Reflectance confocal microscopy (RCM) has seen increasing clinical application for noninvasive diagnosis of skin cancer. Identifying the location of the dermal-epidermal junction (DEJ) in the image stacks is key for effective clinical imaging. For example, one clinical imaging procedure acquires a dense stack of 0.5x0.5mm FOV images and then, after manual determination of DEJ depth, collects a 5x5mm mosaic at that depth for diagnosis. However, especially in lightly pigmented skin, RCM images have low contrast at the DEJ which makes repeatable, objective visual identification challenging. We have previously published proof of concept for an automated algorithm for DEJ detection in both highly- and lightly-pigmented skin types based on sequential feature segmentation and classification. In lightly-pigmented skin the change of skin texture with depth was detected by the algorithm and used to locate the DEJ. Here we report on further validation of our algorithm on a more extensive collection of 24 image stacks (15 fair skin, 9 dark skin). We compare algorithm performance against classification by three clinical experts. We also evaluate inter-expert consistency among the experts. The average correlation across experts was 0.81 for lightly pigmented skin, indicating the difficulty of the problem. The algorithm achieved epidermis/dermis misclassification rates smaller than 10% (based on 25x25 mm tiles) and average distance from the expert labeled boundaries of ~6.4 μm for fair skin and ~5.3 μm for dark skin, well within average cell size and less than 2x the instrument resolution in the optical axis.

One of the major structural proteins in human skin is collagen. Collagen and its crosslinks are essential for the mechanical stability of the skin. Looking at extrinsically aged human skin (photo damaged skin) we find a decrease of mature collagen crosslinks. Immature crosslinks an indicator of the collagen turnover are decreasing as well in extrinsically aged skin. Hence, we assume that a certain range of mature and immature crosslinks reflect a 'good quality' of collagen in terms of photoaging. In this study we established in vitro models of reduced crosslinking. We found that reduced collagen crosslinking resulted in a higher Second Harmonic Generation (SHG) intensity. Furthermore, we found a higher fibril diameter after crosslink reduction without an increase in collagen concentration. SHG is generated by a non-linear effect of femtosecond laser irradiation on collagen molecules. This effect might be influenced by the interspaces of the collagen molecules within the collagen fibril. From these findings the following hypothesis was introduced: reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril resulting in an enhanced SHG signal. Furthermore, in this study the fluorescence lifetime (FLIM) of collagen fluorescence was found to decrease in the in vitro models of reduced crosslinking. We speculate on possible mechanisms being responsible for the decrease in lifetime. Future in vivo measurements of the two parameters (SHG and FLIM) could lead to information about the collagen crosslink status, and therefore the status of photoaging of the skin.

High voltage electrical injuries may lead to irreversible tissue damage or even death. Research on tissue injury following high voltage shock is needed and may yield stage-appropriate therapy to reduce amputation rate. One of the mechanisms by which electricity damages tissue is through Joule heating, with subsequent protein denaturation. Previous studies have shown that blood flow had a significant effect on the cooling rate of heated subcutaneous tissue. To assess the thermal damage in tissue, this study focused on monitoring changes of temperature and optical properties of skin next to high voltage wounds. The burns were created between left fore limb and right hind limb extremities of adult male Sprague-Dawley rats by a 1000VDC delivery shock system. A thermal camera was utilized to record temperature variation during the exposure. The experimental results were then validated using a thermal-electric finite element model (FEM).

We present the development of advanced automatic target recognition (ATR) algorithms for the hair follicles identification in digital skin images to accurately direct the laser beam to remove the hair. The ATR system first performs a wavelet filtering to enhance the contrast of the hair features in the image. The system then extracts the unique features of the targets and sends the features to an Adaboost based classifier for training and recognition operations. The ATR system automatically classifies the hair, moles, or other skin lesion and provides the accurate coordinates of the intended hair follicle locations. The coordinates can be used to guide a scanning laser to focus energy only on the hair follicles. The intended benefit would be to protect the skin from unwanted laser exposure and to provide more effective skin therapy.

Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses responsible for skin homeostasis. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of psoriasis remains under investigated. To elucidate the spatial-temporal morphological evolution of psoriasis we undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to non-invasively document dermal alterations due to immune cell infiltration and epidermal hyperplasia in an Imiquimod (IMQ) induced model of psoriasis-like inflammation in DBA2/C57Bl6 hybrid mice. Quantitative appraisal of dermal architectural changes was achieved through a three parameter fit of OCT axial scans in the dermis of the form A(z) = ρ exp(-mu;z +ε(z)). Ensemble averaging of the fit parameters over 2000 axial scans per mouse in each treatment arm revealed that the local dermal reflectivity ρ, decreased significantly in response to 6 day IMQ treatment (p = 0.0001), as did the standard deviation of the attenuation fluctuation std(ε(z)), (p = 0.04), in comparison to cream controls and day 1 treatments. No significant changes were observed in the average dermal attenuation rate, μ. Our results suggest these label-free OCT-based metrics can be deployed to investigate new therapeutic targets in animal models as well as aid in clinical staging of psoriasis in conjunction with the psoriasis area and severity index.

We present an extended focus OCT system for dermatologic applications that maintains high lateral resolution over a large depth range by using Bessel beam illumination. More, Bessel beams exhibit a self-reconstruction property that is particularly useful to avoid shadowing from surface structures such as hairs. High lateral resolution and high-speed measurement, thanks to a rapidly tuning swept source, allows not only for imaging of small skin structures in depth but also for comprehensive visualization of the small capillary network within the human skin in-vivo. We use this information for studying temporal vaso-responses to hypothermia. In contrast to other perfusion imaging methods such as laser Doppler imaging (LDI), OCT gives specific access to vascular responses in different vascular beds in depth.

Port-wine Stain (PWS) is a vascular malformation characterized by ectasia of superficial dermal capillaries. The flash-lamp pumped pulsed dye laser (PDL) treatment has been the mainstay of PWS for the last decade. Despite the success of the PDL in significantly fading the PWS, the overall cure rate is less than 10%. The precise efficacy of an individual PDL treatment is hard to evaluate and the treatment outcome is measured by visual observation of clinical fading. A hand-held multi-spectral imaging system was developed to image PWS before and after PDL treatment. In an NIH-funded pilot study multi-spectral camera was used to image PWS in children (2- 17 years). Oxygen saturation (S) and blood content (B) of PWS before and after the treatment was determined by analysis of the reflectance spectra. The outcome of the treatment was evaluated during follow up visits of the patients. One of the major causes of failure of laser therapy of port-wine stains (PWS) is reperfusion of the lesion after laser treatment. Oxygen saturation and blood content maps of PWS before and after treatment can predict regions of reperfusion and subsequent failure of the treatment. The ability to measure reperfusion and to predict lesions or areas susceptible to reperfusion, will help in selection of patients/lesions for laser treatment and help to optimize laser dosimetry for maximum effect. The current studies also should provide a basis for monitoring of future alternative therapies or enhancers of laser treatment in resistant cases.

We have designed and developed an optical fiber-probe for spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multidimensional approach. Concerning fluorescence spectroscopy, the excitation is provided by two laser diodes, one emitting in the UV (378 nm) and the other emitting in the visible (445 nm). These two lasers are used to selectively excite fluorescence from NADH and FAD, which are among the brightest endogenous fluorophores in human tissues. For Raman and NIR spectroscopy, the excitation is provided by a third laser diode with 785 nm excitation wavelength. Laser light is delivered to the tissue through the central optical fiber of a fiber bundle. The surrounding 48 fibers of the bundle are used for collecting fluorescence and Raman and for delivering light to the spectrograph. Fluorescence and Raman spectra are acquired on a cooled CCD camera. The instrument has been tested on fresh human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin, finding an optimal correlation with the subsequent histological exam. In some cases our examination was not in agreement with the clinical observation, but it was with the histological exam, demonstrating that the system can potentially contribute to improve clinical diagnostic capabilities and hence reduce the number of unnecessary biopsies.

Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit ^{wsh/ w-sh} mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

Pulsed photothermal radiometry (PPTR) allows noninvasive determination of temperature depth profiles induced by pulsed laser irradiation of strongly scattering biological tissues and organs, including human skin. In present study, we evaluate the potential of this technique for investigational characterization and possibly quantitative evaluation of laser tattoo removal. The study involved 5 healthy volunteers (3 males, 2 females), age 20-30 years, undergoing tattoo removal treatment using a Q-switched Nd:YAG laser. There were four measurement and treatment sessions in total, separated by 2-3 months. Prior to each treatment, PPTR measurements were performed on several tattoo sites and one nearby healthy site in each patient, using a 5 ms Nd:YAG laser at low radiant exposure values and a dedicated radiometric setup. The laser-induced temperature profiles were then reconstructed by applying a custom numerical code. In addition, each tatoo site was documented with a digital camera and measured with a custom colorimetric system (in tristimulus color space), providing an objective evaluation of the therapeutic efficacy to be correlated with our PPTR results. The results show that the laser-induced temperature profile in untreated tattoos is invariably located at a subsurface depth of 300 μm. In tattoo sites that responded well to laser therapy, a significant drop of the temperature peak was observed in the profiles obtained from PPTR record. In several sites that appeared less responsive, as evidenced by colorimetric data, a progressive shift of the temperature profile deeper into the dermis was observed over the course of consecutive laser treatments, indicating that the laser tattoo removal was efficient.

Skin cancer is the most common and most rapidly increasing form of cancer in the world. Optimal treatment of skin cancer before it reaches metastasis depends critically on early diagnosis. Usually physicians will measure some outward features to diagnose malignancy of pigmented skin lesion. These are mostly morphological features like border irregularity, size, shape, and color. Valuable information can be obtained from the analysis of skin roughness. Previously, we developed a hemispherical imaging Stokes polarimeter to monitor skin cancer based on a roughness assessment of the epidermis. In this study, Stokes images were analyzed to measure polarization properties of skin samples such as the principal angle of the polarization ellipse and the degree of polarization. A processing algorithm based on morphological operators was also developed and applied on Stokes images to extract shape information. Finally, an appropriate classifier was designed to determine the type of lesion based on morphological features as well as the roughness information. Clinical evaluation of the technique was performed on patients with benign nevi, melanocytic nevi, melanoma, and normal skin.

Pressure waves generated by laser pulses can permeabilize biological barriers, such as the skin or cellular membranes. The characteristics of the absorbing materials are decisive in determining the shape and amplitude of pressure impulse transients. Based on the physics and photochemistry of light-to-pressure conversion, we generate high intensity broadband ultrasound capable of transiently permeabilizing biological barriers. We show evidence that no damage is done to cells exposed to such pressure waves and that skin recovers its protective function some minutes after exposure to the pressure waves. The ability of the pressure waves to promote transient skin permeabilization is assessed by the increase of transepidermal water loss (TEWL) immediately after the application of pressure waves, and by the full recovery of the skin to the normal TEWL values in the following minutes.

The laser scattering in tissue is significant in diagnostic and therapeutic purposes of laser. Many studies have been conducted to minimize laser scattering in tissue and therefore, to maximize the clinical efficacy by enhancing photon density. Optical clearing agents (OCAs) have been employed for optical tissue clearing (OTC). This study was aimed to investigate the optimal concentration of an OCA, glycerol, in topical application,, so that it can be utilized for clinical diagnosis and therapy in dermatology. Glycerol was topically applied to avoid possible edema caused by dermal injection. The effect of OTC was quantitatively evaluated as a function of the concentration of glycerol with various methods. Optical methods such as optical coherence tomography (OCT) and an integrating sphere were used to assess the enhancement of light penetration depth and refractive index matching. In addition, a non-optical method, ultrasound scanner, was utilized to evaluate quantitatively collagen dissociation. The results revealed that 70 % glycerol was the optimal concentration of OTC for topical application. This study may provide a guideline regarding to the use of glycerol for optimal diagnostic and therapeutic effects in dermatology.

Optical imaging modalities have been utilized as important tools to evaluate skin lesions. Accurate analysis of hyper-pigmentation is important in the evaluation of sun damage, inflammation, and other skin disorders. In this study, both cross-polarization and fluorescent color images were obtained at identical facial skin region and hyper-pigmentation regions were extracted by applying a series of image processing. Cross-polarization image provides subsurface skin information without specular reflection and fluorescence image emphasizes pigmentation in epidermal region. The results were quantitatively evaluated and compared to investigate the difference between two imaging modalities in the evaluation of hyper-pigmentation regions.

Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

Optical diagnostics of bruised skin might provide important information for characterization and age determination of such injuries. Hyperspectral imaging is one of the optical techniques that have been employed for bruise characterization. This technique combines high spatial and spectral resolution and makes it possible to study both chromophore signatures and -distributions in an injury. Imaging and spectroscopy in the visible spectral range have resulted in increased knowledge about skin bruises. So far the SWIR region has not been explored for this application. The main objective of the current study was to characterize bruises in the SWIR wavelength range. Hyperspectral images in the SWIR (950-2500nm ) and VNIR (400-850nm) spectral range were collected from 3 adult volunteers with bruises of known age. Data were collected over a period of 8 days. The data were analyzed using spectroscopic techniques and statistical image analysis. Preliminary results from the pilot study indicate that SWIR hyperspectral imaging might be an important supplement to imaging in the visible part of the spectrum. The technique emphasizes local edema and gives a possibility to visualize features that cannot easily be seen in the visible part of the spectrum.

Skin cancer is most commons type of cancer in United States that occur on sun-exposed cosmetically sensitive areas like face, neck, and forearms. Surgical excision of skin cancer is challenging as more than one-third the actual margins extend beyond the clinically determined margins. Polarized light camera (polCAM) provides images of the superficial layers of the tissue with enhanced contrast which was used to image skin cancer margins. In a NIH-funded pilot study polCAM was used to image skin cancer in patients undergoing Mohs micrographic surgery for skin cancer. Polarized light imaging utilizes the polarization properties of light to create an image of a lesion comprised only of light scattering from the superficial layers of the skin which yields a characteristic "fabric pattern" of the putative lesion and the surrounding normal tissue. In several case studies conducted with a system developed for the clinic, we have found that skin cancer disrupts this fabric pattern, allowing the doctor a new means of identifying the margins of the lesion. Data is acquired before the patient underwent surgery. The clinically determined skin cancer margins were compared with margins determined by examination of the polCAM images. The true margins were provided by the dermatophathologist on examination of the frozen sections. Our initial data suggests that the contrast due to polarization changes associated with cancerous lesions can elucidate margins that were not recognized by the surgeon under normal conditions but were later confirmed by the pathologist.

Colorimetry has been used as an objective measure of perceived skin color by human eye to document and score physiological responses of the skin from external insults. CIE color space values (L*, a* and b*) are the most commonly used parameters to correlate visually perceived color attributes such as L* for pigment, a* for erythema, and b* for sallowness of the skin. In this study, we investigated the relation of Lab color scale to the amount of major skin chromophores (oxy-, deoxyhemoglobin and melanin) calculated from diffuse reflectance spectroscopy. Thirty two healthy human subjects with ages from 20 to 70 years old, skin types I-VI, were recruited for the study. DRS and colorimetry measurements were taken from the left and right cheeks, and on the right upper inner arm. The melanin content calculated from 630-700 nm range of DRS measurements was shown to correlate with the lightness of skin (L*) for most skin types. For subjects with medium-to-light complexion, melanin measured at the blue part spectrum and hemoglobin interfered on the relation of lightness of the skin color to the melanin content. The sallowness of the skin that is quantified by the melanin contribution at the blue part spectrum of DRS was found to be related to b* scale. This study demonstrates the importance of documenting skin color by assessing individual skin chromophores with diffuse reflectance spectroscopy, in comparison to colorimetry assessment.

Most of today's intratissue tattoo pigments are unknown nanoparticles. So far, there was no real control of their use due to the absence of regulations. Some of the tattoo pigments contain carcinogenic amines e.g. azo pigment Red 22. Nowadays, the European Union starts to control the administration of tattoo pigments. There is an interest to obtain information on the intratissue distribution, their interaction with living cells and the extracellular matrix, and the mechanisms behind laser tattoo removal. Multiphoton tomographs are novel biosafety and imaging tools that can provide such information non-invasively and without further labeling. When using the spectral FLIM module, spatially-resolved emission spectra, excitation spectra, and fluorescence lifetimes can pr provided. Multiphoton tomographs are used by all major cosmetic comapanies to test the biosafety of sunscreen nanoparticles.

One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

Optical coherence tomography (OCT) is a tool currently used for noninvasive diagnosis of human disease as well as for monitoring treatment during or after therapy. In this study, OCT was used to examine penetration and accumulation of cosmetic creams on human hand skin. The samples varied in collagen content with one formulation containing soluble collagen as its primary active ingredient. Collagen is a major connective tissue protein that is essential in maintaining health vitality and strength of many organs. The penetration and localization of collagen in cosmetic creams is thought to be the main determinant of the efficacy of new collagen synthesis. Detection and quantification of collagen in cosmetic creams applied to skin may thus help predict the eventual efficacy of the product in skin collagen regeneration. We hypothesize that the topically applied collagen may be detectable by OCT through its modulation of skin scattering properties. To test this hypothesis, we used a FDML swept-source optical coherence tomography (SS-OCT) system. A particular location on the skin of two male adult volunteers was used to investigate 4 different cosmetic creams. The duration of OCT monitoring of cosmetic penetration into skin ranged from 5 minutes to 2 hours following topical application. The results showed that OCT can discriminate between a cream with collagen and other collagen-free formulations. Thus it seems feasible that OCT intensity can monitor the in vivo effects of topical application of collagen contained in cosmetic formulations.

This work demonstrated the discrimination among basal cell carcinoma (BCC) and normal human skin in vivo using near-infrared Raman spectroscopy. Spectra were obtained in the suspected lesion prior resectional surgery. After tissue withdrawn, biopsy fragments were submitted to histopathology. Spectra were also obtained in the adjacent, clinically normal skin. Raman spectra were measured using a Raman spectrometer (830 nm) with a fiber Raman probe. By comparing the mean spectra of BCC with the normal skin, it has been found important differences in the 800-1000 cm^-1 and 1250-1350 cm^-1 (vibrations of C-C and amide III, respectively, from lipids and proteins). A discrimination algorithm based on Principal Components Analysis and Mahalanobis distance (PCA/MD) could discriminate the spectra of both tissues with high sensitivity and specificity.

Successful identification and preservation of the cavernous nerves (CN), which are responsible for sexual function, during prostate cancer surgery, will require subsurface detection of the CN beneath a thin fascia layer. This study explores optical nerve stimulation (ONS) in the rat with a fascia layer placed over the CN. Two near-IR diode lasers (1455 nm and 1550 nm lasers) were used to stimulate the CN in CW mode with a 1-mm-diameter spot in 8 rats. The 1455 nm wavelength provides an optical penetration depth (OPD) of ~350 μm, while 1550 nm provides an OPD of ~1000 μm (~3 times deeper than 1455 nm and 1870 nm wavelengths previously tested). Fascia layers with thicknesses of 85 - 600 μm were placed over the CN. Successful ONS was confirmed by an intracavernous pressure (ICP) response in the rat penis at 1455 nm through fascia 110 μm thick and at 1550 nm through fascia 450 μm thick. Higher incident laser power was necessary and weaker and slower ICP responses were observed as fascia thickness was increased. Subsurface ONS of the rat CN at a depth of 450 μm using a 1550 nm laser is feasible.

Complications from polypropylene mesh after surgery for female stress urinary incontinence (SUI) may require tedious surgical revision and removal of mesh materials with risk of damage to healthy adjacent tissue. This study explores selective laser vaporization of polypropylene suture/mesh materials commonly used in SUI. A compact, 7 Watt, 647-nm, red diode laser was operated with a radiant exposure of 81 J/cm², pulse duration of 100 ms, and 1.0-mm-diameter laser spot. The 647-nm wavelength was selected because its absorption by water, hemoglobin, and other major tissue chromophores is low, while polypropylene absorption is high. Laser vaporization of ~200-μm-diameter polypropylene suture/mesh strands, in contact with fresh urinary tissue samples, ex vivo, was performed. Non-contact temperature mapping of the suture/mesh samples with a thermal camera was also conducted. Photoselective vaporization of polypropylene suture and mesh using a single laser pulse was achieved with peak temperatures of 180 and 232 °C, respectively. In control (safety) studies, direct laser irradiation of tissue alone resulted in only a 1 °C temperature increase. Selective laser vaporization of polypropylene suture/mesh materials is feasible without significant thermal damage to tissue. This technique may be useful for SUI procedures requiring surgical revision.

Recently, thulium (Tm) fiber lasers have been investigated for use in surgical procedures, especially in urology, because of their numerous advantages over existing laser systems. Lockheed Martin Aculight has recently developed the first truly dual-mode Tm laser, which can be operated in either CW or in short-pulsed mode to produce high peak power. The goal of this study was to assess both the soft tissue ablation performance of this laser in vitro and the feasibility of using it for lithotripsy. Ablation tests were performed on liver tissue, chicken breast, and porcine skin, using a 100μm or 200μm delivery fiber, and operated in CW mode or pulsed (~350ns pulse widths) at 10kHz or 1kHz. Ablation efficiencies for long (3 minutes) exposures and collateral damage zones for short (3-5 seconds) exposures were determined for the different pulse modes and a range of pulse energies. In all tissues, the most energy-efficient ablation occurred for the 10kHz pulsed mode operating just above ablation threshold, while the highest mass removal rate occurred in 10kHz pulsed mode operating at max energy (2.1 mJ). In histological sections from short exposures, 10kHz pulsed exposures created slightly smaller thermal coagulation zones than energy-matched CW exposures, while 1kHz deliveries had substantially smaller thermal damage zones. In addition, using a 100μm fiber, the 10kHz mode was able to fragment samples of uric acid stones.

Introduction: Developments in laparoscopic partial nephrectomy (LPN) opened a demand for surgical tools compatible with laparoscopic manipulations to make laser assisted technique safe, feasible and reproducible. Warm ischemia and bleeding during laparoscopic partial nephrectomy place technical constraints on surgeons. Therefore it was the aim to develop a safe and effective laser assisted partial nephrectomy technique without need for ischemia. Patients and methods: A diode laser emitting light at 1318nm in cw mode was coupled into a bare fibre (core diameter 600 μm) thus able to transfer up to 100W to the tissue. After dry lab experience, a total of 10 patients suffering from kidney malformations underwent laparoscopic/retroperitoneoscopic partial nephrectomy. Clinically, postoperative renal function and serum c-reactive protein (CRP) were monitored. Laser induced coagulation depth and effects on resection margins were evaluated. Demographic, clinical and follow-up data are presented. Using a commercial available fibre guidance instrument for lanringeal intervention, the demands on an innovative laser fibre guidance instrument for the laser assisted laparoscopic partial nephrectomy (LLPN) are summarized. Results: Overall, all laparascopic intervention were succesfull and could be performed without conversion to open surgery. Mean operative time and mean blood loss were comparable to conventional open and laparascopic approaches. Laser assisted resection of the kidney tissue took max 15min. After extirpation of the tumours all patients showed clinical favourable outcome during follow up period. Tumour sizes were measured to be up 5cm in diameter. The depth of the coagulation on the removed tissue ranged between <1 to 2mm without effect on histopathological evaluation of tumours or resection margin. As the surface of the remaining kidney surface was laser assisted coagulated after removal. The sealing of the surface was induced by a slightly larger coagulation margin, but could not measured so far. Based on this experiences a simple and easy to use instrument described serving also for suction and rinsing. Conclusion: This prospective in-vivo feasibility study shows that laser assisted partial nephrectomy seems to be a safe and promising medical technique which could be provided either during open surgery as well as laparascopically. This application showed good haemostasis and minimal parenchymal damage. Further investigations and development are needed for on-line detection of the remain coagulation margin. An optimised treatment equipment will support the applicability of laser assisted laparoscopic partial nephrectomy.

Introduction and objectives: Greenlight laser vaporization (LV) of the prostate is characterized by simultaneous vaporization and coagulation of prostatic tissue resulting in tissue ablation together with excellent hemostasis during the procedure. It has been reported that bipolar plasma vaporization (BPV) of the prostate might be an alternative for LV. So far, it has not been shown that BPV is as effective as LV in terms of tissue ablation or hemostasis. We performed transrectal three-dimensional ultrasound investigations to compare the efficiency of tissue ablation between LV and BPV. Methods: Between 11.2009 and 5.2011, 50 patients underwent pure BPV in our institution. These patients were matched with regard to the pre-operative prostate volume to 50 LV patients from our existing 3D-volumetry-database. Transrectal 3D ultrasound and planimetric volumetry of the prostate were performed pre-operatively, after catheter removal, 6 weeks and 6 months. Results: Median pre-operative prostate volume was not significantly different between the two groups (45.3ml vs. 45.4ml; p=1.0). After catheter removal, median absolute volume reduction (BPV 12.4ml, LV 6.55ml) as well as relative volume reduction (27.8% vs. 16.4%) were significantly higher in the BPV group (p<0.001). After six weeks (42.9% vs. 33.3%) and six months (47.2% vs. 39.7%), relative volume reduction remained significantly higher in the BPV group (p<0.001). Absolute volume reduction was non-significantly higher in the BPV group after six weeks (18.4ml, 13.8ml; p=0.051) and six months (20.8ml, 18ml; p=0.3). Clinical outcome parameters improved significantly in both groups without relevant differences between the groups. Conclusions: Both vaporization techniques result in efficient tissue ablation with initial prostatic swelling. BPV seems to be superior due to a higher relative volume reduction. This difference had no clinical impact after a follow-up of 6M.

Introduction: The da Vinci surgical system requires the use of electrosurgical instruments. The re-use of such instruments creates the potential for stray electrical currents from capacitive coupling and/or insulation failure with subsequent injury. The morbidity of such injuries may negate many of the benefits of minimally invasive surgery. We sought to evaluate the rate and nature of electrosurgical injury (ESI) associated with this device. Methods: The Manufacturer and User Facility Device Experience (MAUDE) database is administered by the US Food and Drug Administration (FDA) and reports adverse events related to medical devices in the United States. We analyzed all incidents in the context of robotic surgery between January 2001 and June 2011 to identify those related to the use of electrosurgery. Results: In the past decade, a total of 605 reports have been submitted to the FDA with regard to adverse events related to the da Vinci robotic surgical platform. Of these, 24 (3.9%) were related to potential or actual ESI. Nine out of the 24 cases (37.5%) resulted in additional surgical intervention for repair. There were 6 bowel injuries of which only one was recognized and managed intra-operatively. The remainder required laparotomy between 5 and 8 days after the initial robotic procedure. Additionally, there were 3 skin burns. The remaining cases required conservative management or resulted in no harm. Conclusion: ESI in the context of robotic surgery is uncommon but remains under-recognized and under-reported. Surgeons performing robot assisted surgery should be aware that ESI can occur with robotic instruments and vigilance for intra- and post-operative complications is paramount.

A noninvasive approach to vasectomy may eliminate male fear of complications related to surgery and increase its acceptance. Noninvasive laser thermal occlusion of the canine vas deferens has recently been reported. In this study, optical coherence tomography (OCT) and high-frequency ultrasound (HFUS) are compared for monitoring laser thermal coagulation of the vas in an acute canine model. Bilateral noninvasive laser coagulation of the vas was performed in 6 dogs (n=12 vasa) using a Ytterbium fiber laser wavelength of 1075 nm, incident power of 9.0 W, pulse duration of 500 ms, pulse rate of 1 Hz, and 3-mm-diameter spot. Cryogen spray cooling was used to prevent skin burns during the procedure. An OCT system with endoscopic probe and a HFUS system with 20-MHz transducer were used to image the vas immediately before and after the procedure. Vasa were then excised and processed for gross and histologic analysis for comparison with OCT and HFUS images. OCT provided high-resolution, superficial imaging of the compressed vas within the vas ring clamp, while HFUS provided deeper imaging of the vas held manually in the scrotal fold. Both OCT and high HFUS are promising imaging modalities for real-time confirmation of vas occlusion during noninvasive laser vasectomy.

Real-time imaging with confocal laser endomicroscopy (CLE) probes that fit in standard endoscopes has emerged as a clinically feasible technology for optical biopsy of bladder cancer. Confocal images of normal, inflammatory, and neoplastic urothelium obtained with intravesical fluorescein can be differentiated by morphologic characteristics. We compiled a confocal atlas of the urinary tract using these diagnostic criteria to be used in a prospective diagnostic accuracy study. Patients scheduled to undergo transurethral resection of bladder tumor underwent white light cystoscopy (WLC), followed by CLE, and histologic confirmation of resected tissue. Areas that appeared normal by WLC were imaged and biopsied as controls. We imaged and prospectively analyzed 135 areas in 57 patients. We show that CLE improves the diagnostic accuracy of WLC for diagnosing benign tissue, low and high grade cancer. Interobserver studies showed a moderate level of agreement by urologists and nonclinical researchers. Despite morphologic differences between inflammation and cancer, real-time differentiation can still be challenging. Identification of bladder cancer-specific contrast agents could provide molecular specificity to CLE. By using fluorescently-labeled antibodies or peptides that bind to proteins expressed in bladder cancer, we have identified putative molecular contrast agents for targeted imaging with CLE. We describe one candidate agent - anti-CD47 - that was instilled into bladder specimens. The tumor and normal urothelium were imaged with CLE, with increased fluorescent signal demonstrated in areas of tumor compared to normal areas. Thus, cancer-specificity can be achieved using molecular contrast agents ex vivo in conjunction with CLE.

Background: One of the most important conditions where there is loss of normal bladder function is spinal cord injury (SCI). Currently, evaluation of bladder function is limited to periodic invasive urodynamic testing (UDS). The purpose of this study was to assess the feasibility and usefulness of near-infrared spectroscopy (NIRS) in monitoring bladder function in patients with SCI during bladder filling and emptying and to investigate the correlations of NIRS measures with simultaneous UDS parameters. NIRS is a non-invasive optical method to study tissue oxygenation, hemodynamics and function by monitoring changes in the chromophore concentrations of oxygenated (O₂Hb), deoxygenated (HHb) and total hemoglobin (tHb). Methods: 10 adult paraplegic patients with neurogenic bladder dysfunction who were referred for regular urodynamic evaluation were recruited. Changes in O₂Hb, HHb and tHb, and tissue saturation index (TSI%) in the detrusor were monitored and recorded by a wireless NIRS system during the urodynamic evaluation. Time points of urgency and urinary leakage were marked and patterns of change in NIRS parameters were compared to standard urodynamic pressure tracings. Results: Strong consistency between changes in NIRS-derived tHb and changes in intravesical pressure were observed during filling across the subjects. During bladder filling a gradual increase in O₂Hb and tHb with minimal changes in HHb was observed. Interestingly, a drop in TSI% was detected seconds before strong urgency and urinary leakage. Conclusions: Our preliminary data suggest a relationship between noninvasive NIRS measures and UDS parameters during bladder filling in SCI patients.

Bladder cancer is the most expensive cancer to treat due to the high rate of recurrence. Though white light cystoscopy is the gold standard for bladder cancer surveillance, the advent of fluorescence biomarkers provides an opportunity to improve sensitivity for early detection and reduced recurrence resulting from more accurate excision. Ideally, fluorescence information could be combined with standard reflectance images to provide multimodal views of the bladder wall. The scanning fiber endoscope (SFE) of 1.2mm in diameter is able to acquire wide-field multimodal video from a bladder phantom with fluorescence cancer "hot-spots". The SFE generates images by scanning red, green, and blue (RGB) laser light and detects the backscatter signal for reflectance video of 500-line resolution at 30 frames per second. We imaged a bladder phantom with painted vessels and mimicked fluorescent lesions by applying green fluorescent microspheres to the surface. By eliminating the green laser illumination, simultaneous reflectance and fluorescence images can be acquired at the same field of view, resolution, and frame rate. Moreover, the multimodal SFE is combined with a robotic steering mechanism and image stitching software as part of a fully automated bladder surveillance system. Using this system, the SFE can be reliably articulated over the entire 360° bladder surface. Acquired images can then be stitched into a multimodal 3D panorama of the bladder using software developed in our laboratory. In each panorama, the fluorescence images are exactly co-registered with RGB reflectance.

The Thulium Fiber Laser (TFL) is being studied as an alternative to the Holmium:YAG (Ho:YAG) laser for lithotripsy. The diode-pumped TFL may be electronically modulated to operate with variable parameters for studying the influence of pulse train mode on stone ablation rates. In this study, the TFL was operated at 1908 nm, 35-mJ pulse energy, and 500-μs pulse duration, in a train of 5 micro-pulses, with macro-pulse rates of 10 Hz, compared with TFL operation at 10-50 Hz. TFL energy was delivered through 100-μm-core fibers in contact with human uric acid (UA) and calcium oxalate monohydrate (COM) stones. Mass removal rates, optical coherence tomography, and light microscopy were used to analyze ablation craters. Stone retropulsion and fiber tip degradation was also studied for these laser parameters. TFL operation in micro-pulse train (MPT) mode resulted in a two-fold increase in the ablation rate of 414 ± 94 μg/s and 122 ± 24 μg/s for the UA and COM stones, respectively, compared to 182 ± 69 μg/s and 60 ± 14 μg/s with standard pulse trains delivered at 50 Hz (P < 0.05). Stone retropulsion remained minimal for both pulse modes. Fiber burnback was significant for both pulse modes and was higher for COM stones than UA stones. TFL operation in MPT mode results in increased stone ablation rates which, with further optimization, may approach rates comparable to Ho:YAG laser lithotripsy in the clinic.

Holmium:YAG laser lithotripsy is effective for all stone compositions. However, stone retropulsion is a big concern during laser ablation. The use of metal based anti-retropulsion devices reduces stone retropulsion during laser treatment. However, Ho:YAG laser can also ablate metal based antiretropulsion devices such as metal basket or metal wires and cause the failure of anti-retropulsion device. BackStop is a novel reverse thermosensitive polymer-based anti-retropulsion device. Response of Backstop polymer to pulsed Ho:YAG radiation is unknown, but predicted to withstand energy better than metal based anti-retropulsion devices since BackStop retains a solid form at increased temperature. Further, a solid shape, form, and function should not be compromised even if an ablation crater may be created. This study tests the ability of BackStop polymer to withstand shape and function in response to Ho:YAG laser energy at various pulse energies. Laser polymer interaction is characterized using fast flash imaging technique and pressure transient measurement.

Introduction: Prior research shows that Ho:YAG lithotripsy produces tiny dust fragments at low pulse energy (0.2J). However, calcium oxalate monohydrate (COM) stones may not fragment at this low pulse energy setting. Stone composition is rarely known until after surgery and historically, attempts to predict stone composition on the basis of endoscopic stone appearance were unsuccessful. Current endoscopic technology permits visual details that previously were not evident. As COM appears black under ambient light, we attempt to predict COM stone composition at the time of ureteroscopy based on its endoscopic appearance. Methods: Consecutive subjects undergoing ureteroscopy for stone disease were studied. Any portion of the stone that appeared black under endoscopic vision was considered clinical evidence of COM. Predicted stone composition was correlated with post-operative calculus analysis. Results: 46 consecutive ureteroscopic stone cases were analyzed prospectively. 25 of 28 subjects (89%) with black stones had stones later proven to be COM by composition analysis, versus one of 18 patients (6%) with non-black stones that were COM (p<0.0001). A black endoscopic stone appearance had a positive predictive value for COM of 89% and a non-black endoscopic stone appearance had a negative predictive value for COM of 94% (sensitivity 96%, specificity 83%). Conclusions: COM may reasonably be predicted intra-operatively by its black endoscopic appearance. The clinical utility would be to use higher laser pulse energy settings than for non-COM compositions. This data raises the possibility that more sophisticated optical characterization of endoscopic stone appearance may prove to be a useful tool to predict stone composition.

This work evaluated possible alterations in the Raman spectra of human prostate tissues in vitro, by developing a Principal Components Analysis algorithm for discriminating prostate carcinoma (PrCa) and benign prostate tissue. Raman spectrum was measured using a Raman spectrometer (830 nm) with a fiber Raman probe. Most of the samples exhibited a very strong background fluorescence, which was decreased by photobleaching the fragment during 5 min. and the remaining fluorescence was removed by polynomial filtering. The spectra of prostate in the fingerprint region are dominated by bands of proteins (mainly collagen, elastin, actin). By comparing the spectra of PrCa with the benign prostate tissue, we found a very small difference, indicating similar biochemical constitution of benign and malignant prostate tissue. Principal Components Analysis and Mahalanobis distance could discriminate the spectra of both benign and PrCa tissues with reasonable sensitivity and specificity.

The optical coefficients (μ_s, μ_a, μ'_s and g)of human cancerous and normal prostate tissues were investigated and compared in the spectral range of 750nm - 860 nm. The fractal dimensional parameters including fractal dimension (D_f), cutoff diameter (d_max) and the most efficient diameter (d_m) between the cancerous and normal prostate tissues were determined based on the extinction and diffusion reflection intensity measurements and the determination ofμ_s, μ_a, μ'_s and g. The results are in good agreement with prostate cancer evolution defined by Gleason Grades. The difference of fractal dimensional parameters and optic

Dysplastic and cancerous alterations in oral tissue can be detected noninvasively in vivo using optical techniques including autofluorescence imaging, high-resolution imaging, and spectroscopy. Interim results are presented from a longitudinal study in which optical imaging and spectroscopy were used to evaluate the progression of lesions over time in patients at high risk for development of oral cancer. Over 100 patients with oral potentially malignant disorders have been enrolled in the study to date. Areas of concern in the oral cavity are measured using widefield autofluorescence imaging and depth-sensitive optical spectroscopy during successive clinical visits. Autofluorescence intensity patterns and autofluorescence spectra are tracked over time and correlated with clinical observations. Patients whose lesions progress and who undergo surgery are also measured in the operating room immediately prior to surgery using autofluorescence imaging and spectroscopy, with the addition of intraoperative high-resolution imaging to characterize nuclear size, nuclear crowding, and tissue architecture at selected sites. Optical measurements are compared to histopathology results from biopsies and surgical specimens collected from the measured sites. Autofluorescence imaging and spectroscopy measurements are continued during post-surgery followup visits. We examined correlations between clinical impression and optical classification over time with an average followup period of 4 months. The data collected to date suggest that multimodal optical techniques may aid in noninvasive monitoring of the progression of oral premalignant lesions, biopsy site selection, and accurate delineation of lesion extent during surgery.

Introduction: Thyroid cancer is the most common endocrine malignancy. The current gold standard for diagnosis, fine-needle aspiration (FNA) biopsy, yields 10-25% of indeterminate cytology results, leading to patients undergoing thyroidectomy for diagnosis. We assessed the technical potential of a miniaturized in vivo ESS (elastic light scattering spectroscopy) probe, built into an FNA needle assembly, to differentiate benign from malignant thyroid nodules. Methods: Under IRB approval, 15 patients in the endocrine clinic undergoing FNAB of a thyroid nodule had collection of ESS data using our novel miniaturized FNA probe. Using final surgical pathology as our gold standard, data post processing and visual inspection was completed. Results: 225 spectra were grouped and analyzed (120 benign, 30 malignant and 75 from indeterminate cytology). ESS probes demonstrated excellent reproducibility in use. Initial analysis of these preliminary data is promising, indicating distinction of spectral ESS features between malignant and benign conditions. Conclusion(s): An in vivo trial of an invasive miniaturized integrated ESS biopsy probe is acceptable to patients, and collection of ESS data is feasible and reliable. With development of a disease-specific algorithm, ESS could potentially be used as an in-situ real time intra-operative diagnostic tool or as a minimally invasive adjunct to conventional FNA cytology.

A system based on near-infrared light intensity was used to monitor mouse model of traumatic brain injury (TBI) noninvasively. The measurement system was controlled by microcontroller. Light from a 760/850nm dual-wavelength light emitting diode was coupled to a 0.6-mm-diameter optical fiber. The collection fibers were coupled to optoelectronic detectors, which were placed in the different distance from the source fiber. The system consisted of a constant current bias, a circuit lock-in amplifier (including band pass filter, lock-in amplifier, and low pass filter), a PCI 6240 data acquisition card and a multi-core-processor computer. The modified Lambert Beer law was used to calculate the concentration of _ΔHbO₂ and _ΔHb. The sensitivity matrix was defined to evaluate the region of effective detection of optical probe. Five groups of TBI mouse models were built by Feeney's free-falling method. The data measured by system show after TBI the concentration of _ΔHbO₂ decreased and that of _ΔHb increased. It can be concluded that the system can be used to monitor the changes of TBI of mouse non-invasively.

Vocal-fold vibration is a key dynamic event in voice production, and the vibratory characteristics of the vocal fold correlate closely with voice quality and health condition. Laryngeal imaging provides direct means to observe the vocal fold vibration; in the past, however, available modalities were either too slow or impractical to resolve the actual vocal fold vibrations. This limitation has now been overcome by high-speed digital imaging (HSDI) (or high-speed digital phonoscopy), which records images of the vibrating vocal folds at a rate of 2000 frames per second or higher- fast enough to resolve a specific, sustained phonatory vocal fold vibration. The subsequent image-based functional analysis of voice is essential to better understanding the mechanism underlying voice production, as well as assisting the clinical diagnosis of voice disorders. Our primary objective is to develop a comprehensive analytical platform for voice analysis using the HSDI recordings. So far, we have developed various analytical approaches for the HSDI-based voice analyses. These include Nyquist plots and associated analysese that are used along with FFT and Spectrogram in the analysis of the HSDI data representing normal voice and specific voice pathologies.

Objectives: Laser cordectomy is very popular nowadays and become one of the treatments of choice for early glottis carcinoma. Transoral laser microsurgery has many advantages comparing conventional open surgery or radiation therapy. In this study, we examined the oncologic results of laser cordectomy for early glottic cancer and analyzed the prognostic impact on the survival of the several tumor-related and treatment-related factors. Methods: Patients who were diagnosed as early glottic squamous cell carcinoma, treated by laser cordectomy with curative intent were analyzed. Patients with preivous radiation therapy were included. From June 1988 to March 2005, 202 patients from five hospitals were analyzed (174 T1, 28 T2). Results: Five-year overall survival and disease-free survival were 98.4% and 84.9%. Twenty two patients developed local recurrence. Total laryngectomy was done in 6 patients and laryngeal preservation rate was 97%. Recurrence was higher in the patients with anterior commissure involvement (9/39) than without anterior commissure involvement (13/163). Recurrence was higher in T1b (4/15) than T1a (13/159). Previous radiation was also highly related to the recurrence (7/20 vs 15/182). Twenty patients with local recurrence after radiation therapy were treated by salvage laser cordectomy. Of them, 7 patients developed local recurrence and 5 year disease-free survival was 57%. Complication was rare with one case of hemorrhage. Tracheotomy was not necessary in all patients. Conclusions: Laser cordectomy for early glottic carcinoma showed high survival, laryngeal preservation rate and low complication rate. The prognostic factors were anterior commissure involvement, both vocal fold involvement and previous radiotherapy.

The quantitative characterization of vocal fold (VF) motion can greatly enhance the diagnosis and treatment of speech pathologies. The recent availability of high-speed systems has created new opportunities to understand VF dynamics. This paper presents quantitative methods for analyzing VF dynamics with high-speed digital phonoscopy, with a focus on expected VF changes during childhood. A robust method for automatic VF edge tracking during phonation is introduced and evaluated against 4 expert human observers. Results from 100 test frames show a subpixel difference between the VF edges selected by algorithm and expert observers. Waveforms created from the VF edge displacement are used to created motion features with limited sensitivity to variations of camera resolution on the imaging plane. New features are introduced based on acceleration ratios of critical points over each phonation cycle, which have the potential for studying issues related to impact stress. A novel denoising and hybrid interpolation/extrapolation scheme is also introduced to reduce the impact of quantization errors and large sampling intervals relative to the phonation cycle. Features extracted from groups of 4 adults and 5 children show large differences for features related to asymmetry between the right and left fold and consistent differences for impact acceleration ratio.

Minimally invasive surgical (MIS) techniques, such as laparoscopic surgery and endoscopy, provide reliable disease control with reduced impact on the function of the diseased organ. Surgical lasers can ablate, cut and excise tissue while sealing small blood vessels minimizing bleeding and risk of lymphatic metastases from tumors. Lasers with wavelengths in the IR are readily absorbed by water causing minimal thermal damage to adjacent tissue, ideal for surgery near critical anatomical structures. MIS techniques have largely been unable to adopt the use of lasers partly due to the difficulty in bringing the laser into the endoscopic cavity. Hollow waveguide fibers have been adapted to bring surgical lasers to endoscopy. However, they deliver a beam that diverges rapidly and requires careful manipulation of the fiber tip relative to the target. Thus, the principal obstacle for surgical lasers in MIS procedures has been a lack of effective control instruments to manipulate the laser in the body cavity and accurately deliver it to the targeted tissue. To overcome this limitation, we have designed and built an endoscopic laser system that incorporates a miniature dual wedge beam steering device, a video camera, and the control system for remote and /or robotic operation. The dual wedge Risley device offers the smallest profile possible for endoscopic use. Clinical specifications and design considerations will be presented together with descriptions of the device and the development of its control system.

The line-scanning confocal microscope is simpler than a point-scanning confocal microscope and allows for a smaller and lower cost footprint, making it attractive for endoscopic clinical use. The optical configuration affects image fidelity. Here, we present a benchtop version of an endoscopic line-scanning confocal microscope for intraoral imaging, with a divided pupil and optimal detection configuration (magnification, pixel-to-resolution ratio) to enhance image fidelity. Improved sectioning performance and reduction of "speckle" noise are demonstrated. A topology for use of a deformable MEMs mirror-based optical axial focus control for imaging in depth is presented. Preliminary images of human oral mucosa in vivo demonstrate feasibility for clinical application.

Reflectance confocal microscopy with a line scanning approach potentially offers a smaller, simpler and less expensive approach than traditional methods of point scanning for imaging in living tissues. With one moving mechanical element (galvanometric scanner), a linear array detector and off-the-shelf optics, we designed a compact (102x102x76mm) line scanning confocal reflectance microscope (LSCRM) for imaging human tissues in vivo in a clinical setting. Custom-designed electronics, based on field programmable gate array (FPGA) logic has been developed. With 405 nm illumination and a custom objective lens of numerical aperture 0.5, lateral resolution was measured to be 0.8 um (calculated 0.64 um). The calculated optical sectioning is 3.2 um. Preliminary imaging shows nuclear and cellular detail in human skin and oral epithelium in vivo. Blood flow is also visualized in the deeper connective tissue (lamina propria) in oral mucosa. Since a line is confocal only in one dimension (parallel) but not in the other, the detection is more sensitive to multiply scattered out of focus background noise than in the traditional point scanning configuration. Based on the results of our translational studies thus far, a simpler, smaller and lower-cost approach based on a LSCRM appears to be promising for clinical imaging.

Synthesis of new PDT triads that incorporate a tumor-killing porphyrin with large two-photon cross-section for 150 fs laser pulses (2000 GM) in the Near-infrared (NIR) at 840 nm, a NIR imaging agent, and a small peptide that targets over-expressed EGF receptors on the tumor surface. This triad formulation has been optimized over the past year to treat FADU Head and Neck SCC xenograft tumors in SCID mice. Effective PDT triad dose (1-10 mg/Kg) and laser operating parameters (840 nm, 15-45 min, 900 mW) have been established. Light, dark and PDT treatment toxicities were determined, showing no adverse effects. Previous experiments in phantom and mouse models indicate that tumors can be treated directly through the skin to effective depths between 2 and 5 cm. Treated mice demonstrated rapid tumor regression with some complete cures in as little as 15-20 days. No adverse effects were observed in any healthy tissue through which the focused laser beam passed before reaching the tumor site, and excellent healing occurred post treatment including rapid hair re-growth. Not all irradiation protocols lead to complete cures. Since two-photon PDT is carried out by rastering focused irradiation throughout the tumor, there is the possibility that as the treatment depth increases, some parts of the tumor may escape irradiation due to increased scattering, thus raising the possibility that tumor re-growth could be triggered by small islands of untreated cells, especially at the rapidly growing tumor margins, a problem we hope to alleviate by using image-guided two-photon PDT.

In the clinical work with patients in a medical voice clinic it is important to have a normal updated reference for the data used. Several new parameters have to be correlated to older traditional measures. The older ones are stroboscopy, eventually coordinated with electroglottography (EGG), the Multi- Dimensional-Voice Program and airflow rates. Long Time Averaged Spectrograms (LTAS) and phonetograms (voice profiles) are calculating the range and dynamics of tones of the patients. High-speed films, updated airflow measures as well as area calculations of phonotograms add information to the understanding of the glottis closure in single movements of the vocal cords. A multivariate analysis was made to study the connection between the measures. This information can be used in many connections, also in the otolaryngological clinic.

Recurrent Respiratory Papillomatosis (RRP) is a devastating disorder- especially in a performing professional voice user. The mainstay of treatment is based on immaculate serial removal of regrowing papillomas, usually with a laser. Repetitive laser excisions can cause significant scarring and webbing. The risks of post-operative sequela are exponentially increased with anterior location of papilloma clusters. The resultant dysphonia is not amenable to physiological voice therapy protocols. Additional or adjunctive treatments are eagerly sought by patients to avoid complications. Many of these treatments remain unproven. Recently, bevacizumab (Avastin) has been advocated as potentially useful. Consequently, we report a case treated with KTP lasering of papillomas with adjunctive intralesional bevacizumab injections. Current outcome of the case is analyzed with both traditional LVS and High Speed Digital Imaging (HSDI).

We present excerpts from three archival ultra high-speed films on the function of the human larynx by Paul Moore, Ph. D. and Hans von Leden, M.D. The films received two awards for best scientific cinematography from two different international film festivals in Italy in 1957. These films present ultra high-speed cinematographic accounts on the workings of the human vocal folds during various phonatory and ventilatory activities. These films were captured at speeds of 2000 to 5000 frames-per-second via an ingeniously arranged laryngeal mirror viewing device. Such speeds were revolutionary six decades ago. Technology currently allows us to film laryngeal behavior at speeds of up to 16,000 frames-per-second using digital recordings. However, the ultra high-speed films by Paul and Hans remain a beacon for anyone sincerely interested in how the smallest instrument of sound production works, and how it is subjected to failure by intrinsic or extrinsic factors.

The differential motion of the organ of Corti has been expected as a result of the outer hair cell force, believed to be necessary for the cochlear amplifier. In vitro experiments have been performed to demonstrate this motion but the in vivo data was unavailable due to the technical difficulties. Using a specially-designed time-domain optical coherence tomography system, we performed in vivo imaging and vibration measurement at the sensitive base of the guinea pig cochlea. This technique, for the first time, provides in vivo information about the internal vibration of the organ of Corti. At low sound level, when the cochlea is more sensitive, top surface of the organ of Corti, the reticular lamina (RL) showed tuning at a higher frequency than of the bottom surface, basilar membrane (BM) and its vibration amplitude is 2-3 times of that of the BM. Corresponding to the frequency difference, the phase of RL vibration is lead to that of the BM. Both the amplitude gain and the phase lead on RL is level dependent. This suggests that they are related to the cochlear amplification. The amplitude gain at the RL is an enhancement of the BM motion for stimulating the stereocillia. The advance in time of RL vibration can prepare proper timing of stereocillia stimulation for the cochlear amplification.

The transcanal LLLT was found to recover noise induced hearing loss (NIHL) but the LLLT was performed immediately after the induction of NIHL. The aim of this study was to find an optimal window time to treat and recover a NIHL with LLLT. Bilateral ears of 6SD rats (12ears) were exposed to noise. Left ears of the rats were irradiated with a LLLT (830 nm, 594 J/cm² per day) for 12 days, starting 3 days and 7 days post exposure to noise. Right ears were used as control ears. The hearing levels were measured at each frequency of 4, 8, 12, 16, and 32 kHz before and after the noise exposure and post 12th irradiations. The initial hearing levels in all frequencies before and after the noise exposure were 26.5, 24.5, 24.0, 24.0 and 24.5 dB SPL and 63.5, 64, 71.5, 73.5 and 67.5 dB SPL in 4, 8, 12, 16 and 32 kHz, respectively in 6 ears. After 12th irradiation, the thresholds of the LLLT treated left ears of the 3-day group recovered significantly compared to those of the untreated right. However, for the 7 day group, the recovery of the LLLT treated left ears was not significantly improved compared to that of the untreated right. The results of this study suggest that the optimal window time to treat NIHL with LLLT was within 3 days from the exposure to noise but the hearing failed to recover if the LLLT was started 7 days post exposure to noise.

Clinical diagnosis of cochlear dysfunction typically remains incomplete due to a lack of proper diagnostic methods. Medical imaging modalities can only detect gross changes in the cochlea, and non-invasive in vivo cochlear measurements are scarce. As a result, extensive efforts have been made to adapt optical coherence tomography (OCT) techniques to analyze and study the cochlea. Herein, we detail the methods for measuring vibration using OCT. We used spectral domain OCT with ~950 nm as the center wavelength and a bandwidth of ~80 nm. The custom spectrometer used was based on a high speed line scan camera which is capable of line rates up to 28 kHz. The signal-to- noise ratio of the system was ~90 dB. The data collection and processing software was written in LabVIEW and MATLAB. We tested whether streaming directly from the camera, writing the data to multiple hard drives in the RAID- 0 configuration, and processing using the GPU shortened experiment times. We then analyzed the A-line phase noise over several hundred milliseconds and growth curves from a piezoelectric element. We believe this is the first step towards a diagnostic device which generates vibration information of cochlear structures.

We describe a novel method for the detection of the tiny motions of the middle ear (ME) ossicles and their morphological features with a spectral-domain phase sensitive optical coherence tomography (PS-OCT). Laser Doppler Vibrometry (LDV) and its variations are the most extensively used methods for studding the vibrational modes of the ME. However, most techniques are limited to single point analysis methods, and do not have the ability to provide depth resolved simultaneous imaging of multiple points on the ossicles especially with the intact eardrum. Consequently, the methods have the limited ability to provide relative vibration information at these points. In this study, we demonstrated the feasibility of using PS-OCT for simultaneous depth resolved imaging of both vibration information and morphological features in a cadaver human middle ear with high sensitivity and resolution. This technique has the potential to provide meaningful vibration of ossicles with a vibration sensitivity of ~0.5nm at 1kHz acoustic stimulation. To the best of our knowledge, this is the first demonstration of depth-resolved vibration imaging of ossicles with a PS-OCT system at sub-nanometer scale.

HSDI provides a whole new way to investigate visually intra-laryngeal behavior and posturing during phonation by providing detailed real-time information about laryngeal biomechanics that include observations about mucosal wave, wave motion directionality, glottic area wave form, asymmetry of vibrations within and across vocal folds and contact area of the glottis including posterior commissure closure. These observations are fundamental to our understanding and modeling of both normal and disordered phonation. In this preliminary report we focus on direct HSDI in vivo observations of not only the glottic region, but also on the entire supraglottic laryngeal posturing during fry, breathy/hiss and over-pressured phonation modes produced in a non-pathological settings. Analysis included spatio-temporal vibration patterns of vocal folds, multi-line kymograms, spectral PFFT analysis, and Nyquist spatio-temporal plots. The presented examples reveal that supraglottic contraction assists in prolonged closed phase of the vibratory cycle, and that prolonged closed phase is longest in fry and overpressure and shortest albeit complex in hiss. Hiss also allows for vocal fold vibration despite glottis separation. These findings need to be compared to pathologic phonation representing the three voice modes to derive at better differential diagnosis.

Beside the good image quality with the confocal laser scanning microscope (HRTII) and the Rostock Cornea Module (RCM), this technology can not be used to investigate the human larynx in vivo. To accomplish this, a rigid custom-made endoscope (KARL STORZ GmbH & Co. KG; Tuttlingen Germany) was developed. A connector was developed to connect the scanner head of the HRTII to the rigid endoscope. With the connector, the starting plane can be set manually. To achieve optical sectioning of the laryngeal tissue (80 μm per volume scan), the scanning mechanism of the HRTII needs to be activated using a foot switch. The devices consisting of the endoscope, HRTII, and the connector supply images of 400 x 400 μm and reach average penetration depths of 100-300 μm (λ/4 plate of the scanner head of the HRTII was removed). The lateral and axial resolutions are about 1-2 μm and 2 μm, respectively. In vivo rigid confocal endoscopy is demonstrated with an acquisition time for a volume scan of 6 s. The aim of this study was to differentiate pre-malignant laryngeal lesions from micro-invasive carcinoma of the larynx. 22 patients with suspicious lesions of the true vocal cords were included. This pilot study clearly demonstrates the possibility to detect dysplastic cells close to the basal cell layer and within the subepithelial space in lesions with small leukoplakia (thin keratin layer). These findings may have an impact on microlaryngoscopy to improve the precision for biopsy and on microlaryngoscopic laser surgery of the larynx to identify the margins of the pre-malignant lesion.

We present data on how stress is manifested in the human voice by analyzing acoustic and phonetic structure of life recordings from police 997 emergency call center number in Poland. From the data corpus comprising thousands of authentic Police 997 emergency phone calls, a few hundred were automatically selected according to their duration selection criteria (calls shorter than 3-4 seconds were omitted), and finally from this corpus, voices of 45 speakers were chosen for acoustic analysis and were contrasted to neutral samples. Statistical measurements for stressed and neutral speech samples showed relevance of the arousal dimension in stress processing. The MDVP analysis confirmed statistical significance of following parameters in voice stress detection: fundamental frequency and pitch variation, noise-to-harmonic-ratio, sub-harmonics and voice irregularities. In highly stressful conditions a systematic over-one-octave shift in pitch was observed. Linear Discriminant Analysis based on nine acoustic features showed that it is possible to categorize the speech samples into one of the following classes: male stressed or neutral, or female stressed or neutral.

This paper presents an overview of neoglottic visualization using HSDI in a clinical environment. Three patients who underwent a tracheoesophageal puncture (TEP) for voice restoration following Total Laryngectomy were examined. Images of the entire neoglottis as well as point specific vibratory information (kymograms) were generated. Experiences and interpretations gleaned from this undertaking were compared to what was found in the literature. HSDI appears to be a very effective instrument in any comprehensive examination of neoglottic function. Appreciation of the vibratory characteristics of this sphincter may eventually allow neoglottic voice enhancement procedures to be developed.

The objective was to provide a comprehensive overview of the advanced microscopes (light and electron) and to implicate how laryngeal science can benefit. The Core Facility for Integrated Microscopy (CFIM) has a wide range of state-of the art light and electron microscopes for users of all levels of experience and from any discipline. To explore the increasing findings with the high-speed film, researchers need to know more about the underlying pathology (tissue changes at cellular level). The scientists need to have access to state-of-the-art light and electron microscopes ready for use in their research, as well as the necessary technical assistance and support.

This study examined efficacy of the innate immune defence via the mannose binding lectin (MBL) in a cohort of 55 dystonic patients prospectively referred to the clinic with laryngeal mucosal complaints, who were placed on local steroids (budesonid inhaler, 400 μg 2 times daily) and antihistamines (fexofenadin 180 mg mostly 3 times daily) with adjuvant lifestyle corrections. Treatment efficacy of the larynx was assessed based on mucosal findings of the vocal folds examined with High speed mucosa studies comprising simultaneous high speed digital imagines (HSDI), kymography, electroglottography (EGG) and voice acoustics combined with a visual score of arytenoids oedema, as these measures are indicative of the magnitude of laryngitis. Lactose and gluten intolerance and immunological analyses of the innate system were made systematically. Results showed that the genetic aspects of immunology did not reveal a role for the innate immune system, represented by the mannose binding lectin (MBL). An unexpected positive effect of the larynx treatment on dystonia symptoms was found evidenced by reduction of dystonic complaints and more normative results of High speed mucosa, and a reduction of oedema of the inter arytenoids region. Symptoms relieve and better quality of life was observed on follow up for the dystonia complaints.

Australian didgeridoo is a reed-less hollow conically shape wooden tubular wind instrument typically measuring up to 150 cm in length, with distal and proximal diameters ranging from 150 to 30 mm. This tube allows a player to produce only a narrow variety of sound and sounds effects because it is coupled directly to the player's vocal tract. The typical frequency of the tube typically called the drone, is approximately within 60 to 100 Hz range. This tone generation modulated by lip vibration is supported by circular breathing, allowing for an uninterrupted (indefinite) length of sound generation. Inhalation introduces sound pulsation, while specific tonal effects can be consciously created by manipulation of the player's lips and/or the vocal tract, including conscious phonation using vocal folds vibration, all used to enrich both the sound and the artistic meaning of the played sequence. Though the results of the research on the acoustics of this instrument are often reported in the literature, physiologic data regarding vocal tract configurations, and especially on the behavior of the vocal folds in regulation of ventilation and in phonation, remain less than underreported. The data presented here comprises (as far as we were able to determine) the first ever physiologic account of vocal fold activity in a didgeridoo player observed with help of trans-nasal endoscopy. Our focus was to reveal the work of t

In this study we investigated laryngeal behaviors involved during vocal production of highest female vocal ranges in Flute in M3 Register, in Whistle Register and in a newly formulated by us, Hiss Register. Observations were carried with stroboscopy and High Speed Digital Imaging and with spectrographic and psycho-acoustic analysis by means of a software system having a wide spectral range (0-20.000 Hz). Results indicate that at the highest pitch vocal folds vibration is absent or significantly reduced, glottic contact is incomplete. These acoustic form of extreme pitch levels comprised intra-harmonic noise and overtones within 10 to 18 kHz range.

Introduction: So far various laser systems have been used for volume reduction of hyperplastic nasal turbinates. In case of endonasal application, fiber controlled diode lasers are preferred due to reasons of cost and practicability. The aim of this clinical study was to compare the coagulative tissue effects using either λ=1470nm vs. λ=940nm emitting lasers in treatment of hyperplastic inferior nasal turbinates in an intraindividual manner. Patients and methods: This prospective, randomized, double-blind, clinical feasibility trial included 20 patients suffering from hyperplastic inferior nasal turbinates. In each case, one nasal cavity was treated using 1470nm laser at 4- 5W, the other one with 940nm laser at 12W. Treatment was performed endoscopically controlled in non-contact mode. Clinical presentation and patients symptoms were documented preoperatively and on day 1, 3, 7, 14 and 21 postoperatively using rhinomanometry, standardized questionnaires including SNOT 20 GAV (German adapted version), and separate endoscopic examination respectively. Results: None of the patients showed infections, hemorrhages or other complications occurred intra- or postoperatively. The mean operation time was significantly shorter using the 1470nm diode laser as compared to the 940nm laser, thus lower energy was applied. There was a significant reduction of nasal obstruction on day 21 postoperatively compared to the preoperative condition on both sides regardless of the laser system used. Evaluation of the SNOT-Scores as assessed before and three weeks after surgery showed significant subjective improvements. Conclusion: Compared with standard application of 940nm diode laser, 1470nm diode laser application provides an equivalent tissue reduction in shorter operation time using less total energy and a comparable relief of nasal obstruction postoperatively.

High-speed digital imaging (HSDI) of the glottis provides a direct means to capture the actual vocal-fold vibrations. Subsequent image-based analyses can be used for objective and quantitative assessment of voice kinematics in healthy and diseased states. HSDI generates massive visual data array, yet the development of effective software for handling such massive image data has lagged behind. To obtain a robust and clinically relevant analysis, we have implemented a software system that includes the processing of AVI image sequences from HSDI recordings, a s we l l a s the spatiotemporal analysis of glottal area waveform (GAW) and vocal fold displacements extracted from these image sequences. The software contains the following three modules: 1) Import and View Module- to read AVI video data, edit/compile and save selected image data, and make image montages using DirectShow technology; 2) Process Module- to perform frame-by-frame image segmentation to delineate the glottis, and to extract GAW and bilateral vocal fold displacements; 3) Analysis Module- to adopt Nyquist plot displays that involve the Hilbert transform based analysis of GAW, and to provide instantaneous frequency and amplitude distributions. Upon rigorous testing of this software using numerous clinical data samples, we demonstrate the validity of this software in delivering accurate and useful vibratory characteristics of the vocal folds that may correlate with voice condition.

Preneoplastic lesions of the bronchial tree have a high probability of developing into malignant tumours. Currently the best method for localizing them for further treatment is a combined white light and autofluorescence bronchoscopy (WLB+AFB). Unfortunately the average specificity from large clinical trials for this combined detection method is low at around 60%, which can result in many false positives. However a recent pilot study showed that adding a point laser Raman spectroscopy (LRS) measurement improved the specificity of detecting lesions with high grade dysplasia or carcinoma in situ to 91% with a sensitivity of 96% compared to WLB+AFB alone. Despite this success, there is still room for much improvement. One constant need is to find better ways to measure the inherently weak Raman emissions in vivo which will result in even better diagnostic sensitivity and specificity. With this aim in mind a new generation Raman system was developed. The system uses the latest charge coupled device (CCD) with low noise, and fast cool down times. A spectrometer was incorporated that was able to measure both the low and high frequency Raman emissions with high resolution. The Raman catheter was also redesigned to include a visible light channel to facilitate the accurate indication of the area being measured. Here the benefits in the adjunct use of LRS to WLB + AFB are presented, and description of the new system and the improvements it offers over the old system are shown.

To develop new treatment possibilities for patients with severe lung diseases it is crucial to understand the lung function on an alveolar level. Optical coherence tomography (OCT) in combination with intravital microscopy (IVM) are used for imaging subpleural alveoli in animal models to gain information about dynamic and morphological changes of lung tissue during mechanical ventilation. The image content suitable for further analysis is influenced by image artifacts caused by scattering, refraction, reflection, and absorbance. Because the refractive index varies with each air-tissue interface in lung tissue, these effects decrease OCT image quality exceedingly. The quality of OCT images can be increased when the refractive index inside the alveoli is matched to the one of tissue via liquid-filling. Thereby, scattering loss can be decreased and higher penetration depth and tissue contrast can be achieved. To use the advantages of liquid-filling for in vivo imaging of small rodent lungs, a suitable breathing fluid (perfluorodecalin) and a special liquid respirator are necessary. Here we show the effect of liquid-filling on OCT and IVM image quality of subpleural alveoli in a mouse model.

In the context of protective artificial ventilation strategies for patients with severe lung diseases, the contribution of ventilator settings to tissue changes on the alveolar level of the lung is still a question under debate. To understand the impact of respiratory settings as well as the dynamic process of respiration, high-resolution monitoring and visualization of the dynamics of lung alveoli are essential. An instrument allowing 3D imaging of lung tissue as well as imaging of functional constituents, such as elastin fibers, in in situ experimental conditions is presented in this study using a combination of Fourier domain optical coherence tomography (FD-OCT) and fiber-guided two photon microscopy. In a comparative study, fixed lung tissue, stained with sulforhodamine B for elastin fibers, was used to illustrate the ability of fiber-guided two photon excitation and single photon excitation for the visualization of elastin fibers within the tissue. Together with the fast 3D imaging capability of OCT, a new tool is given for the monitoring of alveolar lung dynamics in future in vivo experiments.

Ultramicroscopy allows imaging tissue three dimensionally with high optical resolution. To work, it requires optically clearing the tissue sample (e.g. by substitution of tissue-liquids with a liquid that reveals the same refractive index as the tissue's protein). In ultramicroscopy, illumination of the tissue with a thin sheet of light enables recording sectional images that can form a 3D-stack. Our preliminary data shows that it is feasible to create highly translucent lung tissue preparations of whole lungs with mechanical properties that are close to in vivo. We conclude that using this method, three dimensional image stacks of an entire lung can be recorded at different states of lung distension. This would ultimately help raising knowledge about fundamental lung function on the cellular and the alveolar level.

We have been investigating ultrahigh resolution optical coherence tomography (UHR-OCT) imaging of lung tissues using fiber super continuum sources. The high power, low-noise, Gaussian shaped supercontinuum generated with ultrashort pulses and optical fibers at several wavelengths were used as the broadband light sources for UHR-OCT. For the 800 nm wavelength region, the axial resolution was 3.0 um in air and 2.0 um in tissue. Since the lung consists of tiny alveoli which are separated by thin wall, the UHR-OCT is supposed to be effective for lung imaging. The clear images of alveoli of rat were observed with and without index matching effects by saline. In this work, we investigated the UHR-OCT imaging of lung disease model. The lipopolysaccharide (LPS) induced acute lung injury / acute respiratory distress syndrome (ALI/ARDS) model of rat was prepared as the sample with disease and the UHR-OCT imaging of the disease part was demonstrated. The increment of signal intensity by pleural thickening was observed. The accumulation of exudative fluid in alveoli was also observed for two samples. By the comparison with normal lung images, we can obviously show the difference in the ALI/ARDS models. Since the lung consists of alveolar surrounded by capillary vessels, the effect of red-blood cells (RBC) is considered to be important. In this work, ex-vivo UHR-OCT imaging of RBC was demonstrated. Each RBC was able to be observed individually using UHR-OCT. The effect of RBC was estimated with the rat lung perfused with PBS.

Motile cilia are cellular organelles that project from different epithelial surfaces including respiratory epithelium. They generate directional fluid flow that removes harmful pathogens and particulate matter from the respiratory system. While it has been known that primary ciliary dyskinesia increases the risk of recurrent pulmonary infections, there is now heightened interest in understanding the role that cilia play in a wide-variety of respiratory diseases. Different optical imaging technologies are being investigated to visualize cilia-driven fluid flow, and quantitative image analysis is used to generate measures of ciliary performance. Here, we demonstrate the quantification of in vivo cilia-driven microfluidic mixing using spatial and temporal measures of Shannon information entropy. Using videomicroscopy, we imaged in vivo cilia-driven fluid flow generated by the epidermis of the Xenopus tropicalis embryo. Flow was seeded with either dyes or microparticles. Both spatial and temporal measures of entropy show significant levels of mixing, with maximum entropy measures of ~6.5 (out of a possible range of 0 to 8). Spatial entropy measures showed localization of mixing "hot-spots" and "cold-spots" and temporal measures showed mixing throughout.In sum, entropy-based measures of microfluidic mixing can characterize in vivo cilia-driven fluid flow and hold the potential for better characterization of ciliary dysfunction.

Smoke inhalation injury is a serious threat to victims of fires and explosions, however accurate diagnosis of patients remains problematic. Current evaluation techniques are highly subjective, often involving the integration of clinical findings with bronchoscopic assessment. It is apparent that new quantitative methods for evaluating the airways of patients at risk of inhalation injury are needed. Optical frequency domain imaging (OFDI) is a high resolution optical imaging modality that enables volumetric microscopy of the trachea and upper airways in vivo. We anticipate that OFDI may be a useful tool in accurately assessing the airways of patients at risk of smoke inhalation injury by detecting injury prior to the onset of symptoms, and therefore guiding patient management. To demonstrate the potential of OFDI for evaluating smoke inhalation injury, we conducted a preclinical study in which we imaged the trachea/upper airways of 4 sheep prior to, and up to 60 minutes post exposure to cooled cotton smoke. OFDI enabled the visualization of increased mucus accumulation, mucosal thickening, epithelial disruption and sloughing, and increased submucosal signal intensity attributed to polymorphonuclear infiltrates. These results were consistent with histopathology findings. Bronchoscopic inspection of the upper airways appeared relatively normal with only mild accumulation of mucus visible within the airway lumen. The ability of OFDI to not only accurately detect smoke inhalation injury, but to quantitatively assess and monitor the progression or healing of the injury over time may provide new insights into the management of patients such as guiding clinical decisions regarding the need for intubation and ventilator support.

Architectural changes in and remodeling of the bronchial and pulmonary vasculature are important pathways in diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. However, there is a lack of methods that can find and examine small bronchial vasculature in vivo. Structural lung airway imaging using optical coherence tomography (OCT) has previously been shown to be of great utility in examining bronchial lesions during lung cancer screening under the guidance of autofluorescence bronchoscopy. Using a fiber optic endoscopic OCT probe, we acquire OCT images from in vivo human subjects. The side-looking, circumferentially-scanning probe is inserted down the instrument channel of a standard bronchoscope and manually guided to the imaging location. Multiple images are collected with the probe spinning proximally at 100Hz. Due to friction, the distal end of the probe does not spin perfectly synchronous with the proximal end, resulting in non-uniform rotational distortion (NURD) of the images. First, we apply a correction algorithm to remove NURD. We then use a speckle variance algorithm to identify vasculature. The initial data show a vascaulture density in small human airways similar to what would be expected.

Through the monitoring of the auto-fluorescent mitochondrial metabolic coenzymes, NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavoprotein Adenine Dinucleotide), the redox state of metabolism can be probed in real time in many intact organs, but its use has not been fully developed in lungs. The ratio of these fluorophores, (NADH/FAD), referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. We have designed a fluorometer that can be used to monitor lung surface NADH and FAD fluorescence in isolated perfused lungs. Surface fluorescence NADH and FAD signals were acquired in the absence (control) and presence of pentachlorophenol (PCP), rotenone, and potassium cyanide (KCN). Rotenone, an inhibitor of complex I, increased RR by 18%, predominantly due to an increase in NADH signal. KCN, an inhibitor of complex IV reduced the chain and resulted in an increase of 33% in RR, as a result of 23% increase in NADH and 8% in FAD . PCP, an uncoupler which oxidizes the respiratory chain, decreased RR by 18% as a result of 14% decrease in NADH signal and 4% increase in FAD signal. These results demonstrate the ability of surface fluorometry to detect changes in lung tissue mitochondrial redox state in isolated perfused lungs.

Recently, we developed the streak-mode Fourier domain optical coherence tomography (OCT) technique in which an area-scan camera is used in a streak-mode to record the OCT spectrum. Here we report the application of this technique to in ovo imaging HH18 embryonic chick hearts with an ultrahigh speed of 1,016,000 axial scans per second. The high-scan rate enables the acquisition of high temporal resolution 2D datasets (1,000 frames per second or 1 ms between frames) and 3D datasets (10 volumes per second), without use of prospective or retrospective gating technique. This marks the first time that the embryonic animal heart has been 4D imaged using a megahertz OCT.

A miniature integrated optical coherence tomography (OCT) - ultrasound (US) probing system for real-time intravascular imaging has been developed. The outer diameter of the integrated probe is 0.69 mm, which is small enough for imaging in human coronary arteries. This probe, which has high resolution and deep tissue penetration, is designed to identify vulnerable atherosclerotic plaques in coronary arteries. The first in vivo images of a rabbit abdominal aorta obtained by the integrated OCT-US probe are presented.

Coronary heart disease (like myocardial infarction) is caused by atherosclerosis. It cause over 30% of all deaths in North America and are the most common cause of death in European men under 65 years of age and the second most common cause in women. To diagnose this atherosclerosis before it gets rupture is the most effect way to increase the chance of survival for patients who suffer from this disease. The crucial tusk is how to find out vulnerable plaques. In resent years optical coherence tomography (OCT) has become a very useful tool for intravascular imaging, since it has high axial and transverse resolution. OCT can tell the detail structure inside the plaque like the thickness of plaque cap which is an important factor to identify vulnerable plaques. But we still need to find out the biochemical characteristics that is unique for vulnerable plaques (like inflammation). Fluorescence molecular imaging is a standard way to exam the biochemical property of biological samples. So we integrate these two techniques together into one probe. Our probe is comprised of a double-clad fiber (DCF) and a grin lens, and rotates with a micro mirror in front. The single-mode inner core of the DCF transmits both OCT and fluorescence excitation light, and the multimode inner cladding is used to detect fluorescence signal. In vitro result shows that this is a possible way for more accurate diagnose of vulnerable plaques.

Quantitative analysis of OCT data can be strongly hampered by speckle. We propose a new method to reduce speckle, operating on a single Fourier-domain optical coherence tomography (FD-OCT) A-line. The full acquired spectrum is used for image generation, exhibiting fully developed speckle. By subdividing the same acquired spectrum in a number of distinct narrower windows, each with a different center frequency, several independent speckles can be generated. These can be averaged to yield a lower-resolution image with strongly reduced speckle. The full resolution image remains available for human interpretation; the low resolution version can be used for parametric imaging. We demonstrate this technique using in vivo intravascular optical frequency domain imaging (OFDI) data.

Effect of stent surface-scattering properties on the appearance of stent struts in IV-OCT images was examined by simulation of light-stent interaction by an optical design software package. A phantom blood vessel was constructed from a mix of polydimethylsiloxane (PDMS) and titanium dioxide to simulate the elastic and optical scattering properties of the arterial wall. A Cordis CYPHER^® sirolimus-eluting stent was deployed within the phantom vessel and high resolution Micro-CT images of the stent strut were recorded to create a three-dimensional representation that was imported into software. A Gaussian surface-scattering model (bi-directional scattering distribution function) was assumed for the strut. Simulation of IV-OCT catheter and reflection of light from the stent strut was implemented for different surface scattering properties. A model of IV-OCT catheter was defined in the optical model and the rotation of the light beam over the stent strut was simulated. The measured parameters included: fraction of the reflected rays returning to the catheter and coordinate locations on the stent struts of returned rays. The results indicate that when the surface scattering of the strut increases, reflectivity is higher, while the angular spread of the light beam that is reflected back to the catheter is wider.

Arterial wall is composed of three layers: intima, media and adventitia. Intima-media thickness (IMT) is an important prognostic indicator of atherosclerotic diseases. Although intravascular ultrasound (IVUS) imaging is a commonly used method for delineation of the layered structures, it is inferior to the optical absorption contrast offered by intravascular photoacoustic (IVPA) imaging. We introduce an integrated miniature probe that combines the capabilities of IVUS and IVPA imaging for the evaluation of arterial wall layered structures. Healthy rabbit aorta was imaged ex vivo. IVPA results showed superior contrast over IVUS in identifying the layered structures of arterial wall.

We investigated the influences on smooth muscle cells after our novel short-duration thermal angioplasty, Photo-thermo Dynamic Balloon Angioplasty (PTDBA), to reveal the mechanism that can suppress neo-intimal hyperplasia after PTDBA. We obtained the sufficient arterial dilatations by short-duration heating (≤15 s, <70°C) and low dilatation pressure (<0.4 MPa) without arterial injuries in our previous in vivo studies. Smooth muscle cells, which play most important role in chronic treatment effects, were heated during PTDBA and stretch-fixed after PTDBA. The dead cell rate by heating, estimated by Arrhenius equation with A=2.5x10¹⁶ s^-1 and E_a=1.17×105 J mol^-1, was 15.7±2.2% after PTDBA. The measured deformation rate of smooth muscle cells' nuclei was 1.6±0.1 after PTDBA in vivo. We found that the expression of smooth muscle cells' growth factor after PTDBA was inhibited 0.52 fold compared to that after the conventional balloon angioplasty in vivo. The measured neo-intimal hyperplasia occupancy rate was less than 20% after PTDBA in vivo. We prospect that the inhibition of the growth factor's expression by stretch-fixing may result to suppress the neo-intimal hyperplasia. In addition, the decrease of smooth muscle cells' density in the vessel media by heating might be another reason for the neo-intimal hyperplasia suppression.

We proposed a short-duration heating balloon angioplasty. We designed a prototype short-duration heating balloon catheter that can heat artery media to 60-70°C within 15-25 s with a combination of laser-driven heat generation and continuous fluid irrigation in the balloon. The purpose of this study was to investigate chronic artery lumen patency as well as histological alteration of artery wall after the short-duration heating balloon dilatation with porcine healthy iliac artery. The short-term heating balloon dilated sites were angiographically patent in acute (1 hour) and in chronic phases (1 and 4 weeks). One week after the dilatation, smooth muscle cells (SMCs) density in the artery media measured from H&E-stained specimens was approx. 20% lower than that in the reference artery. One and four weeks after the dilatations, normal structure of artery adventitia was maintained without any incidence of thermal injury. Normal lamellar structure of the artery media was also maintained. We found that the localized heating restricted to artery media by the short-duration heating could maintain adventitial function and artery normal structure in chronic phase.

Objective: When a stereotactic biopsy is taken to enable histopathological diagnosis of a suspected brain tumor, it is essential to i) do this safely, that is not injure a major blood vessel and ii) to obtain relevant vital material from the tumor. We are investigating the suitability of Indocyanine Green (ICG) fluorescence for blood vessel recognition and 5- Aminolevulinic acid (5-ALA) induced Protoporphyrin IX (PpIX) fluorescence for identification of proliferative brain tumor tissue. Methods: A fiber-optic endoscopic approach was studied to generate and detect both fluorescence signals. PpIX concentrations in brain tumors have been measured by chemical extraction. Preliminary equipment was studied in a mouse model. Results: PpIX-concentrations in glioblastoma tissue showed high inner- and inter-patient variability, but each patient out of 15 with interpretable data showed at least one sample with a PpIX-concentration exceeding 2.4 μmol/l, which is easily detectable by state-of-the-art fiberoptic fluorescence spectroscopy and imaging. The imaging fluoroscope with 30,000 pixels resolution could be introduced through a position controlled stereotactic needle. ICG-fluorescence from vessels with diameters ≥ 0.1 mm can be detected with a contrast of 2-2.5 against surrounding tissue. Conclusion: Fluorescence detection during stereotactic biopsy might increase safety and precision of the procedure significantly.

Raman spectroscopy enables label-free assessment of brain tissues and tumors based on their biochemical composition. Combination of the Raman spectra with the lateral information allows grading of tumors, determining the primary tumor of brain metastases and delineating tumor margins - even during surgery after coupling with fiber optic probes. This contribution presents exemplary Raman spectra and images collected from low grade and high grade regions of astrocytic gliomas and brain metastases. A region of interest in dried tissue sections encompassed slightly increased cell density. Spectral unmixing by vertex component analysis (VCA) and N-FINDR resolved cell nuclei in score plots and revealed the spectral contributions of nucleic acids, cholesterol, cholesterol ester and proteins in endmember signatures. The results correlated with the histopathological analysis after staining the specimens by hematoxylin and eosin. For a region of interest in non-dried, buffer immersed tissue sections image processing was not affected by drying artifacts such as denaturation of biomolecules and crystallization of cholesterol. Consequently, the results correspond better to in vivo situations. Raman spectroscopic imaging of a brain metastases from renal cell carcinoma showed an endmember with spectral contributions of glycogen which can be considered as a marker for this primary tumor.

Monitoring cerebral blood flow (CBF) during neurosurgery can provide important physiological information for a variety of surgical procedures. Although multiple intraoperative vascular monitoring technologies are currently available, a quantitative method that allows for continuous monitoring is still needed. Laser speckle contrast imaging (LSCI) is an optical imaging method with high spatial and temporal resolution that has been widely used to image CBF in animal models in vivo. In this pilot clinical study, we adapted a Zeiss OPMI Pentero neurosurgical microscope to obtain LSCI images by attaching a camera and a laser diode. This LSCI adapted instrument has been used to acquire full field flow images from 10 patients during tumor resection procedures. The patient's ECG was recorded during acquisition and image registration was performed in post-processing to account for pulsatile motion artifacts. Digital photographs confirmed alignment of vasculature and flow images in four cases, and a relative change in blood flow was observed in two patients after bipolar cautery. The LSCI adapted instrument has the capability to produce real-time, full field CBF image maps with excellent spatial resolution and minimal intervention to the surgical procedure. Results from this study demonstrate the feasibility of using LSCI to monitor blood flow during neurosurgery.

Rupture of intracranial aneurysm is a common cause of subarachnoid hemorrhage. An aneurysm may undergo microscopic morphological changes or remodeling of the vessel wall prior to rupture, which could potentially be imaged. In this study we present methods of tissue sample preparation of intracranial aneurysms and correlation between optical coherence tomography imaging and routine histology. OCT has a potential future in the assessment of microscopic features of aneurysms, which may correlate to the risk of rupture.

Carotid Artery Stenting (CAS) is a procedure that treats carotid atherosclerosis which should be monitored by in vivo high resolution imaging for the quality of the procedure and potential complications. The purpose of this pilot study is to evaluate the ability of optical coherence tomography to construct high resolution two and three dimensional images of stenting in porcine carotid artery for high accuracy diagnostic purposes. Four Yorkshire pigs were anaesthetized and catheterized. A state-of-the-art optical coherence tomography (OCT) system (Lightlab Imaging, St. Jude Medical Inc.) and an automated injector were used to obtain both healthy and stented porcine carotid artery images. Data obtained were then processed for visualization. The state-of-the-art OCT system was able to capture high resolution images of both healthy and stented carotid arteries. High quality three dimensional images of stented carotid arteries were constructed, clearly depicting stent apposition and thrombus formation over different stents. The results demonstrated that current state-of-the-art OCT system can be used to generate high quality three dimensional images of carotid arterial stents for accurate diagnosis of stent apposition and complications under appropriate imaging conditions.

Regular monitoring of brain perfusion at the bedside in neurointensive care is desirable. Currently used imaging modalities are not suited for constant monitoring and often require a transport of the patient. Noninvasive near infrared spectroscopy (NIRS) in combination with an injection of a safe dye (indocyanine green, ICG) could serve as a quasi-continuous brain perfusion monitor. In this work, we evaluate prerequisites for the development of a brain perfusion monitor using continuous wave (cw) NIRS technique. We present results from a high-resolution diffuse optical tomography (HR-DOT) experiment in humans demonstrating the separation of signals from skin from the brain. This technique can help to monitor neurointensive care patients on a regular basis, detecting changes in cortical perfusion in time.

During glioblastoma surgery, delineation of the brain tumour margins remains difficult especially since infiltrated and normal tissues have the same visual appearance. This problematic constitutes our research interest. We developed a fibre-optical fluorescence probe for spectroscopic and time domain measurements. First measurements of endogenous tissue fluorescence were performed on fresh and fixed rat tumour brain slices. Spectral characteristics, fluorescence redox ratios and fluorescence lifetime measurements were analysed. Fluorescence information collected from both, lifetime and spectroscopic experiments, appeared promising for tumour tissue discrimination. Two photon measurements were performed on the same fixed tissue. Different wavelengths are used to acquire two-photon excitation-fluorescence of tumorous and healthy sites.

Hypothermia, in which the brain is cooled to 32-33 °C, has been shown to be neuroprotective for brain injury caused by hypoxia-ischemia, head trauma, or neonatal asphyxia. Neuroprotective effect of Hypothermia is partly due to suppression of brain metabolism and cerebral blood flow (CBF). The ability to measure CBF at the bedside provides a means of detecting, and thereby preventing, secondary ischemia during neuro intensive care before brain injury occurs. The purpose of the present study is to investigate the ability of a time-resolved near-infrared (TR-NIR) bolus-tracking method using indocyanine green as an intravascular flow tracer to measure CBF during cooling in a newborn animal model. For validation, CBF was independently measured by computed tomography (CT) perfusion. The results show a good agreement between CBF obtained with the two methods (R² ≈ 0.84, Δ ≈ 5.84 ml. min ^-1.100 g ^-1, 32-38.5 °C), demonstrating the ability of the TR-NIR technique to non-invasively measure absolute CBF in-vivo during dynamic hypothermia. The TR-NIR technique reveals that CBF decreases from 54.3 ± 5.4 ml. min ^-1.100 g ^-1, at normothermia (T_brain of 38.5 °C), to 33.8 ± 0.9 ml. min ^-1.100 g ^-1 at T_brain of 32 °C during the hypothermia treatment.

Introduction: 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 the GFP indicator gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells. Materials and Methods: U251 monolayers were incubated in AlPcS_2a for 18 h. The monolayers were incubated with non-viral vectors for either 4 or 18 hrs. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched PEI or protomine sulfate (PS), were used with the plasmid construct (GFP-PTEN). Results: PS was much less toxic to the gliomas cells compared to BPEI but was highly inefficient at gene transfection. PCI resulted in a 5-10 fold increase in GFP protein expression compared to controls. Conclusions: Collectively, the results suggest that AlPcS_2a-mediated PCI can be used to enhance transfection of tumor suppressor genes in glioma cells.

Introduction: Dynamic Contrast-Enhanced-Magnetic Resonance Imaging (DCE-MRI) appears to provide an unambiguous means of tracking the outcome of photodynamic therapy (PDT) of brain tumors with the photosensitizer Pc 4. The increase in Gd enhancement observed after Pc 4-PDT may be due to a temporary opening of the blood-brain-barrier which, as noted by others, may offer a therapeutic window. Methods: We injected 2.5 x 10⁵ U87 cells into the brains of 9 athymic nude rats. After 8-9 days peri-tumor DCE-MRI images were acquired on a 7.0 T microMRI scanner before and after the administration of 150 μL Gd. DCE-MRI scans were repeated three times following Pc 4-PDT. Results: The average, normalized peak enhancement in the tumor region, approximately 30-90 seconds after Gd administration, was 1.31 times greater than baseline (0.03 Standard Error [SE]) prior to PDT and was 1.44 (0.02 SE) times baseline in the first Post-PDT scans (Day 11), a statistically significant (p ≈ 0.014, N=8) increase over the Pre- PDT scans, and was 1.38 (0.02 SE) times baseline in the second scans (Day 12), also a statistically significant (p ≈ 0.008, N=7) increase. Observations were mixed in the third Post-PDT scans (Day 13), averaging 1.29 (0.03 SE) times baseline (p ≈ 0.66, N=7). Overall a downward trend in enhancement was observed from the first to the third Post-PDT scans. Discussion: DCE-MRI may provide an unambiguous indication of brain tumor PDT outcome. The initial increase in DCE-MRI signal may correlate with a temporary, PDT-induced opening of the blood-brain-barrier, creating a potential therapeutic window.

Early neurovascular coupling (NVC) changes in Alzheimer's disease can potentially provide imaging biomarkers to assist with diagnosis and treatment. Previous efforts to quantify NVC with intrinsic signal imaging have required assumptions of baseline optical pathlength to calculate changes in oxy- and deoxy-hemoglobin concentrations during evoked stimuli. In this work, we present an economical spatial frequency domain imaging (SFDI) platform utilizing a commercially available LED projector, camera, and off-the-shelf optical components suitable for imaging dynamic optical properties. The fast acquisition platform described in this work is validated on silicone phantoms and demonstrated in neuroimaging of a mouse model.

A prototype neurosurgical hand-held optical coherence tomography (OCT) imaging probe has been developed to provide micron resolution cross-sectional images of subsurface tissue during open surgery. This new ergonomic hand-held probe has been designed based on our group's previous work on electrostatically driven optical fibers. It has been packaged into a catheter probe in the familiar form factor of the clinically accepted Bayonet shaped neurosurgical non-imaging Doppler ultrasound probes. The optical design was optimized using ZEMAX simulation. Optical properties of the probe were tested to yield an ~20 um spot size, 5 mm working distance and a 3.5 mm field of view. The scan frequency can be increased or decreased by changing the applied voltage. Typically a scan frequency of less than 60Hz is chosen to keep the applied voltage to less than 2000V. The axial resolution of the probe was ~15 um (in air) as determined by the OCT system. A custom-triggering methodology has been developed to provide continuous stable imaging, which is crucial for clinical utility. Feasibility of this probe, in combination with a 1310 nm swept source OCT system was tested and images are presented to highlight the usefulness of such a forward viewing handheld OCT imaging probe. Knowledge gained from this research will lay the foundation for developing new OCT technologies for endovascular management of cerebral aneurysms and transsphenoidal neuroendoscopic treatment of pituitary tumors.

We present a near-infrared spectroscopy study of the instantaneous phase difference between spontaneous oscillations of cerebral deoxy-hemoglobin and oxy-hemoglobin concentrations ([Hb] and [HbO], respectively) in the low-frequency range, namely 0.04-0.12 Hz. We report phase measurements during the transitions between different sleep stages in a whole-night study of a human subject. We have found that the phase difference between [Hb] and [HbO] low-frequency oscillations tends to be greater in deep sleep (by ~96° on average) and REM sleep (by ~77° on average) compared to the awake state. In particular, we have observed progressive phase increases as the subject transitions from awake conditions into non-REM sleep stages N1, N2, and N3. Corresponding phase decreases were recorded in the reversed transitions from sleep stages N3 to N2, and N2 to awake. These results illustrate the physiological information content of phase measurements of [Hb] and [HbO] oscillations that reflect the different cerebral hemodynamic conditions of the different sleep stages, and that can find broader applicability in a wide range of near-infrared spectroscopy brain studies.

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

Photoacoustic tomography (PAT) and ultrasonography (US) of biological tissues usually rely on transducer arrays for the detection of ultrasound. Obtaining the best sensitivity requires a physical contact with the tissue using an intermediate coupling fluid (water or gel). This type of contact is a major drawback for several applications such as neurosurgery. Laser-ultrasonics is an established optical technique for the non-contact generation and detection of ultrasound in industrial materials. In this paper, the non-contact detection scheme used in laser-ultrasonics is adapted to allow probing of ultrasound in biological tissues while remaining below laser exposure safety limits. Both non-contact PAT (NCPAT) and non-contact US (NCUS) are demonstrated experimentally using a single-frequency detection laser emitting suitably shaped pulses and a confocal Fabry-Perot interferometer. It is shown that an acceptable sensitivity is obtained while remaining below the maximum permissible exposure (MPE) of biological tissues. Results obtained ex vivo with a calf brain specimen show that sub-mm endogenous and exogenous inclusions can be detected at depths exceeding 1 cm. When fully developed, the technique could be a unique diagnostic tool in neurosurgery providing deep imaging of blood vessels, blood clots and blood oxygenation.

Ca²⁺ plays a vital role as second messenger in signal transduction and the intracellular Ca²⁺ ([Ca²⁺]_i) change is an important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum at >3mm under the brain surface) and image [Ca²⁺]_i changes with cellular resolutions. Here, we present two micro-probe approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of neuronal [Ca²⁺]_i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca²⁺]_i dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca²⁺]_i changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their different mean [Ca²⁺]_i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition, microlens images can characterize the different [Ca²⁺]_i dynamics of D1 and D2 neurons in response to cocaine, including new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.

Characterization of cerebral hemodynamic and oxygenation metabolic changes, as well neuronal function is of great importance to study of brain functions and the relevant brain disorders such as drug addiction. Compared with other neuroimaging modalities, optical imaging techniques have the potential for high spatiotemporal resolution and dissection of the changes in cerebral blood flow (CBF), blood volume (CBV), and hemoglobing oxygenation and intracellular Ca ([Ca²⁺]_i), which serves as markers of vascular function, tissue metabolism and neuronal activity, respectively. Recently, we developed a multiwavelength imaging system and integrated it into a surgical microscope. Three LEDs of λ1=530nm, λ₂=570nm and λ₃=630nm were used for exciting [Ca²⁺]_i fluorescence labeled by Rhod2 (AM) and sensitizing total hemoglobin (i.e., CBV), and deoxygenated-hemoglobin, whereas one LD of λ₁=830nm was used for laser speckle imaging to form a CBF mapping of the brain. These light sources were time-sharing for illumination on the brain and synchronized with the exposure of CCD camera for multichannel images of the brain. Our animal studies indicated that this optical approach enabled simultaneous mapping of cocaine-induced changes in CBF, CBV and oxygenated- and deoxygenated hemoglobin as well as [Ca²⁺]_i in the cortical brain. Its high spatiotemporal resolution (30μm, 10Hz) and large field of view (4x5 mm²) are advanced as a neuroimaging tool for brain functional study.

Optical Coherence Tomography (OCT) provides images at near histological resolution, which allows for the identification of micron sized morphological tissue structures. Optical coherence elastography (OCE) measures tissue displacement and utilizes the high resolution of OCT to generate high-resolution stiffness maps. In this work, we explored the potential of measuring shear wave propagation using OCE. A swept-source OCT system was used in this study. The laser had a center wavelength of 1310 nm and a bandwidth of ~110 nm. The lateral resolution was approximately 13 μm in the samples. Acoustic radiation force was applied to two different phantoms using a 20 MHz circular 8.5 mm diameter piezoelectric transducer element (PZT, f-number 2.35) transmitting sine-wave bursts of 400 μs. The first phantom consisted of a 355 μm glass sphere (dark) embedded in gelatin that was used to characterize the ultrasound pushing beam. The second consisted of gelatin mixed with titanium dioxide, which provided uniform acoustic and optical scattering. The OCT signal from this second set of phantoms was used for the measurement of the shear wave speed and viscosity. For both sets of experiments phase analysis was applied to B-mode and M-mode OCT images which were obtained while the ultrasound transducer was generating the "push" in the phantom. The experiments are the first step towards imaging shear wave propagation in tissue and characterization of tissue mechanical properties using OCE, with the eventual goal of developing OCE as a diagnostic tool for the assessment of pathological lesions with different mechanical tissue property.

Here we present the first use of intraneural and intrafascicular infrared neural stimulation (INS) with early-generation Utah Slanted Optrode Arrays (USOAs) to produce highly selective, artifact-free stimulation of peripheral nerves. USOAs utilize technology previously developed for Utah Slanted Electrode Arrays, and contain 100 silicon optrodes of 0.5 to 1.5 mm length, spaced 400 μm apart in a 10 x 10 grid. The optrodes penetrate into the nerve and closely abut nerve fibers, thus providing multiple, independent, focal sites of stimulation. We first demonstrated that intraneural (but extrafascicular) infrared (IR) stimulation of cat sciatic nerve with conventional optical fibers coupled to a Lockheed Martin Aculight Capella laser produced stronger and more selective neural and muscle compound action potentials (CAPs) than did extraneural INS. We next tested INS through individual USOA optrodes (e.g., wavelength 1873 nm, 5- ms stimulus pulse, < 1 mJ at optrode tip). In contrast to extraneural INS, intrafascicular INS evoked relatively strong and highly selective, optrode-specific responses. Further, there were no observable stimulus artifacts, thereby allowing adjacent electrical recordings. These initial results indicate that intrafascicular INS via USOAs may provide a more efficient, more selective, high-optrode-count means of activating axons, plus greater access to interior nerve fibers.

We present an early-generation Utah Slant Optrode Array (USOA) for infrared (IR) neural stimulation. Intrafascicular IR stimulation with the early prototype in the cat sciatic nerve produced highly selective and artifact-free responses, which outperformed extraneural IR stimulation. We characterized the light delivery and loss mechanisms of the device in order to facilitate design optimization. Fabrication of the USOA takes advantage of the extensive research in the development of the Utah Slant Electrode Array (USEA). An undoped (ρ > 20 ω ρ cm) c-Si (100) substrate was used to produce a 10 x 10 array of optrodes with lengths from 0.5 mm to 1.5 mm in a 400-μm pitch. This substrate is able to transmit IR (λ > 1.1μm) with negligible absorption losses. The optrodes were coupled to the laser source via fibers of different core diameters through in-coupling interfaces of varying refractive indices. The effect of these factors on optrode transmission efficiency was investigated. At 1550nm, transmittance for a butt-coupled 50-μm multimode fiber through a medium of index n = 1.66 was measured as 34.7%, which was the maximum value obtained. When the refractive index of the intervening medium was lowered, transmission decreased according to Fresnel reflection theory. Above 100-μm core size, transmitted power decreases by 40% with each doubling of the fiber core diameter. Transmission was also found to be dependent on the optrode length, where shorter and more tapered optrodes provided less output power. The results suggest that Fresnel, coupling, and radiation losses are the primary loss mechanisms.

Neuronal optical excitation can provide non-contacting tools to explore brain circuitry and a durable stimulation interface for cardiac pacing and visual as well as auditory sensory neuronal stimulation. To obtain accurate absorption spectra, we scan the transmission of neurons in cell culture medium, and normalize it by subtracting out the absorption spectrum of the medium alone. The resulting spectra show that the main neuronal absorption peaks are in the 3000- 6000nm band, although there is a smaller peak near 1450nm. By coupling the output of a 3μm interband cascade laser (ICL) into a mid-IR fluorozirconate fiber, we can effectively deliver more than 1J/cm² photon intensity to the excitation site for neuronal stimulation.

The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we developed a system integrating an optical tweezers and a laser dissector to manipulate the sample. A sub-nanosecond pulsed UVA laser was used to inflict a partial damage to the axon of mouse hippocampal neurons at early days in vitro. Partial axonal transections were performed in a highly controlled and reproducible way without affecting the regeneration process. Force spectroscopy measurements, during and after the ablation of the axon, were performed by optical tweezers with a bead attached to the neuronal membrane. Thus, the release of tension in the neurite could be analyzed in order to quantify the inflicted damage. After dissection, we monitored the viscoelastic properties of the axonal membrane, the cytoskeleton reorganization, and the dynamics of the newly formed growth cones during regeneration. In order to follow cytoskeleton dynamics in a long time window by tracking a bead attached to the neuron, we developed a real-time control of the microscope stage position with sub-millisecond and nanometer resolution. Axonal regeneration was documented by long-term (24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.

In this paper we induced sub-axotomy axonal damage and repair by use of the laser microbeam in cultured retinal ganglion cells. When growth cones are near the damaged site of an axon, some cases will respond to the damage by extending filopodia towards the damaged axon. In some cases, the filopodia touch the damaged site. These experiments suggest that guidance cues that are sensed by the growth cones are released from the damaged site. These results also indicate that axon growth cone have a role in repair of the neuronal damage.

Cellular exposure to nanosecond pulsed electric fields (nsPEF) are believed to cause immediate creation of nanopores in the plasma membrane. These nanopores enable passage of small ions, but remain impermeable to larger molecules like propidium iodide. Previous work has shown that nanopores are stable for minutes after exposure, suggesting that formation of nanopores in excitable cells could lead to prolonged action potential inhibition. Previously, we measured the formation of nanopores in neuroblastoma cells by measuring the influx of extracellular calcium by preloading cells with Calcium Green-AM. In this work, we explored the impact of changing the width of a single nsPEF, at constant amplitude, on uptake of extracellular calcium ions by primary hippocampal neurons (PHN). Calcium Green was again used to measure the influx of extracellular calcium and FM1-43 was used to monitor changes in membrane conformation. The observed thresholds for nanopore formation in PHN by nsPEF were comparable to those measured in neuroblastoma. This work is the first study of nsPEF effects on PHN and strongly suggests that neurological inhibition by nanosecond electrical pulses is highly likely at doses well below irreversible damage.

Since development of optogenetic stimulation paradigm, there has been several attempts to red shift the excitation maximum of the efficient blue-sensitive opsins. While there has been some success at the cost of altered light-activation kinetics, near-infrared optogenetic probe will be ideal for in-depth cell-specific stimulation of excitable cells in an organ. However, single-photon near-infrared optogenetics based stimulation will still limit precise probing and modulation of in-vivo neural circuits. In contrast, by virtue of non-linear nature of ultrafast light-matter interaction, high spatial precision in optogenetic activation can be achieved in addition to inherent cellular specificity and temporal resolution provided by the opsins. Here, we report use of non-linear optogenetics for stimulation of neurons in-vivo in mouse models. Advantage of using non-linear optogenetics for probing neuronal circuitry is discussed. Further, effectiveness of the non-diffracting optogenetic Bessel beam over classical Gaussian beam in a layered mouse-brain geometry is demonstrated using Monte Carlo (MC) simulation. This is corroborated by electrophysiological measurements in in-vivo mouse models. The large propagation distance, characteristics of Bessel beam is better suited for in-depth single as well as two-photon optogenetic stimulation.

The rise of optogenetic neural stimulation has opened new opportunities for neuroprosthesis such as visual cortical prosthesis, which necessitates an efficient delivery of light into the cortex. New forms of photosensitizing channelrhodopsin are reducing the required light intensities for stimulation, but implantable systems need to be highly efficient. Such efficiency calls for low loss in the transmission path, high coupling efficiency between the optic delivery system and optical emitter, as well as emitting efficiency from the light emitting diode. In this paper, we perform simulation results based on ray optics and illuminating theory as to the best strategy to attachment of optrode structures to Gallium Nitride-μLED arrays so as to maximize the efficiency of light delivery to the target neural tissue.Our results show that it is feasible to connect optrode elements and GaN-μLEDarrays for cortical stimulation and describe the optimisation requirements.

While pulsed laser beams have been used for stimulation of neurons, cellular specificity during optical stimulation is achieved by photo-sensitization of genetically-targeted cells by optogenetic means. However, till date, the process of optogenetic-sensitization primarily involves use of viral vectors. In rare occasions, electroporation has been used. Here, we report an all-optical method in which pulsed laser beam is used for delivery of genes, encoding optogenetic probes, to spatially-targeted cells, followed by optogenetic stimulation and optical detection of the activation process. Use of laser microbeam enabled highly precise spatially-patterned delivery of optogenes, as confirmed by expression of conjugated fluorescent protein. Light-activation of opsin-expressing cells was confirmed by calcium-imaging. The laser-assisted expression of optogenetic probes in spatially-targeted regions in combination with light-assisted activation and optical detection of neural activity will help in better understanding of the neuronal circuitry.

Myelination, i.e. the wrapping of axons in multiple layers of lipid-rich membrane, is a unique phenomenon in the nervous systems of both vertebrates and invertebrates, that greatly increases the speed and efficiency of signal transmission. In turn, disruption of axo-myelinic integrity underlies disability in numerous clinical disorders. The dependence of myelin physiology on nanometric organization of its lamellae makes it difficult to accurately study this structure in the living state. We expected that fluorescent probes might become highly oriented when partitioned into the myelin sheath, and in turn, this anisotropy could be interrogated by controlling the polarization state of the exciting laser field used for 2-photon excited fluorescence (TPEF). Live ex vivo myelinated rodent axons were labeled with a series of lipohilic and hydrophilic fluorescenct probes, and TPEF images acquired while laser polarization was varied at the sample over a broad range of ellipticities and orientations of the major angle [see Brideau, Micu & Stys, abstract this meeting]. We found that most probes exhibited strong dependence on both the major angle of polarization, and perhaps more surprisingly, on ellipticity as well. Lipophilic vs. hydrophilic probes exhibited distinctly different behavior. We propose that polarization-dependent TPEF microscopy represents a powerful tool for probing the nanostructural architecture of both myelin and axonal cytoskeleton in a domain far below the resolution limit of visible light microscopy. By selecting probes with different sizes and physicochemical properties, distinct aspects of cellular nanoarchitecture can be accurately interrogated in real-time in living tissue.

Recent advent of optogenetics has enabled activation of genetically-targeted neuronal cells using low intensity blue light with high temporal precision. Since blue light is attenuated rapidly due to scattering and absorption in neural tissue, optogenetic treatment of neurological disorders may require stimulation of specific cell types in multiple regions of the brain. Further, restoration of certain neural functions (vision, and auditory etc) requires accurate spatio-temporal stimulation patterns rather than just precise temporal stimulation. In order to activate multiple regions of the central nervous system in 3D, here, we report development of an optogenetic prosthetic comprising of array of fibers coupled to independently-controllable LEDs. This design avoids direct contact of LEDs with the brain tissue and thus does not require electrical and heat isolation, which can non-specifically stimulate and damage the local brain regions. The intensity, frequency, and duty cycle of light pulses from each fiber in the array was controlled independently using an inhouse developed LabView based program interfaced with a microcontroller driving the individual LEDs. While the temporal profile of the light pulses was controlled by varying the current driving the LED, the beam profile emanating from each fiber tip could be sculpted by microfabrication of the fiber tip. The fiber array was used to stimulate neurons, expressing channelrhodopsin-2, in different locations within the brain or retina. Control of neural activity in the mice cortex, using the fiber-array based prosthetic, is evaluated from recordings made with multi-electrode array (MEA). We also report construction of a μLED array based prosthetic for spatio-temporal stimulation of cortex.

In this proceedings we will present a research project financed by Piedmont regional government (Italy) and finalized to the realization and commercialization of functional devices for cellular bio-sensing based on diamond. Partners of the project are: Crisel Instruments, Torino University, Torino Polytechnic, INRIM, Politronica, Bionica Tech, Ulm University Here the main features of the final devices will be briefly summarized. We envisage an active diamond-based cellular substrate that can simultaneously stimulate and detect a variety of signals (chemical, optical, electrical) to and from neuroendocrine cells, in a fully biocompatible environment for the cellular system under test. Such a device can be realized by fully exploiting the peculiar properties of diamond: optical transparency, biocompatibility, chemical inertness, accessibility to a conductive graphite-like phase; properties that will be further explored and tested during the project.

As advances in fiber optic probe design move Raman spectroscopy into the clinic, there remain important practical problems. Most in-vivo non-invasive applications employ specialized fiber optic probes. Much effort has been devoted to minimizing Raman and fluorescence background from fiber. Less attention has been paid to the need to generate reference Raman signals proportional to delivered laser power without direct measurement of tissue albedo. Knowledge of laser power is needed for quantification of changes in tissue composition. The need is especially acute in diffuse Raman tomography, where accurate modeling of light transport through the tissue is required for accurate reconstruction of subsurface features. We describe a fiber optic probe that incorporates a transparent polymer cap at the end of each excitation fiber. As laser light propagates through the cap it generates Raman bands whose intensity can directly measure power delivered to the tissue of interest. Our first implementation uses a fluorinated ethylene-propylene copolymer (FEP) cap that is attached to the ferrule at the distal (delivery) end of each excitation fiber. FEP is transparent and functions as a waveguide with only a small insertion loss, about 5%. Importantly, there are few overlaps between the Raman bands of FEP and the bands of tissue constituents. The cap increases the diameter of the structure in contact with the specimen, but with extensive photon diffusion this makes little difference in performance. We present here latest non-invasive bone spectroscopy results with the calibrator. In addition, extensive enhancement of the calibration signal using a fluorocarbon optical fiber is discussed.

Over the past two decades, osteoporosis has been recognized among the most serious public health problems. Fortunately with the growing awareness of osteoporosis, new treatments have been developed for the prevention of fracture. At the same time, there is a rapid improvement in diagnostic methods. In this study biomedical photoacoustics (PA) is applied to the analysis of bone mineral concentration. The PA signal depends on optical as well as mechanical properties of the object and therefore has the potential to provide higher sensitivity to density variations compared with standard diagnostic methods, like ultrasound. A laser source with 800 nm wavelength and different ultrasonic transducers with resonance frequencies in the range 1 to 5 MHz were employed. The CW or frequency-domain (FD) PA radar method was utilized with linear frequency modulation chirps to provide temporal gating control over the transmitted signal and higher sensitivity in the detected signal. The laser intensity was set below the safety standards for skin exposure. The preliminary studies showed adequate optical absorption by cortical bone to generate measurable PA signals and the transmission of laser light through this layer. Experiments are focused on detection and evaluation of PA signals from in-vitro animal cortical bones with and without a trabecular sublayer. The trabecular layer is then diluted by chemical etching and differences in the PA signals are discussed.

A chirped pulsed photothermal radiometric radar is introduced for the diagnosis of biological samples, especially bones with tissue and skin overlayers. The constraints imposed by the laser safety (maximum permissible exposure, MPE) ceiling on pump laser energy and the strong attenuation of thermal-wave signals in tissues significantly limit the photothermally active depth in most biological specimens to a level which is normally insufficient for practical applications (approx. 1 mm below the skin surface). A theoretical approach for improvement of signal-to-noise ratio (SNR), minimizing the static (dc) component of the photothermal signal and making use of the photothermal radiometric nonlinearity has been introduced and verified by comparing the SNR of four distinct excitation wave forms (sine-wave, square-wave, constant- width and constant duty-cycle pulses) for chirping the pump laser, under constant exposure energy. At low frequencies fixed-pulsewidth chirps of large peak power were found to be superior to all other equal-energy modalities, with an SNR improvement up to two orders of magnitude. Distinct thickness-dependent characteristic delay times in a goat bone were obtained, establishing an active depth resolution range of ca. 2.8 mm in a layered skin-fat- bone structure, a favorable result compared to the maximum reported pulsed photothermal radiometric depth resolution < 1 mm in turbid biological media. Compared to radar peak delay and amplitude, the long-delayed radar output amplitude is found to be more sensitive to subsurface conditions. Two-dimensional spatial plots of this parameter depicting the back surface conditions of bones with and without fat-tissue overlayers are presented.

Energy transport in diffusion-wave fields is gradient driven and therefore diffuse, yielding depth-integrated responses with poor axial resolution. Using matched-filter principles, we propose a methodology enabling these parabolic diffusion-wave energy fields to exhibit energy localization akin to propagating hyperbolic wave-fields. This not only improves the axial resolution, but also allows for deconvolution of individual responses of superposed axially discrete sources, opening a new field of depth-resolved subsurface thermal coherence tomography using diffusion waves. The depth resolved nature of the developed methodology is verified through experiments carried out on phantoms and biological samples. The results suggest that thermal coherence tomography can resolve deep structural changes in hard dental and bone tissues. The sensitivity of the developed diagnostic imaging system is compared to that of polarized Raman spectroscopy.

Raman spectroscopy of bone has been used to characterize chemical changes occurring in diseases such as osteoporosis, osteoarthritis and osteomyelitis. Metastasis of cancer into bone causes changes to bone quality that are similar to those observed in osteoporosis, such as decreased bone strength, but with an accelerated timeframe. In particular, osteolytic (bone degrading) lesions in bone metastasis have a marked effect on patient quality of life because of increased risk of fractures, pain, and hypercalcemia. We use Raman spectroscopy to examine bone from two different mouse models of osteolytic bone metastasis. Raman spectroscopy measures physicochemical information which cannot be obtained through standard biochemical and histological measurements. This study was reviewed and approved by the University of Michigan University Committee on the Care and Use of Animals. Two mouse models of prostate cancer bone metastasis, RM1 (n=3) and PC3-luc (n=4) were examined. Tibiae were injected with RM1 or PC3-luc cancer cells, while the contralateral tibiae received a placebo injection for use as controls. After 2 weeks of incubation, the mice were sacrificed and the tibiae were examined by Raman microspectroscopy (λ=785 nm). Spectroscopic markers corresponding to mineral stoichiometry, bone mineralization, and mineral crystallinity were compared in spectra from the cancerous and control tibiae. X-ray imaging of the tibia confirmed extensive osteolysis in the RM1 mice, with tumor invasion into adjoining soft tissue and moderate osteolysis in the PC3-luc mice. Raman spectroscopic markers indicate that osteolytic lesions are less mineralized than normal bone tissue, with an altered mineral stoichiometry and crystallinity.