This PDF file contains the front matter associated with SPIE Proceedings Volume 9303, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

Melanoma is the most dangerous form of skin cancer and is the most difficult to diagnose and stage. Knowing the thickness of melanoma and its level of invasion into cutaneous tissue is the most important factor [1, 2] in determining a patient’s prognosis [3, 4]. Detection of melanoma is typically done by clinical inspection of lesion morphology, followed by lesion excision and histological assessment of the resected specimen. To improve assessment and diagnosis of melanoma and other pigmented lesions, various non-invasive imaging techniques, including photoacoustic (PA) imaging, have been investigated. PA imaging is a non-invasive imaging modality which combines laser light with ultrasound, and can be used to image pigmented skin lesion morphology [5-7] due the high absorption of melanin in the visible and near-infrared wavelength rage. In this study we investigate the clinical usefulness of PA imaging in diagnosing and assessing pigmented skin lesions such as melanoma and melanocytic nevi. Pre-operative PA images of patients with suspected cases of cutaneous melanoma were taken with the Vevo Lazr® 2100 PA imaging system at several wavelengths. The distribution and maximum thickness of suspect lesions was determined by imaging at 700 nm, and the surrounding vasculature was imaged at 900 nm. Information obtained from the PA images was compared with histological examination of resected surgical specimens.

We apply diffuse reflectance spectroscopy (DRS) and pulsed photothermal radiometry (PPTR) for characterization of the bruise evolution process. While DRS provides information in a wide range of visible wavelengths, the PPTR enables extraction of detailed depth distribution and concentration profiles of selected absorbers (e.g. melanin, hemoglobin). In this study, we simulate experimental DRS spectra and PPTR signals using the Monte Carlo technique and focus on characterization of a suitable fitting approach for their analysis. We find inverse Monte Carlo to be superior to the diffusion approximation approach for the inverse analysis of DRS spectra. The analysis is then augmented with information obtainable by the fitting of the PPTR signal. We show that both techniques can be coupled in a combined fitting approach. The combining of two complementary techniques improves the robustness and accuracy of the inverse analysis, enabling a comprehensive quantitative characterization of the bruise evolution dynamics.

Minimally-invasive cosmetic surgeries such as injection of subdermal fillers have become very popular in the past decade. Although rare, some complications may follow injections such as tissue necrosis and even blindness. There exist two hypothesis regarding source of these complications both of which include microvasculature. The first hypothesis is that fillers in between the tissue structures and compress microvasculature that causes blockage of tissue neutrition and oxygen exchange in the tissue. In another theory, it is hypothesized that fillers move inside major arteries and block the arteries/veins. In this paper, we study these hypotheses using optical coherence tomography and optical microangiography technologies with different hyaluronic-acid fillers in a mouse ear model. Based on our observations, the fillers eventually block arteries/veins if injected directly into them that eventually causes tissue necrosis.

A faster healing process was observed in superficial skin wounds after irradiation with the EMOLED photocoagulator. The instrument consists of a compact handheld photocoagulation device, useful for inducing coagulation in superficial abrasions. In this study, living animals were mechanically abraded in four regions of their back: two regions were left untreated, the other two were treated with EMOLED, healthy skin surrounding the wounds was used as a control. The treatment effect on skin was monitored by visual observations, histopathological analysis, immuno-histochemical analysis, and non-linear microscopic imaging performed 8 days after the treatment, finding no adverse reactions and no thermal damage in both treated areas and surrounding tissues. In addition, a faster healing process, a reduced inflammatory response, a higher collagen content, and a better-recovered skin morphology was evidenced in the treated tissue with respect to the untreated tissue. These morphological features were characterized by means of immuno-histochemical analysis, aimed at imaging fibroblasts and myofibroblasts, and by SHG microscopy, aimed at characterizing collagen organization, demonstrating a fully recovered aspect of dermis as well as a faster neocollagenesis in the treated regions. This study demonstrates that the selective photothermal effect we used for inducing immediate coagulation in superficial wounds is associated to a minimal inflammatory response, which provides reduced recovery times and improved healing process.

Certified clinical multiphoton tomographs are employed to perform rapid label-free high-resolution in vivo histology. Novel tomographs include a flexible 360° scan head attached to a mechano-optical arm for autofluorescence and SHG imaging as well as rigid two-photon GRIN microendoscope. Mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged with submicron resolution in human skin. The system was employed to study the healing of chronic wounds (venous leg ulcer) and acute wounds (curettage of actinic or seborrheic keratosis) on a subcellular level. Furthermore, a flexible sterile foil as interface between wound and focusing optic was tested.

Assessing collagen alignment is of interest when evaluating a therapeutic strategy and evaluating its outcome in scar management. In this work we introduce a theoretical and experimental methodology for the quantification of collagen and birefringent media alignment based on polarized light transport. The technique relies on the fact that these materials exhibit directional anisotropy. A polarized Monte Carlo model and a spectro-polarimetric imaging system were devised to predict and measure the impact of birefringence on an impinging polarized light beam. Experiments conducted on birefringent phantoms, and biological samples consisting of highly packed parallel birefringent fibers, showed a good agreement with the analytical results.

Acne is a common skin disease in society and often leads to scarring. In this paper, we demonstrate the capabilities of swept-source optical coherence tomography (SS-OCT) in detecting specific features of acne lesion initiation and scarring on human facial skin in vivo over 30 days. Optical microangiography (OMAG) technique made it possible to image 3D tissue microvasculature changes up to 1 mm depth in vivo without the need of exogenous contrast agents in ~10 seconds. The presented results show promise to facilitate clinical trials of treatment and prognosis of acne vulgaris by detecting cutaneous microvasculature and structural changes within human skin in vivo.

We present a non-contact, reflective photoplethysmogram (PPG) imaging method and a prototype system for identifying the presence of dermal burn wounds during a burn debridement surgery. This system aims to provide assistance to clinicians and surgeons in the process of dermal wound management and wound triage decisions. We examined the system variables of illumination uniformity and intensity and present our findings. An LED array, a tungsten light source, and eventually high-power LED emitters were studied as illumination methods for our PPG imaging device. These three different illumination sources were tested in a controlled tissue phantom model and an animal burn model. We found that the low heat and even illumination pattern using high power LED emitters provided a substantial improvement to the collected PPG signal in our animal burn model. These improvements allow the PPG signal from different pixels to be comparable in both time-domain and frequency-domain, simplify the illumination subsystem complexity, and remove the necessity of using high dynamic range cameras. Through the burn model output comparison, such as the blood volume in animal burn data and controlled tissue phantom model, our optical improvements have led to more clinically applicable images to aid in burn assessment.

Previous research has shown that the stepwise multi-photon activation fluorescence (SMPAF) of melanin, activated and excited by a continuous-wave (CW) mode near infrared (NIR) laser, is a low-cost and reliable method for detecting melanin. We have developed a device utilizing the melanin SMPAF to guide the ablation of melanin with a 975 nm CW laser. This method provides the ability of targeting individual melanin particles with micrometer resolution, and enables localized melanin ablation to be performed without collateral damage. Compared to the traditional selective photothermolysis, which uses pulsed lasers for melanin ablation, this method demonstrates higher precision and lower cost. Therefore, the SMPAF guided selective ablation of melanin is a promising tool of melanin ablation for both medical and cosmetic purposes.

Due to the often extreme energies employed, contemporary methods of laser delivery utilized in clinical dermatology allow for a dangerous amount of high-intensity laser light to reflect off a multitude of surfaces, including the patient’s own skin. Such techniques consistently represent a clear and present threat to both patients and practitioners alike. The intention of this work was therefore to develop a technique that mitigates this problem by coupling the light directly into the tissue via physical contact with an optical waveguide. In this manner, planar waveguides cladded in silver with thin-film active areas were used to illuminate agar tissue phantoms with nanosecond-pulsed laser light at 532nm. The light then either refracted or optically tunneled through the active area, photoacoustically generating ultrasonic waves within the phantom, whose peak-to-peak intensity directly correlated to the internal reflection angle of the beam. Consequently, angular spectra for energy delivery were recorded for sub-wavelength silver and titanium films of variable thickness. Optimal energy delivery was achieved for internal reflection angles ranging from 43 to 50 degrees, depending on the active area and thin film geometries, with titanium films consistently delivering more energy across the entire angular spectrum due to their relatively high refractive index. The technique demonstrated herein therefore not only represents a viable method of energy delivery for biological tissue while minimizing the possibility for stray light, but also demonstrates the possibility for utilizing thin films of high refractive index metals to redirect light out of an optical waveguide.

The surface of the skin plays an important role in the diagnosis of many clinical conditions, in relation to ageing and the acceptance of many consumer products. Considerable resource has been applied to skin in terms of cancer diagnosis but the actual surface finish of the tissue has been frequently overlooked. An optical system and associated imaging processing method has been developed which analyses the speckle pattern, recorded on a basic digital imaging system, and provides a quantitative analysis of the surface roughness. Results demonstrate that these measurements can be linked with more qualitative perceptions of skin quality (roughness).

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD (“redox ratio”) can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

Background: Prior research indicates the epidermal pigment layer of human skin (Melanin) has a significant absorption coefficient in the near infra-red (NIR) region; hence attenuation of light in vivo is a potential confounder for NIR spectroscopy (NIRS). A NIRS method developed for transcutaneous evaluation of bladder function is being investigated as a means of improving the burden of bladder disease in sub-Saharan Africa. This required development of a simple wireless NIRS device suitable for use as a screening tool in patients with pigmented skin where the NIR light emitted would penetrate through the epidermal pigment layer and return in sufficient quantity to provide effective monitoring.

Methods: Two healthy subjects, one with pigmented skin and one with fair skin, were monitored as they voided spontaneously using the prototype transcutaneous NIRS device positioned over the bladder. The device was a self-contained wireless unit with light emitting diodes (wavelengths 760 and 850 nanometres) and interoptode distance of 4cm. The raw optical data were transmitted to a laptop where graphs of chromophore change were generated with proprietary software and compared between the subjects and with prior data from asymptomatic subjects.

Results: Serial monitoring was successful in both subjects. Voiding volumes varied between 350 and 380 cc. In each subject the patterns of chromophore change, trend and magnitude of change were similar and matched the physiologic increase in total and oxygenated hemoglobin recognized to occur in normal bladder contraction during voiding.

Conclusions: Skin pigmentation does not compromise the ability of transcutaneous NIRS to interrogate physiologic change in the bladder during bladder contraction in healthy subjects.

We present a new concept on how to remove unwanted green fluorescence from urine during Photodynamic Diagnostics of tumours in the bladder using cystoscopy. A high power LED based light source (525 nm) has been made in our laboratory. This light source is tailored to match most commercially available rigid cystoscopes. A suitable spectral filter and adapter, for the eyepiece of the cystoscope, has been selected which allows the urologist to observe both red fluorescence from tumours and autofluorescence from healthy tissue at the same time.

Diseases of urinary bladder are a common healthcare problem world over. Diagnostic precision and predicting response to treatment are major issues. This study aims to create an optical cross-sectionional model of a bladder, capable of visually representing the passage of photons through the tissue layers. The absorption, transmission and reflectance data, along with the derived transmission coefficients (of scattering and absorption) were obtained from literature analysis and were used in the creation of a “generic” cross-section optical property model simulating the passage of thousands of photons through the tissue at different wavelengths. Fluorescence spectra of diagnostically relevant biomarkers excited by the UV and blue wavelengths were modelled on the basis of the Monte-Carlo method. Further to this, fluorescence data gathered by the “LAKK-M” system from pig bladders was applied to the model for a specific representation of the photon passage through the tissues. The ultimate goal of this study is to employ this model to simulate the effects of different laser wavelength and energy inputs to bladder tissue and to determine the effectiveness of potential photonics based devices for the diagnosis of bladder pathologies. The model will aid in observing differences between healthy and pathological bladder tissues registered by photonics based devices.

Purpose: Treatment of urethral strictures is a major challenge in urology. For investigation of different treatment methods an animal model was developed by reproducible induction of urethral strictures in rabbits to mimic the human clinical situation. By means of this model the potential of endoluminal LDR brachytherapy using β-irradiation as prophylaxis of recurrent urethral strictures investigated. Material and Methods: A circumferential urethral stricture was induced by energy deposition using laser light application (wavelength λ=1470 nm, 10 W, 10 s, applied energy 100 J) in the posterior urethra of anaesthetized New Zealand White male rabbits. The radial light emitting fiber was introduced by means of a children resectoscope (14F). The grade of urethral stricture was evaluated in 18 rabbits using videourethroscopy and urethrography at day 28 after stricture induction. An innovative catheter was developed based on a β-irradiation emitting foil containing 32P, which was wrapped around the application system. Two main groups (each n=18) were separated. The "internal urethrotomy group" received after 28days of stricture induction immediately after surgical urethrotomy of the stricture the radioactive catheter for one week in a randomized, controlled and blinded manner. There were 3 subgroups with 6 animals each receiving 0 Gy, 15 Gy and 30 Gy. In contrast animals from the “De Nuovo group” received directly after the stricture induction (day 0) the radioactive catheter also for the duration of one week divided into the same dose subgroups. In order to determine the radiation tolerance of the urethral mucosa, additional animals without any stricture induction received a radioactive catheter applying a total dose of 30 Gy (n=2) and 15 Gy (n=1). Cystourethrography and endoscopic examination of urethra were performed on all operation days for monitoring treatment progress. Based on these investigation a classification of the stricture size was performed and documented for correlation. At further 28 days after catheter removal the animals were euthanasized and the urethra tissue was harvested. Histological examination of tissue with assessment of radiation damage, fibrotic and inflammatory changes were performed. After deblinding histological finding were correlated with the applied dose. Results: All animals developed a stricture, while 15/18 (83,3%) showed a significant, high grade stricture with more than 90% lumen narrowing. Histopathological examination including evaluation of urethral inflammation, fibrosis and collagen content were investigated in additional 6 rabbits confirming the former findings. No rabbits died prematurely during the study. The experiments showed that the procedure of the application of radioactive catheter was safe without any problems in contamination and protection handling. The combination of internal urethrotomy and LDR-brachytherapy results in a stricture free rate of 66.7% in the 15-Gy group, compared with only 33.3% among animals from the 0- and 30-Gy groups. Furthermore histological classification of inflammation and fibrosis of 0 Gy and 15 Gy showed similar extent. Conclusion: This new method of laser induced urethral stricture was very efficient and showed a high reproducibility, thus being useful for studying stenosis treatments. The experiments showed that application of local β-irradiation by means of radioactive catheters modulated the stenosis development. This kind of LDR-brachytherapy shows potential for prophylaxis of urethral stricture. As this was an animal pilot experiment a clinical dose response study is needed.

Using a validated in vitro ureter model for laser lithotripsy, the performance of an experimental Thulium fiber laser (TFL) was studied and compared to clinical gold standard Holmium:YAG laser. The Holmium laser (λ = 2120 nm) was operated with standard parameters of 600 mJ, 350 μs, 6 Hz, and 270-μm-core optical fiber. TFL (λ = 1908 nm) was operated with 35 mJ, 500 μs, 150-500 Hz, and 100-μm-core fiber. Urinary stones (60% calcium oxalate monohydrate / 40% calcium phosphate), of uniform mass and diameter (4-5 mm) were laser ablated with fibers through a flexible video-ureteroscope under saline irrigation with flow rates of 22.7 ml/min and 13.7 ml/min for the TFL and Holmium laser, respectively. The temperature 3 mm from tube’s center and 1 mm above mesh sieve was measured by a thermocouple and recorded during experiments. Total laser and operation times were recorded once all stone fragments passed through a 1.5-mm sieve. Holmium laser time measured 167 ± 41 s (n = 12). TFL times measured 111 ± 49 s, 39 ± 11 s, and 23 ± 4 s, for pulse rates of 150, 300, and 500 Hz (n = 12 each). Mean peak saline irrigation temperatures reached 24 ± 1 °C for Holmium, and 33 ± 3 °C, 33 ± 7 °C, and 39 ± 6 °C, for TFL at pulse rates of 150, 300, and 500 Hz. To avoid thermal buildup and provide a sufficient safety margin, TFL lithotripsy should be performed with pulse rates below 500 Hz and/or increased saline irrigation rates. The TFL rapidly fragmented kidney stones due in part to its high pulse rate, high power density, high average power, and reduced stone retropulsion, and may provide a clinical alternative to the conventional Holmium laser for lithotripsy.

Fiber-tip degradation, damage, or burn back is a common problem during the ureteroscopic laser lithotripsy procedure to treat urolithiasis. Fiber-tip burn back results in reduced transmission of laser energy, which greatly reduces the efficiency of stone comminution. In some cases, the fiber-tip degradation is so severe that the damaged fiber-tip will absorb most of the laser energy, which can cause the tip portion to be overheated and melt the cladding or jacket layers of the fiber. Though it is known that the higher the energy density (which is the ratio of the laser energy fluence over the cross section area of the fiber core), the faster the fiber-tip degradation, the damage mechanism of the fibertip is still unclear. In this study, fiber-tip degradation was investigated by visualization of shockwave, cavitation/bubble dynamics, and calculus debris ejection with a high-speed camera and the Schlieren method. A commercialized, pulsed Ho:YAG laser at 2.12 um, 273/365/550-um core fibers, and calculus phantoms (Plaster of Paris, 10x10x10 mm cube) were utilized to mimic the laser lithotripsy procedure. Laser energy induced shockwave, cavitation/bubble dynamics, and stone debris ejection were recorded by a high-speed camera with a frame rate of 10,000 to 930,000 fps. The results suggested that using a high-speed camera and the Schlieren method to visualize the shockwave provided valuable information about time-dependent acoustic energy propagation and its interaction with cavitation and calculus. Detailed investigation on acoustic energy beam shaping by fiber-tip modification and interaction between shockwave, cavitation/bubble dynamics, and calculus debris ejection will be conducted as a future study.

Introduction and objective: In Europe, nearly every sixth couple in the reproductive age is involuntarily childless. In about 30%, both male and female reveal fertility problems. In about 10% of infertile men, azoospermia is the underlying cause. As conventional therapeutic options are limited, surgical testicular sperm extraction (TESE) is necessary to obtain sperms for assisted reproductive techniques. Regarding the females, up to 30% of all idiopathic infertilities are due to alterations of the uterine tube So far, no imaging technique, which does not require any labelling, is available to evaluate the male and female genital tract at a microscopic level under in vivo conditions. Thus, the aim of this study was to investigate the potential of optical coherence tomography (OCT) as a non-invasive diagnostic tool in gynaecology and andrology. Material and Methods: Tissues samples from the bovine testis, epididymis, vas deferens, ovary, oviduct (ampulla and isthmus) and uterus were obtained immediately after slaughter (14 cows aged 3 to 8 years and 14 bulls aged 3 to 6 years; breeds: Holstein- Friesian, and Deutsches Fleckvieh). Imaging was done by using the US Food and Drug Administration (FDA) approved probe-based Niris Imaging System (Imalux, Cleveland, Ohio, USA) and the Telesto 1325 nm OCT System and Ganymede 930 nm OCT System (Thorlabs Inc., Dachau, Germany). All images obtained were compared to histological images after paraffin embedding and HE staining. Results: OCT imaging visualized the microarchitecture of the testis, epididymis, spermatic duct and the ovary, oviduct and uterus. Using the Thorlabs systems a axial resolution of approx. 5μm and lateral resolution of 8- 15μm could be achieved. Different optical tissue volumes could be visualized, which depends on the optical penetration depth of the wavelength of the system used. While the tissue volume observed by probe based Imalux-OCT is similar to the used Thorlabs systems, the optical resolution is reduced. By means of the microscopic OCT-system differentiation of testical tissue structures like content and diameter of seminiferous tubules and the epididymal duct was possible. Structures of the female oviduct, like the primary, secondary and tertiary folds including the typical epithelium consisting of secretory and ciliated cells were identified. Ampulla and isthmus were clearly differentiated by the height of the folds and the thickness of the smooth muscle layer. Imaging was successful both from the outside wall and from the inner lumen. After experience with microscopic OCT-structure identification such structures could also be identified by means of probe based OCT. Conclusions: Technical improvement of probe-based OCT up to a high-resolution level of nowadays-available OCT microscopic systems could open up new ways of in vivo imaging in the reproductive tract. Potential applications could be an OCT-guided testicular biopsy for improving sperm retrieval or microscopic evaluation of the oviduct by OCT-assisted fertiloscopy. The latter would provide a valuable tool to facilitate the decision of which type of assisted reproductive techniques might be preferred.

In clinical practice, histopathological analysis of biopsied tissue is the main method for bladder cancer diagnosis and prognosis. The diagnosis is performed by a pathologist based on the morphological features in the image of a hematoxylin and eosin (HE) stained tissue sample. This manuscript proposes algorithms to perform morphometric analysis on the HE images, quantify the features in the images, and discriminate bladder cancers with different grades, i.e. high grade and low grade. The nuclei are separated from the background and other types of cells such as red blood cells (RBCs) and immune cells using manual outlining, color deconvolution and image segmentation. A mask of nuclei is generated for each image for quantitative morphometric analysis. The features of the nuclei in the mask image including size, shape, orientation, and their spatial distributions are measured. To quantify local clustering and alignment of nuclei, we propose a 1-nearest-neighbor (1-NN) algorithm which measures nearest neighbor distance and nearest neighbor parallelism. The global distributions of the features are measured using statistics of the proposed parameters. A linear support vector machine (SVM) algorithm is used to classify the high grade and low grade bladder cancers. The results show using a particular group of nuclei such as large ones, and combining multiple parameters can achieve better discrimination. This study shows the proposed approach can potentially help expedite pathological diagnosis by triaging potentially suspicious biopsies.

Optical nerve stimulation (ONS) is being explored as an alternative to electrical nerve stimulation (ENS) for use as an intra-operative diagnostic method for identification and preservation of prostate cavernous nerves (CNs) during radical prostatectomy. Nerve priming and fatigue studies were performed to further characterize CNs and provide insight into the different ONS and ENS mechanisms. ONS studies were conducted using a 1455-nm diode laser, coupled to fiber optic probe, and delivering a collimated, 1-mm-diameter laser spot on CNs. For nerve priming studies, laser power was escalated in 5 mW increments (15 - 60 mW) with each stimulation lasting 15 s, until a strong ICP response was observed, and then power was similarly de-escalated. For ONS fatigue studies, a constant laser power was delivered for a period of 10 min. ENS studies were conducted for comparison, with standard parameters (4 V, 5 ms, 16 Hz) for fatigue studies (10 min. duration), but incrementally increasing/decreasing voltage (0.1 - 4.0 V) for priming studies with 15 s stimulations. ONS threshold was approximately 20% higher during initial escalating laser power steps (6.4 W/cm²) than in subsequently de-escalating laser power steps (5.1 W/cm²), demonstrating a nerve priming effect. Evidence of nerve priming during ENS was not observed. For nerve fatigue studies, ONS of CNs showed a peak ICP response at about 60 s, followed by a gradual decay in ICP, while ENS maintained a strong, but cyclical ICP. Nerve priming may allow repetitive ONS of CNs at lower and hence safer laser power settings. Both nerve priming and fatigue studies revealed different mechanisms for ONS and ENS.

We are exploring infrared (IR) lasers as an alternative energy modality to radiofrequency (RF) and ultrasonic (US) devices intended to provide rapid surgical hemostasis with minimal collateral zones of thermal damage and tissue necrosis. Previously, a 1470-nm IR laser sealed and cut ex vivo porcine renal arteries of 1-8 mm in 2 s, yielding burst pressures < 1200 mmHg (compared to normal systolic blood pressure of 120 mmHg) and thermal coagulation zones < 3 mm (including the seal). This preliminary study describes in vivo testing of a laser probe in a porcine model. A prototype, fiber optic based handheld probe with vessel/tissue clasping mechanism was tested on blood vessels < 6 mm diameter using incident 1470-nm laser power of 35 W for 1-5 s. The probe was evaluated for hemostasis after sealing isolated and bundled vasculature of abdomen and hind leg, as well as liver and lung parenchyma. Sealed vessel samples were collected for histological analysis of lateral thermal damage. Hemostasis was achieved in 57 of 73 seals (78%). The probe consistently sealed vasculature in small bowel mesentery, mesometrium, and gastro splenic and epiploic regions. Seal performance was less consistent on hind leg vasculature including saphenous arteries and bundles and femoral and iliac arteries. Collagen denaturation averaged 1.6 mm in 8 samples excised for histologic examination. A handheld laser probe sealed porcine vessels in vivo. With further improvements in probe design and laser parameter optimization, IR lasers may provide an alternative to RF and US vessel sealing devices.

Our laboratory is studying the experimental Thulium fiber laser (TFL) as an alternative lithotripter to clinical gold standard Holmium:YAG laser. Safety studies characterizing undesirable Holmium laser-induced damage to Nitinol stone baskets have been previously reported. Similarly, this study characterizes TFL induced stone basket damage. A TFL beam with pulse energy of 35 mJ, pulse duration of 500 μs, and pulse rates of 50-500 Hz was delivered through 100-μm-core optical fibers, to a standard 1.9-Fr Nitinol stone basket wire. Stone basket damage was graded as a function of pulse rate, number of pulses, and working distance. Nitinol wire damage decreased with working distance and was non-existent at distances greater than 1.0 mm. In contact mode, 500 pulses delivered at pulse rates ≥ 200 Hz (≤ 2.5 s) were sufficient to cut Nitinol wires. The Thulium fiber laser, operated in low pulse energy and high pulse rate mode, may provide a greater safety margin than standard Holmium laser for lithotripsy, as evidenced by shorter non-contact working distances for stone basket damage than previously reported with Holmium laser.

Laser light has been widely used as a surgical tool to treat benign prostate hyperplasia with high laser power. The purpose of this study was to validate the feasibility of photoactive dye injection to enhance light absorption and eventually to facilitate tissue ablation with low laser power. The experiment was implemented on chicken breast due to minimal optical absorption Amaranth (AR), black dye (BD), hemoglobin powder (HP), and endoscopic marker (EM), were selected and tested in vitro with a customized 532-nm laser system with radiant exposure ranging from 0.9 to 3.9 J/cm². Light absorbance and ablation threshold were measured with UV-VIS spectrometer and Probit analysis, respectively, and compared to feature the function of the injected dyes. Ablation performance with dye-injection was evaluated in light of radiant exposure, dye concentration, and number of injection. Higher light absorption by injected dyes led to lower ablation threshold as well as more efficient tissue removal in the order of AR, BD, HP, and EM. Regardless of the injected dyes, ablation efficiency principally increased with input parameter. Among the dyes, AR created the highest ablation rate of 44.2±0.2 μm/pulse due to higher absorbance and lower ablation threshold. Preliminary tests on canine prostate with a hydraulic injection system demonstrated that 80 W with dye injection yielded comparable ablation efficiency to 120 W with no injection, indicating 33 % reduced laser power with almost equivalent performance. In-depth comprehension on photoactive dye-enhanced tissue ablation can help accomplish efficient and safe laser treatment for BPH with low power application.

Three dimensional (3D) imaging of the tympanic membrane (TM) has been carried out using a traditional otoscope equipped with a high-definition webcam, a portable projector and a telecentric optical system. The device allows us to project fringe patterns on the TM and the magnified image is processed using phase shifting algorithms to arrive at a 3D description of the TM. Obtaining a 3D image of the TM can aid in the diagnosis of ear infections such as otitis media with effusion, which is essentially fluid build-up in the middle ear. The high resolution of this device makes it possible examine a computer generated 3D profile for abnormalities in the shape of the eardrum. This adds an additional dimension to the image that can be obtained from a traditional otoscope by allowing visualization of the TM from different perspectives. In this paper, we present the design and construction of this device and details of the imaging processing for recovering the 3D profile of the subject under test. The design of the otoscope is similar to that of the traditional device making it ergonomically compatible and easy to adopt in clinical practice.

In this study, we measure the in vivo apical-turn vibrations of the guinea pig organ of Corti in both axial and radial directions using phase-sensitive Fourier domain optical coherence tomography. The apical turn in guinea pig cochlea has best frequencies around 100 – 500 Hz which are relevant for human speech. Prior measurements of vibrations in the guinea pig apex involved opening the otic capsule, which has been questioned on the basis of the resulting changes to cochlear hydrodynamics. Here this limitation is overcome by measuring the vibrations through bone without opening the otic capsule. Furthermore, we have significantly reduced the surgery needed to access the guinea pig apex in the axial direction by introducing a miniature mirror inside the bulla. The method and preliminary data are discussed in this article.

Aim: The LLLT was found to recover NIHL and ototoxicity induced hearing loss in rats but the optimal LLLT laser dosage to treat NIHL needs to be determined. The aim of this study was to find the optimal laser dosage to recover a NIHL with transmeatal-LLLT. Methods: Bilateral ears of rats were exposed to noise (narrow band noise, 120 dB, 16 kHz, 6 h). Left ears of the rats were irradiated with transmeatal-LLLT (830 nm) of 50, 100, 150, 200, 250, 300 mW for 60 minutes per day for 12 days, starting 1 day post induction of NIHL. Right ears were not irradiated and used as control ears. The hearing levels were measured at each frequency of 8, 12, and 32 kHz before the noise exposure, 1, 3, 8, and 12 days post noise exposure. The differences of hearing levels between left treated ear and right controlled ear at each frequency of different laser dosages (50 – 300 mW) were compared to see the most effective laser dosages to treat NIHL. Results: Hearing levels were most improved by 150 mW, slightly improved by 200 mW, not improved by 50 and 250 mW, and became worse by 300 mW. Conclusion: The results of this study suggest that most effective therapeutic laser dosage window to treat NIHL with transmeatal-LLLT was 150 mW for 12 days and it was not effective by 50, 250, and 300 mW.

Auditory prostheses may benefit from Infrared Neural Stimulation (INS) because optical stimulation allows for spatially selective activation of neuron populations. Selective activation of neurons in the cochlear spiral ganglion can be determined in the central nucleus of the inferior colliculus (ICC) because the tonotopic organization of frequencies in the cochlea is maintained throughout the auditory pathway. The activation profile of INS is well represented in the ICC by multichannel electrodes (MCEs). To characterize single unit properties in response to INS, however, single tungsten electrodes (STEs) should be used because of its better signal-to-noise ratio. In this study, we compared the temporal properties of ICC single units recorded with MCEs and STEs in order to characterize the response properties of single auditory neurons in response to INS in guinea pigs. The length along the cochlea stimulated with infrared radiation corresponded to a frequency range of about 0.6 octaves, similar to that recorded with STEs. The temporal properties of single units recorded with MCEs showed higher maximum rates, shorter latencies, and higher firing efficiencies compared to those recorded with STEs. When the preset amplitude threshold for triggering MCE recordings was raised to twice over the noise level, the temporal properties of the single units became similar to those obtained with STEs. Undistinguishable neural activities from multiple sources in MCE recordings could be responsible for the response property difference between MCEs and STEs. Thus, caution should be taken in single unit recordings with MCEs.

Fiber-optic pressure sensors have been developed for measurements of intracochlear pressure. The present family of transducers includes an 81 μm diameter sensor employing a SLED light source and single-mode optic fiber, and LED/multi-mode sensors with 126 and 202 μm diameter. The 126 μm diameter pressure sensor also has been constructed with an electrode adhered to its side, for coincident pressure and voltage measurements. These sensors have been used for quantifying cochlear mechanical impedances, informing our understanding of conductive hearing loss and its remediation, and probing the operation of the cochlear amplifier.

The supporting cells and hair cells (HCs) in the organ of Corti (OoC) are highly organized. The precise 3D micro-structure is hypothesized to play a critical role in cochlear function. Recently, we combined two techniques to obtain the organ of Corti cytoarchitecture. Two-photon imaging allowed us to perform in situ imaging without subjecting the tissue to other potential distortions, while genetically engineered mTmG mice have a fluorophore embedded in the cell membranes. In this contribution we discuss the parameterization step necessary to compare structures obtained with this technique at different locations and in different specimens.

First, the z-axis is chosen perpendicular to the basilar membrane. Subsequently, base and apex of cells are indicated by landmarks. As such, the cells are approximated as a stick representation. This representation is used to calculate the 3D lengths and angles of all imaged cells. Since the OoC is not straight but spiral-shaped, the radial (y) and longitudinal (x) directions differ at each location. Therefore, circular arcs are fitted through the 3 rows of outer HCs to define the local radial (y) and longitudinal (x) direction. Novel in this approach is the 3D data of the cell position in the organ of Corti. Cell diameters and tissue areas cannot be quantified with this stick representation and need to be measured separately.

Sinus blockages are a common reason for physician visits, affecting 1 out of 7 in the United States. Over 20 million cases of acute bacterial sinusitis become chronic and require medical treatment. Diagnosis in the primary care setting is challenging because symptom criteria (via detailed clinical history) plus objective imaging (CT or endoscopy) is recommended. Unfortunately, neither option is routinely available in primary care. Our previous work demonstrated that low-cost near infrared (NIR) transillumination instruments produced signals that correlated with the bulk findings of sinus opacity measured by CT. We have upgraded the technology, but questions remain such as finding the optimal arrangement of light sources, measuring the influence of specific anatomical structures, and determining detection limits. In order to begin addressing these questions, we have modeled NIR light propagation inside the adult human head using a mesh-based Monte Carlo algorithm (MMCLab) applied to a detailed anatomical head model constructed from CT images. In this application the sinus itself, which under healthy conditions is a void region (e.g., non-scattering), is the region of interest instead of an obstacle to other contrast mechanisms. We report preliminary simulations that characterize the changes in detected intensity due to clear (i.e., healthy) versus blocked sinuses. We also ran simulations for two of our clinical cases and compared results with the measurements. The simulations presented herein serve as a proof of concept that this approach could be used to understand contrast mechanisms and limitations of NIR imaging of the sinus cavities.

Focus is upon our clinical experience in a prospective cohort study on cure of dystonia where the mode of treatment was fexofenadine tablets and local budesonide inhaler in the larynx, and in a randomized controlled trial of lifestyle change related to acid provocation of food and habits in laryngopharyngeal reflux (LPR). The advanced high-speed films is one new tool, another being optical coherence tomography (OCT), which should be used in the future in randomized controlled trials.

We are focusing on OCT of the swallowing process in the oesophagaus and larynx as well as the vocal fold function. It can be shown on OCT how the layer of the vocal folds develop, possibly corresponding to hormonal and paediatric development. The arytenoid area in the larynx should also be focused upon with OCT in pathology. The thyroid function is related to voice and the swallowing function, both hormonally and pathoanatomically. We know too little about voice and thyroid hormones in an updated way as well as the outer anatomic supporting muscular structure of the larynx, related to thyroid immune degeneration and cysts. Also, here OCT analyses might be of value.

Full-Field OCT (FFOCT) produces optical slices of tissue using white light interferometry providing in-depth 2D images, with an isotropic resolution around 1 micrometer. These optical biopsy images are similar to those obtained with established histological procedures, but without tissue preparation and within few minutes. This technology could be useful when diagnosing a lesion or at the time of its surgical management.

Here we evaluate the clinical value of FFOCT imaging in the management of patients with Head and Neck cancers by assessing the accuracy of the diagnosis done on FFOCT images from resected specimen.

FFOCT images from Head and Neck samples were first compared to the gold standard (HES-conventional histology). An image atlas dedicated to the training of pathologists was built and diagnosis criteria were identified.

Then, we performed a morphological correlative study: both healthy and cancerous samples from patients who undergo Head and Neck surgery of oral cavity, pharynx, and larynx were imaged. Images were interpreted in a random way by two pathologists and the FFOCT based diagnostics were compared with HES (gold standard) of the same samples.

Here we present preliminary results showing that FFOCT provides a quick assessment of tissue architecture at microscopic level that could guide surgeons for tumor margin delineation during intraoperative procedure.

Macrophages loaded with gold nanoshells (AuNS), that convert near infrared light to heat, can be used as transport vectors for photothermal hyperthermia of tumors. The purpose of this study was to investigate the effects of combined macrophage mediated photothermal therapy (PTT) and PDT on head and neck squamous cell carcinoma (HNSCC). The results provide proof of concept for the use of macrophages as a delivery vector of AuNS for photothermal enhancement of the effects of PDT on squamous cell carcinoma. A significant synergy was demonstrated with combined PDT and PTT compared to each modality applied separately.

Low-level laser therapy (LLLT) is a non-thermal phototherapy used in several medical applications, including wound healing, reduction of pain and amelioration of oral mucositis. Nevertheless, the effects of LLLT upon cancer or dysplastic cells have been so far poorly studied. Here we report that the effects of laser irradiation on anaplastic thyroid cancer cells leads to hyperplasia. 650nm of laser diode was performed with a different time interval (0, 15, 30, 60J/cm² , 25mW) on anaplastic thyroid cancer cell line FRO in vivo. FRO was orthotopically injected into the thyroid gland of nude mice and the irradiation was performed with the same method described previously. After irradiation, the xenograft evaluation was followed for one month. The thyroid tissues from sacrificed mice were undergone to H&E staining and immunohistochemical staining with HIF-1α, Akt, TGF-β1. We found the aggressive proliferation of FRO on thyroid gland with dose dependent. In case of 60 J/ cm² of energy density, the necrotic bodies were found in a center of the thyroid. The phosphorylation of HIF-1α and Akt was detected in the thyroid gland, which explained the survival signaling of anaplastic cancer cell was turned on the thyroid gland. Furthermore, TGF-β1 expression was decreased after irradiation. In this study, we demonstrated that insufficient energy density irradiation occurred the decreasing of TGF-β1 which corresponding to the phosphorylation of Akt/ HIF-1α. This aggressive proliferation resulted to the hypoxic condition of tissue for angiogenesis. We suggest that LLLT may influence to cancer aggressiveness associated with a decrease in TGF-β1 and increase in Akt/HIF-1α.

Globally, fifteen million babies are born preterm each year, affecting 1 in 8 pregnancies in the US alone. Cervical remodeling includes a biochemical cascade of changes that ultimately result in the thinning and dilation of the cervix for passage of a fetus. This process is poorly understood and is the focus of this study. Our group is utilizing Raman spectroscopy to evaluate biochemical changes occurring in the human cervix throughout pregnancy. This technique has high molecular specificity and can be performed in vivo, with the potential to unveil new molecular dynamics essential for cervical remodeling.

Vulvovaginal candidiasis is a common cause of vaginal infections. This study investigates the efficiency of antimicrobial photodynamic therapy (aPDT) against yeast cells in mice. Methylene blue (MB), malachite green (MG), and a special designed protoporphirin (PpNetNI) were used as photosensitizers. Female BALB-c mice were infected with Candida albicans ATCC 90028. PDT was applied with two different light sources, intravaginal and transabdominal. Vaginal washes were performed and cultivated for microbial quantification. Antimicrobial PDT was able to decrease microbial content with MB and PpNetNI (p<0.05), it was not effective, however, with MG photosensitizer. The results of this study demonstrate that aPDT may be a viable alternative treatment for vaginal candidiasis.

Diffuse optical spectroscopy (DOS) may be advantageous for monitoring tumor response during chemotherapy treatment, particularly in the early treatment stages. In this paper we perform a second analysis on the data of a clinical trial with 25 breast cancer patients that received neoadjuvant chemotherapy. Patients were monitored using delayed contrast enhanced MRI and additionally with diffuse optical spectroscopy at baseline, after 1 cycle of chemotherapy, halfway therapy and before surgery.

In this analysis hemoglobin content between tumor tissue and healthy tissue of the same breast is compared on all four monitoring time points. Furthermore, the predictive power of the tumor-healthy tissue difference of HbO₂ for non-responder prediction is assessed.

The difference in HbO₂ content between tumor and healthy tissue was statistically significantly higher in responding tumors than in non-responding tumors at baseline (10.88 vs -0.57 μM, P=0.014) and after one cycle of chemotherapy (6.45 vs -1.31 μM, P=0.048). Before surgery this difference had diminished. In the data of this study, classification on the HbO₂ difference between tumor and healthy tissue was able to predict tumor (non-)response at baseline and after 1 cycle with an area-under-curve of 0.95 and 0.88, respectively.

While this result suggests that tumor response can be predicted before chemotherapy onset, one should be very careful with interpreting these results. A larger patient population is needed to confirm this finding.

Photoacoustic spectroscopy, a hybrid of optics and acoustics has been gaining popularity in the biomedical field very fast. The main aim in the present study was to apply this technique to detect and distinguish breast tumor tissues from normal and hence develop a tool for clinical applications. There were 224 photoacoustic spectra recorded from 28 normal and 28 breast tumor tissues using PZT detector at 281nm pulsed laser excitations from Nd-YAG laser pumped frequency doubled dye laser system. The recorded time domain photoacoustic spectra were fast Fourier transformed into frequency domain patterns in the frequency region 0-1250kHz and from each pattern, 7 features (mean, median, mode, variance, standard deviation, area under the curve & spectral residual after fitting with 10^th degree polynomial) were extracted using MATLAB algorithms. These features were then tested for their significance between normal and malignant conditions using Student T-test and two of them (variance, std. deviation) showing significant variation were selected for further discrimination analysis using supervised quadratic discriminate analysis (QDA). In QDA, 60 spectra from each of the normal and malignant were used for making the respective calibration sets and the remaining 52 spectra from each were used for the validation. The performance of the analysis tested for the frequency region 406.25 - 625.31 kHz, showed specificity and sensitivity values of 100% and 88.46% respectively suggesting possible application of the technique in breast tumor detection.

Clinical imaging techniques for diagnosing breast cancer mainly include X-ray mammography, ultrasound, and magnetic resonance imaging (MRI), which have respective drawbacks. Multiphoton microscopy (MPM) has become a potentially attractive optical technique to bridge the current gap in clinical utility. In this paper, MPM was used to image normal and ductal cancerous breast tissues, based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our results showed that MPM has the ability to exhibit the microstructure of normal breast tissue, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) lesions at the molecular level comparable to histopathology. These findings indicate that, with integration of MPM into currently accepted clinical imaging system, it has the potential to make a real-time histological diagnosis of mammary ductal carcinoma in vivo.

Abnormal metabolism can be a hallmark of cancer occurring early before detectable histological changes and may serve as an early detection biomarker. The current gold standard to establish breast cancer (BC) diagnosis is histological examination of biopsy. Previously we have found that pre-cancer and cancer tissues in animal models displayed abnormal mitochondrial redox state. Our technique of quantitatively measuring the mitochondrial redox state has the potential to be implemented as an early detection tool for cancer and may provide prognostic value. We therefore in this present study, investigated the feasibility of quantifying the redox state of tumor samples from 16 BC patients. Tumor tissue aliquots were collected from both normal and cancerous tissue from the affected cancer-bearing breasts of 16 female patients (5 TNBC, 9 ER⁺, 2 ER⁺/Her2⁺) shortly after surgical resection. All specimens were snap-frozen with liquid nitrogen on site and scanned later with the Chance redox scanner, i.e., the 3D cryogenic NADH/oxidized flavoprotein (Fp) fluorescence imager. Our preliminary results showed that both NADH and Fp (including FAD, i.e., flavin adenine dinucleotide) signals in the cancerous tissues roughly tripled to quadrupled those in the normal tissues (p<0.05); and the redox ratio Fp/(NADH+Fp) was about 27% higher in the cancerous tissues than in the normal ones (p<0.05). Our findings suggest that the redox state could differentiate between cancer and non-cancer breast tissues in human patients and this novel redox scanning procedure may assist in tissue diagnosis in freshly procured biopsy samples prior to tissue fixation. We are in the process of evaluating the prognostic value of the redox imaging indices for BC.

There is a need for accurate, high-throughput measures to gauge the efficacy of potential drugs in living cells. Metabolism is an early marker of drug response in cells, and NADH and FAD are autofluorescent cellular metabolic coenzymes that can be non-invasively monitored using optical techniques. Relative rates of glycolysis and oxidative phosphorylation in a cell are quantified by the redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD. Microplate readers are high-throughput instruments that can rapidly measure NADH and FAD autofluorescence intensities for hundreds of wells, and are capable of identifying receptor status and resolving drug response in breast cancer cell lines. This study tests the accuracy and repeatability of plate reader experiments measuring the redox ratio in breast cancer cell lines. NADH and FAD fluorescence levels remained constant over the course of multiple measurements (p<0.1), ruling out the incidence of photobleaching. The contribution of media to background fluorescence signal was also investigated. Media fluorescence levels for both coenzymes were significantly lower (p<0.0001) than those from wells containing cells, and replacing the media with saline resulted in the same redox ratio trends among cell lines as initial measurements with media. Following treatment with carbonyl cyanide p-fluorodeoxyphenylhydrazone (FCCP), an oxidative phosphorylation inhibitor, the redox ratio decreased (p<0.05), validating NADH and FAD as the primary fluorescence sources. These findings verify that autofluorescence measurements taken by microplate readers accurately and reliably characterize NADH and FAD fluorescence, validating their promise in the areas of metabolic monitoring and drug development.

Because mammography, the gold standard of breast cancer screening and monitoring treatment efficacy, has limitations, there is a necessity to have a new method for breast cancer patients. Raman spectroscopy is considered as one of the best alternative approaches due to its ability of visualizing (bio)chemical information of a matter. In this study, we hypothesized that the change of biochemical composition occurs earlier than morphological change in breast cancer during chemotherapy, and attempted to prove it by employing fiber-optic Raman spectroscopy for longitudinal Raman measurement in small animal breast cancer model. To confirm the hypothesis, we measured Raman spectra of a tumor breast and the contralateral breast during chemotherapy for 4 fisher 344 female rats longitudinally. Principal component analysis and Raman spectral differences between breast tumor and contralateral normal breast did not show a clear difference between them which may have been caused by interference from skin. Thus, spatially-offset Raman spectroscopy will be employed in order to acquire the Raman signal directly from tumor while suppressing Raman signal from skin for the future study.

Breast cancer is the second most common cancer all over the world. Heterogeneity in breast cancer makes it a difficult task to detect with the existing serum markers at an early stage. With an aim to detect the disease early at the pre-malignant level, MCF-7 cells xenografts were developed using female nude mice and blood serum were extracted on days 0^th, 10^th, 15^th & 20^th post tumor cells injection (N=12 for each time point). Photoacoustic spectra were recorded on the serum samples at 281nm pulsed laser excitations. A total of 144 time domain spectra were recorded from 48 serum samples belonging to 4 different time points. These spectra were then converted into frequency domain (0–1250kHz) using MATLAB algorithms. Subsequently, seven features (mean, median, mode, variance, standard deviation, area under the curve & spectral residuals after 10^th degree polynomial fit) were extracted from them and used for PCA. Further, using the first three Principal components (PCs) of the data, Linear Discriminate Analysis has been carried out. The performance of the analysis showed 82.64% accuracy in predicting various time points under study. Further, frequency-region wise analysis was also performed on the data and found 95 - 203.13 kHz region most suitable for the discrimination among the 4 time points. The analysis provided a clear discrimination in most of the spectral features under study suggesting that the photoacoustic technique has the potential to be a diagnostic tool for early detection of breast tumor development

The cervical cancer screening at a pre-cancer stage is beneficial to reduce the mortality of women. An opto-electronic joint detection system based on DSP aiming at early cervical cancer screening is introduced in this paper. In this system, three electrodes alternately discharge to the cervical tissue and three light emitting diodes in different wavelengths alternately irradiate the cervical tissue. Then the relative optical reflectance and electrical voltage attenuation curve are obtained by optical and electrical detection, respectively. The system is based on DSP to attain the portable and cheap instrument. By adopting the relative reflectance and the voltage attenuation constant, the classification algorithm based on Support Vector Machine (SVM) discriminates abnormal cervical tissue from normal. We use particle swarm optimization to optimize the two key parameters of SVM, i.e. nuclear factor and cost factor. The clinical data were collected on 313 patients to build a clinical database of tissue responses under optical and electrical stimulations with the histopathologic examination as the gold standard. The classification result shows that the opto-electronic joint detection has higher total coincidence rate than separate optical detection or separate electrical detection. The sensitivity, specificity, and total coincidence rate increase with the increasing of sample numbers in the training set. The average total coincidence rate of the system can reach 85.1% compared with the histopathologic examination.

Full-field optical coherence tomography (FFOCT) quickly produces images that resemble conventional pathology images. We examined endometrium in an intra-operative like fashion (more than forty samples). FFOCT-imaged endometrium was recognizable to pathologists and compared favorably with microscopy of the same samples. Additional image enhancements and acquisition techniques were explored and may improve interpretation accuracy. Wider evaluation of images is ongoing, using more pathologist subjects. FFOCT may revolutionize pathology practice in the future by permitting rapid diagnosis and in vivo diagnosis; this is potentially a disruptive new diagnostic technique in pathology.

An increased number of integrated optical acoustic intravascular imaging systems have been researched and hold great hope for accurate diagnosing of vulnerable plaques and for guiding atherosclerosis treatment. However, in any intravascular environment, vascular lumen is filled with blood, which is a high-scattering source for optical and high frequency ultrasound signals. Blood must be flushed away to make images clear. To our knowledge, no research has been performed to find the ideal flushing agent that works for both optical and acoustic imaging techniques. We selected three solutions, mannitol, dextran and iohexol, as flushing agents because of their image-enhancing effects and low toxicities. Quantitative testing of these flushing agents was performed in a closed loop circulation model and in vivo on rabbits.

The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo. Traditional methods are destructive, time- and cost- inefficient, and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and neo-intimal and neo-medial thickening. These results encourage the future application of a catheter-based technique that combines these imaging modalities for nondestructive characterization of vascular grafts in vivo.

We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20 kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment) enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime information. Intensity en face images over the four bands of the instrument were available within seconds whereas lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.

Atherosclerosis, the development of intraluminal plaque, is a fundamental pathology of cardiovascular system and remains the leading cause of morbidity and mortality worldwide. Biomechanical in nature, plaque rupture occurs when the mechanical properties of the plaque, related to the morphology and viscoelastic properties, are compromised, resulting in intraluminal thrombosis and reduction of coronary blood flow. In this report, we describe the first simultaneous application of confocal Brillouin and Raman microscopies to ex-vivo aortic wall samples. Such a non-invasive, high specific approach allows revealing a direct relationship between the biochemical and mechanical properties of atherosclerotic tissue.

Spectroscopic techniques have been researched for intravascular diagnostic imaging of atherosclerotic plaque. Nearinfrared (NIR) light efficiently penetrates of biological tissues, and the NIR region contains the characteristic absorption range of lipid-rich plaques. The objective of this study is to observe atherosclerotic plaque using a NIR multispectral angioscopic imaging. Atherosclerotic plaque phantoms were prepared using a biological tissue model and bovine fat. For the study, we developed an NIR multispectral angioscopic imaging system with a halogen light, mercury-cadmiumtelluride camera, band-pass filters and an image fiber. Apparent spectral absorbance was obtained at three wavelengths, 1150, 1200 and 1300 nm. Multispectral images of the phantom were constructed using the spectral angle mapper algorithm. As a result, the lipid area, which was difficult to observe in a visible image, could be clearly observed in a multispectral image. Our results show that image-enhanced observation and quantification of atherosclerotic plaque by NIR multispectral imaging at wavelengths around 1200 nm is a promising angioscopic technique with the potential to identify lipid-rich plaques.

The ever-evolving medical field continues to trend toward less invasive approaches to the diagnosis and treatment of pathological conditions. Basic sciences research has allowed for improved technologies that are translated to the clinical sciences. Similarly, advancements in imaging modalities continue to improve and their applications become more varied. As such, surgeons and pathologists are able to depend on smaller samples for tissue diagnosis of pathological disease, where once large sections of tissue were needed. Optical coherence tomography (OCT), a high-resolution imaging technique, has been used extensively in different medical fields to improve diagnostic yield. Its use in dental fields, particularly in oral and maxillofacial surgery, remains limited. Our goal is to assess the use of OCT for improving soft tissue analysis and diagnosis, particularly for its applications in the field of oral and maxillofacial surgery. Optical coherence tomography is a modality that uses an optical signal using safe near-infrared light which is reflected off the sub-surface structures. This allows for high-resolution cross-sectional images of the tissue morphology to be obtained. Ophthalmologists have been using OCT to obtain images of the retina to assess for age-related macular degeneration. More recently, OCT has been used by Interventional Cardiology to image coronary arteries, and assess plaque thickness and morphology. This technology is now being investigated in several medical fields as a form of optical biopsy, providing in situ images with high-resolution morphology of tissues. We are particularly interested in its use on epithelial tissues, and therefore performed a literature review on the use of OCT for assessing epithelium. Evaluation of histologically-diagnosed actinic keratosis, for example, was found to correlate well with the imaging discrepancies found on OCT; and the in vivo assessment of atypical keratinocytes was firmly established. Additionally, studies have shown a potential application in that OCT may provide a method for studying the evolution of epithelial lesions OCT’s potential in producing high-resolution images of tissue morphology can prove to be a valuable tool for characterizing different soft tissue pathological disorders. Furthermore, it has been shown to measure changes in light intensity at tissue-fluid interfaces, which can provide surgeons the ability to characterize oral mucosal surfaces noninvasively. OCT can also prove to be valuable in detecting oral cancerous and pre-cancerous lesions, as altered epithelium containing increased dysplasia shows differences in light scattering than normal epithelium. Additionally, OCT has been shown to analyze deeper collagen tissues of the oral mucosa and is not limited to the surface epithelium. This can aid in characterizing such inflammatory conditions that alter these tissues. Several tissue samples from the maxillofacial region were obtained and assessed using an OCT device at our institution. The analysis has shown high-resolution images of soft tissue-bone interface, titanium implant-bone interface, and other anatomical sites within the oral cavity. OCT has been shown to be a valuable modality in different medical fields. Its use in oral and maxillofacial surgery can potentially aid in diagnostic techniques. Alongside traditional histological technique, it can be help characterize tissues at the cellular level, which would improve costs, time, and most importantly, patient care. We aim to introduce OCT and its diagnostic abilities to the field of oral and maxillofacial surgery to help aid clinicians and provide improved care for patients.

Osteoporosis is a major bone disease that connotes the risk of fragility fractures resulting from alterations to bone quantity and/or quality to mechanical competence. Bone strength arises from both bone quantity and quality. Assessment of bone quality and bone quantity is important for prediction of fracture risk. In spite of the two factors contribute to maintain the bone strength, only one factor, bone mineral density is used to determine the bone strength in the current diagnosis of osteoporosis. On the other hand, there is no practical method to measure chemical composition of bone tissue including hydroxyapatite and collagen non-invasively. Raman spectroscopy is a powerful technique to analyze chemical composition and material properties of bone matrix non-invasively. Here we demonstrated Raman spectroscopic analysis of the bone matrix in osteoporosis model rat. Ovariectomized (OVX) rat was made and the decalcified sections of tibias were analyzed by a Raman microscope. In the results, Raman bands of typical collagen appeared in the obtained spectra. Although the typical mineral bands at 960 cm^-1 (Phosphate) was absent due to decalcified processing, we found that Raman peak intensities of amide I and C-C stretching bands were significantly different between OVX and sham-operated specimens. These differences on the Raman spectra were statistically compared by multivariate analyses, principal component analysis (PCA) and liner discrimination analysis (LDA). Our analyses suggest that amide I and C-C stretching bands can be related to stability of bone matrix which reflects bone quality.

Osteogenesis imperfecta (OI) is a genetic disorder resulting in defective collagen or collagen-associated proteins and fragile, brittle bones. To date, therapies to improve OI bone mass, such as bisphosphonates, have increased bone mass in the axial skeleton of OI patients, but have shown limited effects at reducing long bone fragility. Sclerostin antibody (Scl- Ab), currently in clinical trials for osteoporosis, stimulates bone formation and may have the potential to reduce long bone fracture rates in OI patients. Scl-Ab has been investigated as an anabolic therapy for OI in the Brtl/+ mouse model of moderately severe Type IV OI. While Scl-Ab increases long bone mass in the Brtl/+ mouse, it is not known whether material properties and composition changes also occur. Here, we report on the effects of Scl-Ab on wild type and Brtl/+ young (3 week) and adult (6 month) male mice. Scl-Ab was administered over 5 weeks (25mg/kg, 2x/week). Raman microspectroscopy and nanoindentation are used for bone composition and biomechanical bone property measurements in excised bone. Fluorescent labels (calcein and alizarin) at 4 time points over the entire treatment period are used to enable measurements at specific tissue age. Differences between wild type and Brtl/+ groups included variations in the mineral and matrix lattices, particularly the phosphate v₁, carbonate v₁, and the v(CC) proline and hydroxyproline stretch vibrations. Results of Raman spectroscopy corresponded to nanoindentation findings which indicated that old bone (near midcortex) is stiffer (higher elastic modulus) than new bone. We compare and contrast mineral to matrix and carbonate to phosphate ratios in young and adult mice with and without treatment.

By using a novel microfluidic set-up for drug screening applications, this study examines delivery of a novel risedronate based drug formulation for treatment of osteoporosis that was developed to overcome the usual shortcomings of risedronate, such as its low bioavailability and adverse gastric effects. Risedronate nanoparticles were prepared using muco-adhesive polymers such as chitosan as matrix for improving the intestinal cellular absorption of risedronate and also using a gastric-resistant polymer such as sodium alginate for reducing the gastric inflammation of risedronate. The in-vitro characteristics of the alginate encapsulated chitosan nanoparticles are investigated, including their stability, muco-adhesiveness, and Caco-2 cell permeability. Fluorescent markers are tagged with the polymers and their morphology within the microcapsules is imaged at various stages of drug release.

The ultimate goal of this work is to develop a novel photoacoustic (QPA) platform for highly-sensitive and quantitative assessment of bone health. First, the feasibility to perform 3D photoacoustic imaging (PAI) of bone was investigated. Then another two techniques, including thermal photoacoustic measurement (TPAM) and photoacoustic spectral analysis (PASA), both being able to achieve quantitative results were investigated for bone characterization. TPAM, by evaluating the dependence of photoacoustic signal amplitude on the sample temperature, is sensitive to the chemical constituents in tissue and holds promise for assessment of bone mineral density (BMD). PASA characterizes micron size physical features in tissue, and has shown feasibility for objective assessment of bone microarchitecture (BMA). This integrated QPA platform can assess both bone mass and microstructure simultaneously without involving invasive biopsy or ionizing radiation. Since QPA is non-ionizing, non-invasive, and has sufficient penetration in both soft tissue and bone, it has unique advantages for clinical translation.

This study examines the sensitivity and specificity of backscattered ultrasound (US) and backscattering photoacoustic (PA) signals for bone composition variation assessment. The conventional approach in the evaluation of bone health relies on measurement of bone mineral density (BMD). Although, a crucial and probably the most important parameter, BMD is not the only factor defining the bone health. New trends in osteoporosis research, also pursue the changes in collagen content and cross-links with bone diseases and aging. Therefore, any non-invasive method that can assess any of these parameters can improve the diagnostic tools and also can help with the biomedical studies on the diseases themselves. Our previous studies show that both US and PA are responsive to changes in the BMD, PA is, in addition, sensitive to changes in the collagen content of the bone. Measurements were performed on bone samples before and after mild demineralization and decollagenization at the exact same points. Results show that combining both modalities can enhance the sensitivity and specificity of diagnostic tool.

Many areas of the body such as the tibia have minimal tissue thickness overlying bone. Near-infrared (NIR) optical windows may be used to image more deeply to reveal abnormalities hidden beneath tissue. We report on the potential application of a compact Leukos supercontinuum laser source (model STM-2000-IR) with wavelengths in the four NIR optical windows (from 650 nm to 950 nm, 1,100 nm to 1,350 nm, 1,600 to 1,870, and 2,100 nm to 2,300 nm, respectively) and between 200 - 500 microwatt/nm power, with InGaAs (Goodrich Sensors Inc. SU320- 1.7RT) and InSb detectors (Teledyne Technologies) to image microfractures and abnormalities of bone hidden beneath tissue.

Near- infrared (NIR) light with wavelengths from 650 nm to 950 nm (known as the first NIR window) has conventionally been used as a non-invasive technique that can reach deeper penetration depths through media than light at shorter wavelengths. Recently, several novel, NIR, label-free, techniques have been developed to assess Paget’s disease of bone, osteoporosis and bone microfractures. We designed a Bone Optical Analyzer (BOA) which utilizes the first window to measure changes of Hb and HbO₂. Paget’s disease is marked by an increase in vascularization in bones, and this device can enable easy diagnosis and more frequent monitoring of the patient’s condition, without exposing him to a high cumulative dose of radiation. We have also used inverse imaging algorithms to reconstruct 2D and 3D maps of the bone’s structure. This device could be used to assess diseases such as osteoporosis. Using 800 nm femtosecond excitation with two-photon (2P) microscopy, we acquired 2PM images of the periosteum and spatial frequency spectra (based on emission of collagen) from the periosteal regions. This technique can provide information on the structure of the periosteum and can detect abnormalities which may be an indication of disease. Most recently, we showed that longer NIR wavelengths in the second and third NIR windows (1100 nm-1350 nm, 1600 nm-1870 nm), could be used to image bone microfractures. Use of NIR light could allow for repeated studies in patients with diseases such as Paget’s and osteoporosis quickly and non-invasively, and could impact the current management for these diseases.

Every year in the United States approximately 1.5 million people are suffering from bone fractures. Current treatment solutions include surgeries and grafting process; however, it does not show any osteoconductive action, which is an essential character for biomaterials. The main objective of the reported studies is to develop a novel nanocomposite comprising of hydroxyapatite nanospheres (~40nm) and gelatin methacrylate to promote bone regeneration without any osteoinductive factors. To validate our hypothesis that chemical and mechanical properties of nanocomposite get enhanced, we employed various characterization techniques including Brillouin and Raman spectroscopies. The results imply that hydroxyaoatite nanoparticles are capable of enhancing the macroscopic stiffness at the structural level. To the contrary, Brillouin spectroscopy suggests that the microscopic elasticity of the nanocomposite was weakened.

Polarization Raman Spectroscopy has been used to demonstrate microstructural features and collagen fiber orientation in human and mouse bone, concurrently measuring both organization and composition; however, it is unclear as to what extent these measurements explain the mechanical quality of bone. In a cohort of age and gender matched cadaveric cortical bone samples (23-101 yr.), we show homogeneity of both composition and structure are associated with the age related decrease in fracture toughness. 64 samples were machined into uniform specimens and notched for mechanical fracture toughness testing and polished for Raman Spectroscopy. Fingerprint region spectra were acquired on wet bone prior to mechanical testing by sampling nine different microstructural features spaced in a 750x750 μm grid in the region of intended crack propagation. After ASTM E1820 single edge notched beam fracture toughness tests, the sample was dried in ethanol and the osteonal-interstitial border of one osteon was samples in a 32x32 grid of 2μm² pixels for two orthogonal orientations relative to the long bone axis. Standard peak ratios from the 9 separate microstructures show heterogeneity between structures but do not sufficiently explain fracture toughness; however, peak ratios from mapping highlight both lamellar contrast (ν1Phos/Amide I) and osteon-interstitial contrast (ν1Phos/Proline). Combining registered orthogonal maps allowed for multivariate analysis of underlying biochemical signatures. Image entropy and homogeneity metrics of single principal components significantly explain resistance to crack initiation and propagation. Ultimately, a combination of polarization content and multivariate Raman signatures allowed for the association of microstructural tissue heterogeneity with fracture resistance.

Mechanical properties of hard tissues play an important role in understanding underlying biological structures, as well as assessing the quality of artificial bone replacement materials. In this study, we employed Brillouin spectroscopy as a non-invasive approach to probe the microscopic elasticity of hard tissues, such as bones. Brillouin spectra were collected using a background free virtually imaged phased array spectrometer. As a reference, Raman spectra were also acquired for each imaging point. Experimental results reveal a positive correlation between the local concentration of the mineral content and the corresponding tissue stiffness, assessed through a Brillouin shift.