursts of 750 kHz ultrasound at a repetition rate of 300 Hz delivered transabdominally from a highly focused 15 cm aperture array. Transrectal ultrasound imaging provided accurate targeting and real-time monitoring of histotripsy treatment. Prostates were harvested at 0, 7, 28, or 56 days after treatment. Consistent mechanical tissue fractionation and debulking of prostate tissue was seen acutely and at delayed time points without collateral injury. Urothelialization of the treatment cavity was apparent 28 days after treatment. Canine subjects tolerated histotripsy with minimal hematuria or discomfort. Only mild transient lab abnormalities were noted. Histotripsy is a promising non-invasive therapy for prostate tissue fractionation and debulking that appears safe and well tolerated without systemic side effects in the canine model.

Male sterilization (vasectomy) is more successful, safer, less expensive, and easier to perform than female sterilization (tubal ligation). However, female sterilization is more popular, due to male fear of vasectomy complications (e.g. incision, bleeding, infection, and scrotal pain). The development of a completely noninvasive vasectomy technique may allay some of these concerns. Ytterbium fiber laser radiation with a wavelength of 1075 nm, average power of 11.7 W, 1-s pulse duration, 0.5 Hz pulse rate, and 3-mm-diameter spot was synchronized with cryogen cooling of the scrotal skin surface in canine tissue for a treatment time of 60 s. Vas thermal lesion dimensions measured 2.0 ± 0.3 mm diameter by 3.0 ± 0.9 mm length, without skin damage. The coagulated vas bursting pressure measured 295 ± 72 mm Hg, significantly higher than typical vas ejaculation pressures of 136 + 29 mm Hg. Noninvasive thermal coagulation and occlusion of the vas is feasible.

INTRODUCTION: WBPDT has been used to treat resistant superficial bladder cancer, with clinical benefits and associated dose-dependent side effects. OBJECTIVE: The objective of this study was to assess the safety of three sequential WBPDT treatments in patients with resistant non-muscle invasive (NMI) bladder cancer. MATERIALS AND METHODS: 12 males and one female provided written informed consent in this Phase II study. Each patient received intravenous injection of Photofrin® (AXCAN Parma Inc, Canada) at 1.5 mg/kg two days prior to whole bladder laser (630nm) treatment. Assessment of safety and efficacy included weekly urinary symptoms; cystoscopy, biopsy and cytology; and measurement of bladder volume quarterly after each treatment at baseline, six and 12 months. Treatment #2 and/or #3 occurred only in the absence of bladder contracture, and/or disease progression. RESULTS: 13 patients: 12 males and one female have been enrolled and average age of enrollees is 67.1(52 - 87) years. Four patients had Ta-T1/Grade I-III tumors; two patients had CIS associated with T1/GI-III; and seven patients had carcinoma in situ (CIS) only. Three patients received 3/3 treatments, and are evaluable for toxicity; three patients received two treatments only; and seven patients received one treatment only. There was no bladder contracture; transient mild to moderate bladder irritative voiding symptoms of dysuria, urinary frequency, nocturia and urgency occurred in all patients. The three evaluable patients were without evidence of disease at average of 13.1 (7-20) months. CONCLUSION: Three sequential WBPDT treatments might have a favorable toxicity profile in the management of recurrent/ refractory non-muscle invasive bladder cancer.

Photodynamic diagnosis (PDD) is a technique that enhances the detection of tumors during cystoscopy using a photosensitizer which accumulates primarily in cancerous cells and will fluoresce when illuminated by violetblue light. A disadvantage of PDD is the relatively low specificity. In this retrospective study we aimed to identify predictors for false positive findings in PDD. Factors such as gender, age, recent transurethral resection of bladder tumors (TURBT), previous intravesical therapy (IVT) and urinary tract infections (UTIs) were examined for association with the false positive rates in a multivariate analysis. Data of 366 procedures and 200 patients were collected. Patients were instilled with 5-aminolevulinic acid (5-ALA) intravesically and 1253 biopsies were taken from tumors and suspicious lesions. Female gender and TURBT are independent predictors of false positives in PDD. However, previous intravesical therapy with Bacille Calmette-Guérin is also an important predictor of false positives. The false positive rate decreases during the first 9-12 weeks after the latest TURBT and the latest intravesical chemotherapy. Although shortly after IVT and TURBT false positives increase, PDD improves the diagnostic sensitivity and results in more adequate treatment strategies in a significant number of patients.

Objectives: In this study, clinically available pulsed laser systems emitting either in the infrared (IR) - or visible (VIS) spectral region were compared in a standardized manner with respect to their impact on phantom stones in an underwater laboratory set-up. Methods: There were three pulsed laser systems emitting light either in the IR (λ=2100nm: Ho: YAG-laser) or VIS (λ=532nm/1064nm: FREDDY-laser and 598nm: FLPD-laser) spectral range available for this investigation. After determination of the ablation threshold different fragmentation rates were determined in relation to the fluence (depending on pulse energy and fiber diameter) using artificial stones. Results: The threshold value of the laser pulse energy to induce an ablation of artificial stones induced by the different laser systems showed that even the lowest laser settings induced significant ablation with no regards to the repetition rate and fiber diameter. The VIS-lasers showed higher fragmentation rates than the IR-lasers. Conclusions: VIS-lasers are solely useful for laser induced shockwave lithotripsy, while IR-lasers are also in use for other clinical applications (e.g. coagulation and ablation). Investigations on artificial stone fragmentation are useful to compare clinical laser parameter settings but can partially be transferred to clinically urinary stone fragmentation.

Biofilm formation has been demonstrated for many mucosal pathogens such as Haemophilus influenzae. The presence of mucosal biofilms with chronic otitis media with effusion (COME) suggests that bacteria do not clear by antibiotics. Aim: To test the effect of photodynamic therapy (PDT) on H. influenzae biofilm in vitro. Methods: Sixteen biofilms of H. influenzae were maintained on culture chamber with continuous flow cell system. The biofilms were divided into control, laser, photofrin, and PDT groups. For culture group, the biofilms were cultured. For laser group, 7.2 J/cm2 of 632 nm diode laser was irradiated to the biofilms. For photofrin group, photofrins 5 and 25ug/ml were added to the media. For PDT group, photofrins 5 and 25 ug/ml were added to the media following 632 nm diode laser was irradiated (7.2 J/cm2) to the biofilms. Live/Dead (DAPI/PI) stain was performed and biofilms were examined under confocal laser microscope for thickness and density of biofilms. Results: By DAPI/PI staining, significant reduction of biofilms thickness and complete killing of H. influenzae in PDT group with 25µg photofrin was noted while the biofilms were well maintained in the other groups. Conclusion: The results of this study demonstrated that PDT appears to be effective to photoinactivate experimental H. influenzae biofilms in vitro. Clinical implication: PDT can be a possible alternative treatment to antiobiotic treatment on otitis media with biofilm formation.

Introduction: While a variety of materials has been evaluated for replacement of human middle ear ossicles following inflammation, titanium and its alloys have shown excellent sound transmission properties and biocompatibility. However, cartilage thickness at the tympanic membrane interface deteriorates over time, while fibrous tissue formation may dislodge the titanium prosthesis. This study was performed to evaluate the effect of microstructures on titanium surfaces in contact with adjacent biological tissue. Materials and Methods: Titanium samples of 5mm diameter and 0,25mm thickness were structured by means of a Ti:Sapphire femtosecond laser operating at 970nm. The structures applied were lines of parabolic shape (cross-sectional) of 5µm (parallel), 5µm (cross-hatch) and 10µm width (parallel). The inter-groove distance between two maxima was exactly twice the line width. Results: Lines smaller than 5µm were not feasible due to the natural irregularity of the basic material with pits and level changes of up to 2µm. The process showed little debris and constant microstructure shape over the whole structured area (2x2mm). The resulting debris was examined for toxic by-products on human fibrobcytes and chondrocytes. Discussion: The results show that microstructures can be applied on titanium surfaces for human implantation with reproducible and constant shapes. Further studies will focus on cell culture which has suggested a relative selectivity for chondrocyte compared to fibrocyte growth in earlier studies with selected microstructures.

The success of conventional hearing aids and electrical cochlear implants have generally been limited to hearing in quiet situations, in part due to a lack of localized (i.e., frequency specificity) sensorineural activation and subsequent impaired speech discrimination in noise. Laser light is a source of energy that can be focused in a controlled manner and may provide more localized activation of the inner ear, the cochlea. Compound action potentials have been elicited using 2.12 µm laser pulses through activation of auditory nerve fibers (Izzo et al. 2006). Laser stimulation (813 nm) of the cochlea has shown to induce basilar membrane motion and cochlear microphonic potentials (Fridberger et al. 2006). We sought to assess if visible light (green, 532 nm, 10 ns pulses) could be used to consistently activate the cochlea. The laser parameters were selected based on our initial attempt to induce an optoacoustic effect as the energy transfer mechanism to the cochlea. Click evoked auditory brainstem responses (AABRs) were recorded preoperatively in ketamine-anesthetized guinea pigs to confirm normal hearing. The bulla and then the cochlea were exposed. Optically evoked ABRs (OABR) were recorded in response to laser stimulation with a 50 µm optical fiber (532 nm, 10 ns pulses, 500 repetitions, 10 pulses/s; Nd:YAG laser) at the round window (RW) directed towards the basilar membrane (BM). OABRs similar in morphology to acoustically evoked ABRs, except for shorter latencies, were obtained for stimulation through the RW with energy levels between 1.7-30 µJ/pulse. The OABRs increased with increasing energy level reaching a saturation level around 13-15 µJ/pulse. Furthermore the responses remained consistent across stimulation over time, including stimulation at 13 µJ/pulse for over 30 minutes, indicating minimal or no damage within the cochlea with this type of laser stimulation. Overall we have demonstrated that laser light stimulation with 532 nm has potential for a new type of auditory prosthesis that can activate the cochlea without any apparent functional damage. Further studies are needed to determine the optimal laser parameters and fiber placement locations for localized and tonotopic activation.

Objectives: To determine the feasibility and accuracy of the Niris Optical Coherence Tomography (OCT) system in imaging of the mucosal abnormalities of the head and neck. The Niris system is the first commercially available OCT device for applications outside ophthalmology. Methods: We obtained OCT images of benign, premalignant and malignant lesions throughout the head and neck, using the Niris OCT imaging system (Imalux, Cleveland, OH). This imaging system has a tissue penetration depth of approximately 1-2mm, a scanning range of 2mm and a spatial depth resolution of approximately 10-20μm. Imaging was performed in the outpatient setting and in the operating room using a flexible probe. Results: High-resolution cross-sectional images from the oral cavity, nasal cavity, ears and larynx showed distinct layers and structures such as mucosa layer, basal membrane and lamina propria, were clearly identified. In the pathology images disruption of the basal membrane was clearly shown. Device set-up took approximately 5 minutes and the image acquisition was rapid. The system can be operated by the person performing the exam. Conclusions: The Niris system is non invasive and easy to incorporate into the operating room and the clinic. It requires minimal set-up and requires only one person to operate. The unique ability of the OCT offers high-resolution images showing the microanatomy of different sites. OCT imaging with the Niris device potentially offers an efficient, quick and reliable imaging modality in guiding surgical biopsies, intra-operative decision making, and therapeutic options for different otolaryngologic pathologies and premalignant disease.

Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and has been used to image the human larynx during surgical endoscopy. The design of a long GRIN lens based probe capable of capturing images of the human larynx by use of swept-source OCT during a typical office-based laryngoscopy examination is presented. In vivo OCT imaging of the human larynx is demonstrated with 40 fame/second. Dynamic vibration of the vocal folds is recorded to provide not only high-resolution cross-sectional tissue structures but also vibration parameters, such as the vibration frequency and magnitude of the vocal cord, which provide important information for clinical diagnosis and treatment, as well as in fundamental research of the voice. Office-based OCT is a promising imaging modality to study the larynx.

Background: Clinical laser lithotripsy in urology promises a good fragmentation combined with a minimal risk of soft tissue damage and low medical complications. This in vitro study investigates the fragmentation of salivary stones by means of two clinically used laser systems. Materials and Methods: The effects induced by the FREDDY laser (WOM, Germany, λ=532 nm / 1,064 nm, Epulse=120-160 mJ/pulse) and the Ho:YAG (AURIGA, StarMedTec, Germany, λ=2,100 nm,Epulse=300-800 mJ/ pulse) on clinical salivary calculi (n=15) and on salivary gland tissue were investigated using clinical laser parameter settings. All experiments were performed in an under water experimental set-up using flexible fibres (core diameter 230μm) positioned in front of each specimen. In order to assess fragmentation efficacy, each stone was placed on a grating (rhombic mash-diameter 1-3 mm). The fragmentation rate was calculated with respect to the energy applied (mg/J), to the number of pulses (mg/pulse), and to the time needed (mg/minute). In addition the composition of the stones were analysed spectrographically. The soft tissue interaction on human salivary duct mucosa was examined histologically (HE-staining). Results: Spectrographic composition of the salivary stones showed a two component ratio of protein/carbonate apatite varying between 5/95 and 25/75. Stones treated by the Ho:YAG were vaporised in a milling-like process, while using the FREDDY laser stones are cracked into pieces and fragmentation failed in two cases. The fragmentation rates achieved by the FREDDY laser were greater than those of the Ho:YAG laser, but fragments mainly bigger. A dependency on the composition of the stones could not be found. Laser pulse effects on soft tissue were found slightly beyond the mucosa. Conclusion: This study clearly demonstrated the different processes of destroying salivary stones using two different laser systems. While the Ho:YAG vaporises the calculi in a more milling and soft sense, the FREDDY shows a more cracking and explosive destruction. Although both laser systems showed little direct risk to the surrounding tissue, it has to be proven whether cracked and accelerated particles could cause harm to soft tissue. With respect to this, further in vitro studies and clinical treatments in selected cases are needed to proof these results.

Electromechanical reshaping (EMR) of cartilage is a novel technique that has significant potential for use in facial reconstructive surgery. EMR achieves permanent shape change by initiating electrochemical redox reactions in the vicinity of stress concentrations, thereby altering mechanical properties of tissue matrix. This study reports the use of a six electrode needle-based geometric configuration to reshape cartilage. Rectangular samples (24 x 12 x 1 mm) of rabbit nasal septal cartilages were bent at a right angle in a precision-machined reshaping jig. Two parallel arrays of three platinum needle electrodes were each inserted into cartilage along the bend at 3 mm from the bend line. One array served as an anode and the other as cathode. Constant voltage at 1, 2, 4, 6, and 8 volts was applied to the arrays for 2 minutes. The specimens were then removed from the jig and rehydrated for 15 minutes in phosphate buffered saline. Following rehydration, bend angles and thicknesses were measured. Bend angle increased with increasing voltage and application time. No statistically significant bending was observed below 6 volts for 2 minutes application time. Maximum bend angle of 33 ± 8 degrees or reshaping degree of 33% was observed at 8 volts applied for 2 minutes. Current flow was small (< 0.1 A) for each case. Sample thickness was 0.9 ± 0.2 mm. ANOVA analysis showed that cartilage thickness had no significant impact on the extent of reshaping at given voltage and application time. The six needle electrode geometric configuration conforms to the voltage- and time-dependent trends predicted by previous EMR studies. In the future, the reshaping properties of other geometric configurations will be explored.

Optical Coherence Tomography (OCT) is rapidly becoming the method of choice for assessing arterial wall pathology in vivo. Atherosclerotic plaques can be diagnosed with high accuracy, including measurement of the thickness of fibrous caps, permitting an assessment of the risk of rupture. While the OCT image presents morphological information in highly resolved detail, it relies on interpretation of the images by trained readers for the identification of vessel wall components and tissue type. We developed a framework to aid the recognition of these atherosclerotic plaque constituents, based on the optical attenuation coefficient of the tissue. Based on a single-scattering model and using the point spread functions of individually characterized imaging catheters, several parameters need to be set in the analysis that may influence the results. Here, we present a simulation study used to optimize these parameters.

We developed coronary artery phantoms that should be of great use for intravascular optical coherence tomography. Our phantoms mimic the OCT signal profile of coronary arteries, show mechanical properties approaching those of real tissue, and are durable.

Atherosclerotic plaque composition has been associated with plaque instability and rupture. This study investigates the use of fluorescence lifetime imaging microscopy (FLIM) for mapping plaque composition and assessing features of vulnerability. Measurements were conducted in atherosclerotic human aortic samples using an endoscopic FLIM system (spatial resolution of 35 µm; temporal resolution 200 ps) developed in our lab which allows mapping in one measurement the composition within a volume of 4 mm diameter x 250 µm depth. Each pixel in the image represents a corresponding fluorescence lifetime value; images are formed through a flexible 0.6 mm side-viewing imaging bundle which allows for further intravascular applications. Based on previously recorded spectra of human atherosclerotic plaque, fluorescence emission was collected through two filters: f1: 377/50 and f2: 460/60 (center wavelength/bandwidth), which together provides the greatest discrimination between intrinsic fluorophores related to plaque vulnerability. We have imaged nine aortas and lifetime images were retrieved using a Laguerre expansion deconvolution technique and correlated with histopathology. Early results demonstrate discrimination using fluorescence lifetime between early, lipid-rich, and collagen-rich lesions which are consistent with previously reported time-resolved atherosclerotic plaque measurements.

The existing non-invasive optical methods of the hemoglobin (Hb) and myoglobin (Mb) estimation in cardiac tissues imply knowledge of the light pathlength (L) when various modifications of Lambert-Beer law for either spectrophotometry or light diffuse reflectance is applied. For Hb and/or Mb quantification in tissue, a few invasive (biochemical) approaches were applied. For L (differential pathlength factor; DPF) determination in tissue, special optical methods were used. No approaches have been proposed to simultaneously and non-invasively determine Hb/Mb and L in cardiac or other muscle tissues. In the present study, the first derivative of the NIR diffuse reflectance spectrum is shown to be effective in simultaneous determination of Hb+Mb concentration (in mM) and L (in mm) in cardiac tissue in vivo. The results showed that measured in a few minutes in a normal pig heart in vivo the total Hb+Mb concentration was 0.9-1.2 mM of heme, tissue oxygen saturation parameter (OSP) was approximately 65%, and DPF at 700-965 nm was of 2.7-2.8. At the experimental ischemia, total [Hb+Mb] decreased by 25%, OSP reduced to zero, while DPF did not change. These results correlated with the previously published. The method may be applied during open-heart surgery, heart studies ex vivo or to any muscle tissue to continuously and non-invasively monitor the [Hb+Mb] content and oxygenation as well as L, which may reflect the changes in tissue structure.

Myocardial fibrosis, a common sequela of cardiac hypertrophy, has been shown to be associated with arrhythmias in experimental models. Some research has indicated that myocardial fibrosis plays an important role in predisposing patients to atrial fibrillation. Second harmonic generation (SHG) is an optically nonlinear coherent process to image the collagen network. In this presentation, we observe the SHG images of the collagen matrix in atrial myocardium and we analyzed of collagen fibers arrangement by using Fourier-transform analysis. Moreover, comparing the SHG images of the collagen fibers in atrial myocardium between normal sinus rhythm (NSR) and atrial fibrillation (AF), our result indicated that it is possible to realize the relation between myocardial fibrosis and AF.

Cardiovascular disease is the number one cause of death worldwide. It is broadly defined to include anything which adversely affects the heart or blood vessels. One-third of Americans have one or more forms of it. By one estimate, average human life expectancy would increase by seven years if it were eliminated. The mainstream medical model seeks mostly to "manage" cardiovascular disease with pharmaceuticals or to surgically bypass or reopen blocked vessels via angioplasty. These methods have proven highly useful and saved countless lives. Yet drug therapy may be costly and ongoing, and it carries the risk of side effects while often doing little or nothing to improve underlying health concerns. Similarly, angioplasty or surgery are invasive methods which entail risk. Laser therapy¹ regenerates tissue, stimulates biological function, reduces inflammation and alleviates pain. Its efficacy and safety have been increasingly well documented in cardiovascular disease of many kinds. In this article we will explore the effects of laser therapy in angina, atherosclerosis, coronary artery disease, hypertension, hyperlipidemia, myocardial infarction, stroke and other conditions. The clinical application of various methods of laser therapy, including laserpuncture and transcutaneous, supravascular and intravenous irradiation of blood will be discussed. Implementing laser therapy in the treatment of cardiovascular disease offers the possibility of increasing the health and wellbeing of patients while reducing the costs and enhancing safety of medical care.

Laser angioplasty, for example XeCl excimer laser coronary angioplasty (ELCA), has gained more attention for the treatment of serious stenosis blocked by plaque. Low degrees of thermal damage after ablation of atherosclerotic plaques have been achieved by ELCA. However, the large number of risks associated with the procedure, for example, dissections or perforations of the coronary arteries limits its application. A laser treatment technique with high ablation efficiency but low arterial wall injury is desirable. Mid-infrared laser with a wavelength of 5.75 µm is selectively well absorbed in C=O stretching vibration mode of ester bonds in cholesteryl ester. The purpose of this study is to determine the effectiveness of nanosecond pulsed laser at 5.75 µm irradiation for atherosclerotic plaques. We made a study on the irradiation effect to atherosclerotic plaques in tunica intima in a wet condition. In this study, we used a mid-infrared tunable solid-state laser which is operated by difference-frequency generation, with a wavelength of 5.75 µm, a pulse width of 5 ns and a pulse duration of 10 Hz as a treatment light source, and a thoracic aorta of WHHLMI rabbit as an atherosclerosis model. As a result, less-invasive interaction parameters for removing atherosclerotic plaques were confirmed. This study shows that the nanosecond pulsed laser irradiation at 5.75 µm is a powerful tool for selective and less-invasive treatment of atherosclerotic plaques.

In this paper an improved particle swarm optimization algorithm (IPSO) for ultrasound applications is proposed. In order to overcome the drawbacks of standard PSO, some improved mechanisms for velocity updating, the exceeding boundary control, global best perturbation and the simplified quadratic interpolation (SQI) operator are adopted. To show the effectiveness of the proposed algorithm, a selected set of numerical example, concerned with linear as well as planar array, is presented. Simulation results show that the refined pinpointing search ability and the global search ability of the proposed algorithm are significantly improved when compared to the particle swarm optimization (PSO) and Genetic Algorithm (GA).

This study aims to determine the effect of varying gadopentetate dimeglumine (Gd-DTPA) dose on Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) tracking of brain tumor photodynamic therapy (PDT) outcome. Methods: We injected 2.5 x 10⁵ U87 cells (derived from human malignant glioma) into the brains of six athymic nude rats. After 9, 12, and 13 days DCE-MRI images were acquired on a 9.4 T micro-MRI scanner before and after administration of 100, 150, or 200 μL of Gd-DTPA. Results: Tumor region normalized DCE-MRI scan enhancement at peak was: 1.217 over baseline (0.018 Standard Error [SE]) at the 100 μL dose, 1.339 (0.013 SE) at the 150 μL dose, and 1.287 (0.014 SE) at the 200 μL dose. DCE-MRI peak tumor enhancement at the 150 μL dose was significantly greater than both the 100 μL dose (p < 3.323E-08) and 200 μL dose (p < 0.0007396). Discussion: In this preliminary study, the 150 μL Gd-DTPA dose provided the greatest T1 weighted contrast enhancement, while minimizing negative T2* effects, in DCE-MRI scans of U87-derived tumors. Maximizing Gd-DTPA enhancement in DCE-MRI scans may assist development of a clinically robust (i.e., unambiguous) technique for PDT outcome assessment.

We report on a pilot study demonstrating a proof of concept for the passive delivery of nanoshells to an orthotopic tumor where they induce a local, confined therapeutic response distinct from that of normal brain resulting in the photo-thermal ablation of canine Transmissible Venereal Tumor (cTVT) in a canine brain model. cTVT fragments grown in SCID mice were successfully inoculated in the parietal lobe of immuno-suppressed, mixed-breed hound dogs. A single dose of near-infrared absorbing, 150 nm nanoshells was infused intravenously and allowed time to passively accumulate in the intracranial tumors which served as a proxy for an orthotopic brain metastasis. The nanoshells accumulated within the intracranial cTVT suggesting that its neo-vasculature represented an interruption of the normal blood-brain barrier. Tumors were thermally ablated by percutaneous, optical fiber-delivered, near-infrared radiation using a 3.5 W average, 3-minute laser dose at 808 nm that selectively elevated the temperature of tumor tissue to 65.8±4.1ºC. Identical laser doses applied to normal white and gray matter on the contralateral side of the brain yielded sub-lethal temperatures of 48.6±1.1ºC. The laser dose was designed to minimize thermal damage to normal brain tissue in the absence of nanoshells and compensate for variability in the accumulation of nanoshells in tumor. Post-mortem histopathology of treated brain sections demonstrated the effectiveness and selectivity of the nanoshell-assisted thermal ablation.

The highly malignant brain tumor, glioblastoma multiforme (GBM), is difficult to fully delineate during surgical resection due to its infiltrative ingrowth and morphological similarities to surrounding functioning brain tissue. Selectiveness of GBM to 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) is reported by other researchers to visualize tumor margins under blue light microscopy. To allow objective detection of GBM, a compact and portable fiber optic based fluorescence spectroscopy system is developed. This system is able to deliver excitation laser light (405 nm) in both the continuous and pulsed mode. PpIX fluorescence peaks are detected at 635 and 704 nm, using a fiber-coupled spectrometer. It is necessary to optimize the detection efficiency of the system as the PpIX quickly photobleaches during the laser illumination. A light dose of 2.5 mJ (fluence rate = 9 mJ/mm²) is experimentally approved to excite an acceptable level of fluourescence signal arising from glioblastoma. In pulsed illumination mode, an excitation dose of 2.5 mJ, with a dark interval of 0.5 s (duty cycle 50%) shows a significantly shorter photobleaching time in comparison to the continuous illumination mode with the same laser power (p < 0.05). To avoid photobleaching (the remaining signal is more than 90% of its initial value) when measuring with 2.5 mJ delivered energy, the time for continuous and pulsed illumination should be restricted to 2.5 and 1.1 s, respectively.

The utility of photochemical internalization (PCI) for the treatment of malignant gliomas was investigated in vitro using: (1) monolayers consisting of F98 rat glioma cells, and (2) human glioma spheroids established from biopsy-derived glioma cells. In both cases, the cytotoxicity of AlPcS_2a- based PCI of bleomycin was compared to: (1) AlPcS_2a-PDT, and (2) bleomycin. In all cases, monolayers and spheroids were incubated in AlPcS_2a (18 h), bleomycin (4 h), or AlPcS_2a (18 h) + bleomycin (4 h) and were subsequently exposed to 670 nm light. Toxicity was evaluated using colony formation assays or spheroid growth kinetics. Neither F98 rat glioma cells in monolayer nor human glioma spheroids were found to be particularly sensitive to the effects of low irradiance (5 mW cm^-2), low radiant exposure (1.5 J cm^-2) AlPcS_2a -PDT. Bleomycin was found to be moderately toxic to F98 cells in monolayer at relatively low concentrations - incubation of F98 cells in 0.1 μg ml^-1 for 4 hours resulted in 80% survival. Under similar incubation conditions, the effects of bleomycin on human glioma spheroids were negligible. In both in vitro systems investigated, the PCI effect was found to be significant. For example, PCI consisting of a radiant exposure of 1.5 J cm^-2 together with 0.25 μg ml^-1 bleomycin resulted in approximately 20 and 65 % survival of F98 rat glioma cells and human glioma spheroids respectively. These results show that AlPcS_2a-mediated PCI can be used to enhance the efficacy of chemotherapeutic agents such as bleomycin in malignant gliomas.

Introduction: Failure to eradicate infiltrating glioma cells using conventional treatment regimens results in tumor recurrence and is responsible for the dismal prognosis of patients with glioblastoma multiforme (GBM). This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB) which prevents the delivery of most anti-cancer agents. We have evaluated the ability of photochemical internalization (PCI) to selectively disrupt the BBB in rats. This will permit access of anti-cancer drugs to effectively target the infiltrating tumor cells, and potentially improve the treatment effectiveness for malignant gliomas. Materials and Methods: PCI treatment, coupling a macromolecule therapy of Clostridium perfringens (Cl p) epsilon prototoxin with AlPcS_2a-PDT, was performed on non-tumor bearing inbred Fisher rats. T1-weighted post-contrast magnetic resonance imaging (MRI) scans were used to evaluate the extent of BBB disruption which can be inferred from the volume contrast enhancement. Results: The synergistic effect of PCI to disrupt the BBB was observed at a fluence level of 1 J with an intraperitoneal injection of Cl p prototoxin. At the fluence level of 2.5J, the extent of BBB opening induced by PCI was similar to the result of PDT suggesting no synergistic effect evoked under these conditions. Conclusion: PCI was found to be highly effective and efficient for inducing selective and localized disruption of the BBB. The extent of BBB opening peaked on day 3 and the BBB was completed restored by day 18 post treatment.

In the current research, diffuse optical imaging (DOI) is used for the first time towards studies related to sociocommunication impairments, which is a characteristic feature of autism. DOI studies were performed on normal adult volunteers to determine the differences in the brain activation (cognitive regions) in terms of the changes in the cerebral blood oxygenation levels in response to joint and non-joint attention based stimulus (i.e. socio-communicative paradigms shown as video clips). Functional connectivity models are employed to assess the extent of synchronization between the left and right pre-frontal regions of the brain in response to the above stimuli.

In this work we focused on time-resolved measurements in diffusive media performed at small (few millimeters) source-detector distances in reflectance geometry. This configuration has been predicted to have better contrast, better spatial resolution, and lower noise than the typical measurements performed at few centimeters. In our instrumental set-up we exploited a fast-gating (rise-time < 400 ps) front-end electronics enabling a silicon Single- Photon Avalanche Diode (SPAD) for time-correlated single-photon counting. By means of this detector, we can acquire "late" photons of the diffused light collected 2 mm apart from the injection point. This is possible because the fast gated SPAD rejects the huge amount of "early" photons which otherwise would saturate the detection electronic chain. The time resolution of the set-up is 100 ps. The instrument has been validated on both homogeneous and inhomogeneous (high absorbing inclusion at different depths inside) tissue phantoms with different optical properties. We obtained diffused time-resolved curves with dynamic ranges of about 107. Moreover, we demonstrated good agreement between the measured time-resolved contrasts and those calculated by Monte Carlo numerical simulations.

Simultaneously acquiring cortical functional Near Infrared Spectroscopy (fNIRS) during repeated Transcranial Magnetic Stimulation (rTMS) offers the possibility of directly investigating the effects of rTMS on brain regions without quantifiable behavioral changes. In this study, the left motor cortex and subsequently the left prefrontal cortex were stimulated at 1 Hz while fNIRS data was simultaneously acquired. Changes in hemodynamic signals were measured on both ipsilateral and contralateral sides. In each cortex, a significantly larger decrease in the concentration of oxygenated hemoglobin and a smaller increase in the concentration of deoxygenated hemoglobin during the stimulation periods were observed in both the motor and prefrontal cortices. The ipsilateral and contralateral changes showed high temporal consistency. Same experiment was repeated for each subject 2 or 3 days later. The hemodynamic responses associated with the stimulation showed good reproducibility in two sessions. To our knowledge, this is the first report of simultaneous fNIRS measurement of ipsilateral and contralateral changes of either the motor or prefrontal cortex during rTMS stimulation.

Functional near-infrared brain imaging (fNIRI) and event-related potential (ERP) were used simultaneous to detect the prefrontal cortex (PFC) which is considered to execute cognitive control of the subjects while performing the Chinese characters color-word matching Stroop task with event-related design. The fNIRI instrument is a portable system operating at three wavelengths (735nm & 805nm &850nm) with continuous-wave. The event-related potentials were acquired by Neuroscan system. The locations of optodes corresponding to the electrodes were defined four areas symmetrically. In nine native Chinese-speaking fit volunteers, fNIRI measured the hemodynamic parameters (involving oxy-/deoxy- hemoglobin) changes when the characteristic waveforms (N500/P600) were recorded by ERP. The interference effect was obvious as a longer reaction time for incongruent than congruent and neutral stimulus. The responses of hemodynamic and electrophysiology were also stronger during incongruent compared to congruent and neutral trials, and these results are similar to those obtained with fNIRI or ERP separately. There are high correlations, even linear relationship, in the two kinds of signals. In conclusion, the multi-modality approach combining of fNIRI and ERP is feasible and could obtain more cognitive function information with hemodynamic and electrophysiology signals. It also provides a perspective to prove the neurovascular coupling mechanism.

In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RA-SHG) capable of optically recording fast membrane potential events occurring in a wide-field configuration. The RA-SHG microscope in combination with a bulk staining method with FM4-64 was used to simultaneously record electrical activity from clusters of Purkinje cells (PCs) in acute cerebellar slices. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where APs were recorded in a single trial without averaging. These results show the strength of this technique to describe the temporal dynamics of neuronal assemblies.

μMany fluorescent tools have been developed through the past decades in order to better understand the physiology at a cellular level. They are generally used for microscopy or endoscopy, in vivo and in vitro, but they are also usable with fluorescent sensors such as fiber optic sensors. Among these tools, fluorescent ion indicators have been widely used to understand ionic dynamics into living cells. Indicators are mainly designed for Ca²⁺, although K⁺ is also an important target for its role in maintaining cellular membrane potential and regulating many other electrophysiological phenomena. Here we propose a technique to improve the use of a miniature fiber optic sensor to sense potassium dynamics in vivo. Due to the lack of commercially available potassium indicators, we are using a UV excitable indicator (PBFI). The UV excitation light induces unwanted fluorescence and/or luminescence from the optical components including the sensing fiber, leading to a poor signal-to-noise ratio. Our technique uses a UV diode pumped Q-switched source from CrystaLaser, a time gated acquisition system and a delay fiber that improve the signal-to-noise ratio and allow reducing the sensor size and the light intensity provided to the tissue, thus diminishing UV induced photodamage. Using this technique we have been able to record intracellular potassium fluctuations in vivo with fiber optic sensors of 10μm diameter.

Using a near infrared (NIR) light flood-illumination imager equipped with a high-speed CCD camera, we demonstrated reflected light imaging of stimulus-evoked retinal ON and OFF responses in isolated, but intact, frog eye. Both fast and slow transient intrinsic optical signals (IOSs) were observed. Fast optical response occurred immediately after the stimulus onset, and correlated tightly with the ON and OFF edges of the visible light stimulus. High resolution images revealed both positive (increasing) and negative (decreasing) IOSs, and dynamic optical change at individual CCD pixels could often exceed 10% of the background light intensity. Our experiment on isolated eye suggests that further development of fast, high resolution fundus imager will allow robust detection of fast IOSs in vivo, and thus allow noninvasive, three-dimensional evaluation of retinal neural function.

The Prefrontal Cortex (PFC) in response to anagram tasks was investigated using multi-channel, functional Near Infrared Spectroscopy (fNIRS). The study focused on the variability of PFC response with variable anagram durations (executing time). The duration of the anagram ranged from a few seconds to over one minute. The topographic image of PFC showed high lateral consistency across different anagram durations and did not show remarkable discrimination between two difficulty levels: 4-letter and 6-letter anagrams. The superposition principle of a linear system model was employed to investigate the possible non-linearity between three discrete duration subgroups (D1 = 2 ± 0.33, D2 = 4 ± 0.67, and D3 = 8 ± 1.00 sec) and their resultant fNIR readings. Such analysis showed clearly a nonlinear behavior in fNIRS signals within the duration range of less than 4 sec, agreeing with the previous studies on cognitive response measured with functional Magnetic Resonant Imaging (fMRI). To our knowledge, this is the first report on the nonlinearity of cognitive response using fNIRS.

We evaluated frontal brain activation during a working memory task with graded levels of difficulty in a group of 19 healthy subjects, by means of time-resolved fNIRS technique. Brain activation was computed, and was then separated into a "block-related" and a "tonic" components. Load-related increases of blood oxygenation were studied for the four different levels of task difficulty. Generalized Linear Models were applied to the data in order to explore the metabolic processes occurring during the mental effort and, possibly, their involvement in short term memorization. Results attest the presence of a persistent attentional-related metabolic activity, superimposed to a task-related mnemonic contribution. Moreover, a systemic component probably deriving from the extra-cerebral capillary bed was detected.