One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction.

If no fresh skin samples can be obtained or used, it is important for research and industries to have models and stored tissue samples as close to the native state as possible at disposal. One way to preserve tissues for a longer timeframe is to use deep freezing cryo-techniques. Unfortunately much damage can be induced during the cooling and the thawing processes like disruption of cells and extra-cellular matrices due to the formation of ice crystals. This could lead to a disturbance of the united cell structure up to the point of a loss of cell viability. Two-photon microscopy is able to gather information about cells and tissue components via excitation of the autofluorescence deep inside the sample with a high resolution in both, frozen and thawed states. It is possible to monitor the samples before and after and, important, observe events during the freezing process like the formation of ice crystals. To determine the state of skin tissues after slow rate freezing and the quick process of vitrification, the samples were examined with two-photon microscopy. To establish an optimized freezing-protocol for skin tissues, morphological changes, changes in autofluorescence of endogenous fluorophores (NADH, keratin, flavins, elastin) or changes in second harmonic generation of collagen fibres could provide information about the quality of the used freezing parameters and protective additives and lead to an optimized freezing-protocol with a new set of parameters to obtain mostly intact tissue samples. Multiphoton microscopy has been established as a useful tool for optical in situ quality control of frozen tissues.

The eradication of Trichophyton rubrum has been attempted via laser irradiation because it could result advantageous relative to current clinical therapies. Anticipating that the necessary thermal effects could unintentionally damage the underlying toe dermal layer, we have explored two auxiliary approaches: (a) laser irradiation under vacuum pressure, with and without water dousing and, (b) cooling followed by laser heating (thermal shock). The rationale is that at low pressures, the temperature necessary to achieve water evaporation/boiling is significantly reduced, thus requiring lower fluences. Similarly, a thermal shock induced by cooling followed by laser irradiation may require lower fluences to achieve fungus necrosis. For all experiments presented we use a Cooltouch, model CT3 plus, 1320 nm laser to irradiate fungi colonies. The vacuum pressure experiments exposed fungi colonies to a subatmospheric pressure of 84.7 kPa (25 inHg) with and without water dousing for 5 min, followed by irradiation with 4.0 J/cm² fluence and 40-90 J total energies. The thermal shock experiments consisted of three sections at 4.8 J/cm²: cooling the fungus to 0 °C at 0.39 °C/min and then irradiating to 45-60 °C; cooling to -20 °C at 1.075 °C/min and irradiating to 45 °C; and cooling to -20 °C at 21.5 °C/min and irradiating to 45 °C. Fungus growth rate over a 1-week period assessed the feasibility of these procedures. Results indicated both approaches hamper the growth rate of fungi colonies relative to untreated control samples, especially water dousing under vacuum conditions and slow cooling rate preceding irradiation for thermal shock effect.

We present in vivo measurements of the excitation-wavelength dependence of the autofluorescence of major endogenous fluorophores of human skin with a multiphoton tomograph. For the investigation high-resolution multiphoton images at different depths inside the skin were recorded and the main fluorophores identified. In particular, for the autofluorescence of the fluorophores keratin, NAD(P)H, elastin and for the second-harmonic-generation light induced by collagen fibers clear trends are shown.

The blue light (~400 nm) emitted by high power Light Emitting Diodes (LED) is selectively absorbed by the haemoglobin content of blood and then converted into heat. This is the basic concept in setting up a compact, low-cost, and easy-to-handle photohaemostasis device for the treatment of superficial skin abrasions. Its main application is in reducing bleeding from superficial capillary vessels during laser induced aesthetic treatments, such as skin resurfacing, thus reducing the treatment time and improving aesthetic results (reduction of scar formation). In this work we firstly present the preliminary modeling study: a Finite Element Model (FEM) of the LED induced photothermal process was set up, in order to estimate the optimal wavelength and treatment time, by studying the temperature dynamics in the tissue. Then, a compact, handheld illumination device has been designed: commercially available high power LEDs emitting in the blue region were mounted in a suitable and ergonomic case. The prototype was tested in the treatment of dorsal excoriations in rats. Thermal effects were monitored by an infrared thermocamera, experimentally evidencing the modest and confined heating effects and confirming the modeling predictions. Objective observations and histopathological analysis performed in a follow-up study showed no adverse reactions and no thermal damage in the treated areas and surrounding tissues. The device was then used in human patients, in order to stop bleeding during Erbium laser skin resurfacing procedure. By inducing LED-based photocoagulation, the overall treatment time was shortened and scar formation was reduced, thus enhancing esthetic effect of the laser procedure.

The Dermoscope is a widespread and essential tool for dermatology. It reveals morphologic characteristics and aid the identification and diagnosis of a skin lesion. The diagnosis though is not always unambiguous and still the use of Dermoscopy doesn't obviate the need for histopathological verification. We report on the development of the Multispectral Dermoscope which employs high luminance LEDs with emission at three distinct spectral regions (470 nm, 530 nm, 625 nm). The illumination is polarized and an analyzer is used for the detection. Subsequent image analysis for enhancing the contrast for single scattered photons, hemoglobin absorption and melanin absorption is performed. Features like the surface texture of the skin, scattering structures in the epidermis, blood vessel morphology even in pigmented lesions and melanin localization have been observed in various skin pathologies. The device has been tested on volunteers and the features revealed have proven to be helpful in the diagnosis of skin lesions. Use of the Multispectral Dermoscope could improve the sensitivity and specificity of Dermoscopy.

Photodynamic Therapy is a recent treatment modality that allows malignant tissue destruction. The technique provides a localized effect and good cosmetic results. The application of Photodynamic Therapy is based on the inoculation of a photosensitizer and the posterior irradiation by an optical source. This radiation chemically activates the drug and provokes reactions that lead to tissue necrosis. Nowadays there are fixed clinical Photodynamic Therapy protocols that make use of a particular optical dose and photosensitizer amount. These parameters are independent of the patient and the lesion. In this work we present a Photodynamic Therapy model that tries to predict the effect of the treatment on the skin. First the results of a clinical study in the Dermatology Department of the Marqués de Valdecilla University Hospital are presented. The most common lesions and some unsuccessful cases are stated. The predictive model proposed is based on a 3D optical propagation of radiation by a Monte Carlo approach. Once the optical energy is obtained, a complex photochemical model is employed. This model takes into account the electronic transitions between molecular levels and particles concentrations. As the process of generation of photosensitizer is not homogeneous, the photosensitizer distribution is also taken into account. The optical power of the source, the exposition time and the optochemical characteristics of the tissue can be varied. This implies that these parameters could be adjusted to the particular pathology we are dealing with, so the unsuccessful cases could be better treated.

In rhytidectomy the postoperative edema (swelling) and ecchymosis (bruising) can influence the cosmetic results. Evaluation of edema has typically been performed by visual inspection by a trained physician using a fourlevel or, more commonly, a two-level grading⁽¹⁾. Few instruments exist capable of quantitatively assessing edema and ecchymosis in skin. Here we demonstrate that edema and ecchymosis can be objectively quantitated in vivo by a multispectral clinical imaging system (MSCIS). After a feasibility study of induced stasis to the forearms of volunteers and a benchtop study of an edema model, five subjects undergoing rhytidectomy were recruited for a clinical study and multispectral images were taken approximately at days 0, 1, 3, 6, 8, 10, 15, 22 and 29 (according with the day of their visit). Apparent concentrations of oxy-hemoglobin, deoxy-hemoglobin (ecchymosis), melanin, scattering and water (edema) were calculated for each pixel of a spectral image stack. From the blue channel on cross-polarized images bilirubin was extracted. These chromophore maps are two-dimensional quantitative representations of the involved skin areas that demonstrated characteristics of the recovery process of the patient after the procedure. We conclude that multispectral imaging can be a valuable noninvasive tool in the study of edema and ecchymosis and can be used to document these chromophores in vivo and determine the efficacy of treatments in a clinical setting.

Atopic Dermatitis (AD) is an inflammatory disease of human skin. Its pathogenesis is still unknown; however, dysfunctions of the epidermal barrier and the immune response are regarded as key factors for the development of AD. In our study we applied intravital multiphoton tomography (5D-IVT), equipped with a spectral-FLIM module for in-vivo and ex-vivo analysis of human skin affected with AD. In addition to the morphologic skin analysis, FLIM technology gain access to the metabolic status of the epidermal cells referring to the NADH specific fluorescence lifetime. We evaluated a characteristic 5D-IVT skin pattern of AD in comparison to histological sections and detected a correlation with the disease activity measured by SCORAD. FLIM analysis revealed a shift of the mean fluorescence lifetime (tau_m) of NADH, indicating an altered metabolic activity. Within an ex-vivo approach we have investigated cryo-sections of human skin with or without barrier defects. Spectral-FLIM allows the detection of autofluorescent signals that reflect the pathophysiological conditions of the defect skin barrier. In our study the tau_m value was shown to be different between healthy and affected skin. Application of the 5D-IVT allows non-invasive in-vivo imaging of human skin with a penetration depth of 150 μm. We could show that affected skin could be distinguished from healthy skin by morphological criteria, by FLIM and by spectral-FLIM. Further studies will evaluate the application of the 5D-IVT technology as a diagnostic tool and to monitor the therapeutic efficacy.

Today it is still clinical practice to determine burns wounds and their depth by visual inspection. However, it was recently shown that burns develop differently from their initial grade depending on the contact time of the source. As this contact time varies it is difficult to assess the burn severity relaying only on a naked eye. Parameters such as oxygen saturation, hematocrit, water presence, and perfusion, can offer a more quantitative approach to wound assessment hence improving diagnosis and treatment. These parameters can be obtained with spectroscopic and flow sensitive techniques. We propose a study of burns dynamic using a combination of spectroscopic and thermal imaging techniques. A spectral camera based on a lenslet array architecture was used to obtain 18 images of the skin, each lenslet was interfaced with a narrowband filter hence 18 spectrally sensitive images were obtained. In this paper the results of a preliminary electrical burns study are presented.

Optical Coherence Tomography (OCT) is a non-invasive imaging modality that acquires cross sectional images of tissue in-vivo. It accelerates skin diagnosis by eliminating invasive biopsy and laborious histology in the process. Dermatologists have widely used it for looking at morphology of skin diseases such as psoriasis, dermatitis, basal cell carcinoma etc. Skin scientists have also successfully used it for looking at differences in epidermal thickness and its underlying structure with respect to age, body sites, ethnicity, gender, and other related factors. Similar to other in-vivo imaging systems, OCT images suffer from a high degree of speckle and noise content, which hinders examination of tissue structures. Most of the previous work in OCT segmentation of skin was done manually. This compromised the quality of the results by limiting the analyses to a few frames per area. In this paper, we discuss a region growing method for automatic identification of the upper and lower boundaries of the epidermis in living human skin tissue. This image analysis method utilizes images obtained from a frequency-domain OCT. This system is high-resolution and high-speed, and thus capable of capturing volumetric images of the skin in short time. The three-dimensional (3D) data provides additional information that is used in the segmentation process to help compensate for the inherent noise in the images. This method not only provides a better estimation of the epidermal thickness, but also generates a 3D surface map of the epidermal-dermal junction, from which underlying topography can be visualized and further quantified.

Underlying optical properties linked to the visual appearance of skin was studied by obtaining reflectance images using a multispectral imaging system. The analysis of the resulting reflectance spectra yields the melanin content M (volume fraction of melanosome in the pigmented epi-dermis), the blood content B (average volume fraction of whole blood in skin), oxygen saturation level S, water content W (average volume fraction of water in tissue) and the reduced scattering μ'_s500 at 500 nm. The spatial map of the optical properties can now be linked to the visual appearance of the skin.

Single-wall carbon nanotubes (SWNTs) are a new type of nanomaterial with strong optical absorption. SWNTs also have intense Raman signals that facilitate convenient monitoring of their location within tissue thereby enabling noninvasive pharmacokinetic study. We hypothesize that SWNTs can absorb 785-nm laser light and generate significant local hyperthermia to destroy cancer cells and eradicate tumors. In this study a 785-nm diode laser is used for both Raman excitation and photothermal therapy. SWNTs are made water-soluble by functionalizing with polyethylene glycol (PEG) and administrated by intratumoral injection. C3H/HeN mice were injected subcutaneously with 2 million mouse squamous cell carcinoma (SCCVII) cells to create the tumor model. We conducted experiments with 100 mice divided into 10 different groups: control, SWNT only, 100 mW/cm² laser irradiation only, 200 mW/cm² laser irradiation only, and 6 treatment groups with different drug and light dose combinations (SWNTs 0.1, 0.5. 1 mg/ml, laser 100 and 200 mW/cm²). The treatment time was 10 minutes. The temperatures of the tumors irradiated by laser were monitored by an IR thermometer. Mice survival was observed for 45 days. The study revealed that the temperature within the tumors increased in a light- and drug-dose dependent manner. The optimized light and drug dose combinations (1 mg/ml + 120 J/cm²) resulted in tumor temperature elevation of 18.5°C and successful eradication of the tumors. This light dose is moderate and is as low as 1/10 of other published studies using nanomaterials. The Raman spectroscopy measurements suggest that SWNTs persisted within the tumor tissue for months.

It is known that effectiveness of acne treatment increases when the lesions are detected earlier, before they could progress into mature wound-like lesions, which lead to scarring and discoloration. However, little is known about the evolution of acne from early signs until after the lesion heals. In this work we computationally characterize the evolution of inflammatory acne lesions, based on analyzing cross-polarized images that document acne-prone facial skin over time. Taking skin images over time, and being able to follow skin features in these images present serious challenges, due to change in the appearance of skin, difficulty in repositioning the subject, involuntary movement such as breathing. A computational technique for automatic detection of lesions by separating the background normal skin from the acne lesions, based on fitting Gaussian distributions to the intensity histograms, is presented. In order to track and quantify the evolution of lesions, in terms of the degree of progress or regress, we designed a study to capture facial skin images from an acne-prone young individual, followed over the course of 3 different time points. Based on the behavior of the lesions between two consecutive time points, the automatically detected lesions are classified in four categories: new lesions, resolved lesions (i.e. lesions that disappear completely), lesions that are progressing, and lesions that are regressing (i.e. lesions in the process of healing). The classification our methods achieve correlates well with visual inspection of a trained human grader.

In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.

Our previous study reported a sensitivity of 96.6% and a specificity of 89.2% in rapidly detecting Basal Cell Carcinomas (BCCs) when nuclei were stained with acridine orange. Squamous Cell Carcinomas (SCCs) and infiltrative BCCs remain difficult to detect. More complete screening can be achieved utilizing both acridine orange for nuclei staining and eosin for cytoplasmic contrast, using two lasers to excite the two stains independently. Nuclear fluorescence is achieved by staining with acridine orange (0.5mM, 60 s), and cytoplasmic fluorescence is achieved by staining with eosin working solution (30 s). This work shows good morphological contrast of SCC and infiltrative BCC with eosin, acridine orange, and reflectance, and presents a means for rapid SCC and infiltrative BCC detection in fresh skin excisions using multimodal confocal microscopy. In addition, digital staining is shown to effectively simulate hematoxylin and eosin (H&E) histology with confocal mosaics.

Skin cancer is the most commonly occurring cancer, with incidence rates rising annually. Realizing favorable outcomes requires early diagnosis, for which the current standard is biopsy followed by histopathology. This process can be invasive, subjective, time consuming, and costly. Optical techniques, including Optical Coherence Tomography (OCT) and Raman Spectroscopy (RS), have been developed to perform non-invasive characterization of skin lesions, however neither is without limitation. Here, we demonstrate a clinical instrument for morphological and biochemical characterization of skin cancers with combined RS-OCT. The portable instrument utilizes independent RS and OCT system backbones, and is integrated in a common clinical probe. The potential of the probe for cancer diagnosis is demonstrated on malignant and non-malignant lesions.

The stratum corneum provides a vital physical barrier that protects against external insults and excessive internal water loss. Water activity is thought as a key factor to maintain proper skin barrier integrity via regulating enzyme activities and lipid phase behavior. Consequently, maintenance of an optimal hydration level in SC becomes an important clinical and cosmetic concern. The objective methods to assess SC hydration are based on either electrical or optical measurements. Electrical techniques used in the current study include high frequency conductance (Skicon), impedance (Nova DPM) and DC I-V curve (Skinsensor). Confocal Raman Microscopy was utilized to document water profile versus depth, and this technique is based on inelastic scattering of monochromatic light from different chemical species of skin. Water patches were applied on the 14 subjects' forearm for 20 minutes and 1.5 hrs. Skin hydration levels for individuals were documented by utilizing the mentioned above instruments in vivo. Results show that patterns of water profiles upon the hydration are significantly different among the individuals and these differences may be related to skin barrier function integrity. The intrinsic water content and water absorption upon the hydration were summed corresponding to different depths (3 μm and 15 μm) from the data obtained by confocal Raman microscopy. These results were correlated to the readings from electrical approaches. Superficial (3 μm) but not deeper layer (15 μm) water contents correlated well with the readings from SkinSensor. Neither depth measurements correlate well with the Skicon. There is strong correlation between the data acquired with Skicon and SkinSensor.

We present the construction of a new multimodal, multiphoton spectroscopic and imaging instrument for in vivo patient use. Utilizing a tunable femtosecond laser, we are able to simultaneously acquire two-photon excited fluorescence, second harmonic generation, and confocal reflectance images at half video rate, while concurrently acquiring two-photon excited fluorescence and second harmonic generation spectra.

Measurement of cutaneous microcirculation is of great importance for clinical evaluations as many biological processes (i.e. inflammation) activate superficial vessels. Diffuse Reflectance Spectroscopy (DRS) is a widely used method to assess cutaneous microcirculation since it is based on the spectral characteristics of skin chromophores, particularly the strong absorption bands of blood in the visible spectral range. However, the DRS system only provides a snapshot of the blood content in tissue from the reflectance detected at a single exposure time, and it fails to demonstrate dynamic changes of blood flow inside the tissues. In the present study, a prototype system for functional DRS (fDRS) has been developed for the sequential acquisition of multiple reflectance spectra in the visible range at sub-second intervals. Twelve healthy subjects with skin phototype I-III were recruited for a UV skin phototest where the subject's back was exposed to an increasing dose of solar-simulated radiation at a maximum of 3 minimum erythema dose (MED). Evaluations included conventional DRS, functional DRS, and a laser Doppler flowmeter/imaging system. In a frequency analysis results, there were two distinctive frequency components. A low-frequency component was found near 0.03-0.1 Hz, and another high-frequency component near 0.9-1.2 Hz which is synchronous with heart pulsations. The magnitude of the high-frequency and the steady-state components of the fDRS signal increased with increase in exposure dose. These results demonstrate the potential of this technique for noninvasive assessment of cutaneous microcirculation.

Zinc oxide nanoparticles (ZnO NPs) are commonly used in sunscreens to reduce the risk of skin cancer by blocking ultraviolet radiation. ZnO NPs absorption through the transdermal route may not cause high health risk as inhalation or ingestion. However, in practical usage of sunscreens and cosmetics, ZnO NPs are topically applied to a large area of skin with long periods hence the potential absorption amount of ZnO NPs is still need to be concerned. Therefore, if the ZnO NPs are able the pass the barrier of normal skin, the pathways of transdermal delivery and the factors of enhancements become important issues. In this work, multiphoton microscopy provides us a non-invasive visualization of ZnO NPs in skin. Moreover, we quantitatively analyzed the enhancement of oleic acid and ethanol. Due to the fact that photoluminance of ZnO NPs spectrally overlaps autofluorence from skin stratum corneum (SC) and high turbidity of both ZnO NPs and SC, it is difficult to resolve the distribution of ZnO NPs in skin by using fluorescence microscopy. In this work, the second harmonic generation (SHG) signals from ZnO NPs which double the frequency of excitation source to characterize the delivery pathways and penetration depth in skin. Moreover, we quantitatively compare the ZnO NPs delivery efficiency in normal skin and in skins with three chemically enhancing conditions: ethanol, oleic acid and the combination of ethanol and oleic acid.

In vivo reflectance confocal microscopy shows promise for the early detection of malignant melanoma. One diagnostic trait of malignancy is the presence of pagetoid melanocytes in the epidermis. For automated detection of MM, this feature must be identified quantitatively through software. Beginning with in vivo, noninvasive confocal images from 10 unequivocal MMs and benign nevi, we developed a pattern recognition algorithm that automatically identified pagetoid melanocytes in all four MMs and identified none in five benign nevi. One data set was discarded due to artifacts caused by patient movement. With future work to bring the performance of this pattern recognition technique to the level of the clinicians on difficult lesions, melanoma diagnosis could be brought to primary care facilities and save many lives by improving early diagnosis.

Near infrared Nd:YAG pulsed laser treatment had been proved to be an efficient method to treat large-sized vascular malformations like leg telangiectasia for deep penetrating depth into skin and uniform light distribution in vessel. However, optimal clinical outcome was achieved by various laser irradiation parameters and the key factor governing the treatment efficacy was still unclear. A mathematical model in combination with Monte Carlo algorithm and finite difference method was developed to estimate the light distribution, temperature profile and thermal damage in epidermis, dermis and vessel during and after 1064 nm pulsed Nd:YAG laser irradiation. Simulation results showed that epidermal protection could be achieved during 1064 nm Nd:YAG pulsed laser irradiation in conjunction with cryogen spray cooling. However, optimal vessel closure and blood coagulation depend on a compromise between laser spot size and pulse duration.

Background: Continuous wave near-infrared spectroscopy (NIRS) can monitor chromophore change in the bladder detrusor muscle during voiding; oxygenation and hemodynamic data derived differ in health and disease. Application of wireless NIRS for evaluation of voiding dysfunction would benefit children. Methods: Subjects: 20 children (4-17 yrs) [5 normal, 15 with urinary tract pathology]. Instrumentation: self-contained device weight 84 gm; 3 paired light emitting diodes (760/850 nm) in a spatially resolved configuration; source-detector separation distances (30, 35 and 40 mm); silicon photodiode detector; and Bluetooth®. Procedure: Transcutaneous monitoring (midline abdominal skin 2 cm above pubis) during spontaneous voiding (bladder contraction) of oxygenated (O₂Hb), deoxygenated (HHb) and total hemoglobin (tHb) and tissue oxygen saturation index (TSI %) at 10 Hz. Results: All 20 trials produced clear graphic data with no movement effect evident. Comparison of patterns of chromophore change between normal and symptomatic subjects revealed trend differences in O₂Hb and tHb. (Normal positive; Symptomatic negative, and TSI% fell in symptomatic group). Conclusions: Wireless NIRS is technically feasible in ambulant children. Negative trends in chromophore concentration and falls in TSI% suggest a hemodynamic impairment may underlie some forms of voiding dysfunction, with abnormal physiology involving the microcirculation possibly resulting in muscle fatigue during voiding.

Two-photon microscopy has been successfully used to image several types of tissues, including skin, muscles, tendons. Nevertheless, its usefulness in imaging bladder tissue has not been investigated yet. In this work we used combined twophoton excited fluorescence, second-harmonic generation microscopy, fluorescence lifetime imaging microscopy, and multispectral two-photon emission detection to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa and carcinoma in-situ samples in a good agreement with common routine histology. Cancer cells showed different morphology with respect to the corresponding healthy cells: they appeared more elongated and with a larger nucleus to cytoplasm ratio. From the spectroscopic point of view, differences between the two tissue types in both spectral emission and fluorescence lifetime distribution were found. Even if further analysis, as well as a more significant statistics on a larger number of samples would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well to be implemented in a multi-photon endoscope or in a spectroscopic for in in-vivo imaging applications.

Urolithiasis is a common, disturbing disease with high recurrent rate (60% in five years). Accurate identification of urinary stone composition is important for treatment and prevention purpose. Our previous studies have demonstrated that micro-Raman spectroscopy (MRS)-based approach successfully detects the composition of tiny stone powders after minimal invasive urological surgery. But quantitative analysis of urinary stones was not established yet. In this study, human urinary stone mixed with two compositions of COM, HAP, COD, and uric acid, were analyzed quantitatively by using a 632.98 nm Raman spectrometric system. This quantitative analysis was based on the construction of calibration curves of known mixtures of synthetically prepared pure COM, HAP, COD and uric acid. First, the various concentration (mole fraction) ratio of binary mixtures including COM and HAP, COM and COD, or COM and uric acid, were produced. Second, the intensities of the characteristic bands at 1462cm ^-1(I_RCOM), 1477cm^-1(I_RCOD), 961cm^-1(IRHAP) and 1402cm^-1(I_{Ruric acid}), for COD, COM, HAP and uric acid were used respectively for intensity calculation. Various binary mixtures of known concentration ratio were recorded as the basis for the quantitative analysis. The ratios of the relative intensities of the Raman bands corresponding to binary mixtures of known composition on the inverse of the COM concentration yielded a linear dependence. Third, urinary stone fragments collected from patients after management were analyzed with the use of the calibration curve and the quantitative analysis of unknown samples was made by the interpolation analysis. We successfully developed a MRS-based quantitative analytical method for measuring two composition.

Optical imaging systems utilizing near-infrared light sources such as optical coherence tomography (OCT) have recently been used for imaging the prostate gland. However, the optimal wavelength for deep imaging of the prostate has yet to be determined. The objective of this study is to determine the optimal near-infrared wavelength for OCT imaging of the prostate using a system that has the potential to be used in an in vivo model. An obliqueincidence single point measurement technique using a normal-detector scanning system was implemented to determine the absorption (μ_a) and reduced scattering coefficients (μ_'s) of fresh canine prostate tissue, ex vivo, from the diffuse reflectance profile of near-IR light as a function of source-detector distance. The effective attenuation coefficient (μ_eff) and optical penetration depth (OPD) were then calculated for near-IR wavelengths of 1064, 1307, and 1555 nm. A total of ten canine samples were used for this study. At wavelengths of 1064, 1307, and 1555 nm, the mean absorption coefficients measured 0.08 ± 0.03, 0.12 ± 0.04, and 0.23 ± 0.09 cm^-1, respectively. The mean reduced scattering coefficients measured 16.60 ± 0.95, 14.30 ± 1.14, and 10.98 ± 2.35 cm^-1. The effective attenuation coefficients were calculated to be 2.00, 2.28, and 2.78 cm^-1, yielding OPD's of 0.5, 0.44, and 0.36 cm at 1064, 1307, and 1555 nm. OCT imaging studies of the prostate may benefit from replacement of commonly used 1310 nm broadband light sources with 1064 nm sources.

Prostate cancer is the second leading cause of death in the United States in 2009. Radical prostatectomy (complete removal of the prostate) is the most common treatment for prostate cancer, however, differentiating prostate tissue from adjacent bladder, nerves, and muscle is difficult. Improved visualization could improve oncologic outcomes and decrease damage to adjacent nerves and muscle important for preservation of potency and continence. A novel Stokes polarimetry imaging (SPI) system was developed and evaluated using a dog prostate specimen in order to examine the feasibility of the system to differentiate prostate from bladder. The degree of linear polarization (DOLP) image maps from linearly polarized light illumination at different visible wavelengths (475, 510, and 650 nm) were constructed. The SPI system used the polarization property of the prostate tissue. The DOLP images allowed advanced differentiation by distinguishing glandular tissue of prostate from the muscular-stromal tissue in the bladder. The DOLP image at 650 nm effectively differentiated prostate and bladder by strong DOLP in bladder. SPI system has the potential to improve surgical outcomes in open or robotic-assisted laparoscopic removal of the prostate. Further in vivo testing is warranted.

This paper aims to describe the development of a method to monitor laser interstitial thermo therapy by MR images. The method is based on the texture analysis using fractal geometry features of the images to estimate the size of the induced necrosis. The method was validated by comparing the results to macroscopic measurements. It demonstrates the ability to achieve good estimation of the necrosis in ex-vivo experimentations involving pig liver and in vivo experimentations done on tumors grown on Copenhagen rats.

We consider the cross-polarization OCT (CP OCT) that is focused on comparison of images resulting from cross-polarization and co-polarization scattering simultaneously. This technique provides information about microstructural and biochemical alterations in depolarizing tissue components (collagen). We found that mature type I collagen gives a strong signal in orthogonal polarization. CP OCT images of benign inflammatory processes always feature signal in orthogonal polarization, with layers and borders persisting to be well defined. In the presence of precancerous alterations, signal in orthogonal polarization is available in the image but it is irregular, disappearing in some areas. A CP OCT image of bladder cancer in orthogonal polarization either shows no signal at all or a weak signal.

OBJECTIVES: We evaluated the long term therapeutic efficacy of 80 watt photo-selective vaporization of the prostate (PVP) in patients suffering from lower urinary tract symptoms (LUTS) secondary to prostatic obstruction. MATERIAL & METHODS: 150 unselected patients at the average age 73 (range 51-92) and a mean American Society of Anesthesiologists score of 2.4 (median 2.0), of whom 33% were medicated with acetylsalicylic acid and 5% were anticoagulated with warfarin. Inclusion/exclusion criteria were the same as for TUR-P at our institution. First patient was operated March 2004 and yearly follow-up of all patients has been attempted for 5 years. Follow-up variables have included yearly creatinine, PSA, IPSS, ØOL, post-void residual urin and maximum/average urine flow rate. RESULTS: At 12 and 24 months postoperatively, the following parameters were significantly (p<0.001) improved: trans-rectal ultrasound, international prostate symptom score, quality of life score, post-void residual urine volume, flow max/average, opening pressure, pressure @ flow-max, and micturition resistance. At 48 and 60 months creatinine, PSA, IPSS, ØOL, post-void residual urin and maximum/average urine flow rates were still significantly (p<0.001) improved compared to pre-operative values. CONCLUSION: Up to 5 year follow-up reveals that 80 watt PVP provides significant and stable symptom relief as well as objective improvement in residual urine and flowmetric outcomes.

Introduction: Biochemical recurrence of prostate cancer after definitive therapy with radical prostatectomy (RP) is known to occur between 25-30%. We present the first known case of 1.5T MRI guided ablation using laser interstitial thermal therapy (LITT) for locally recurrent prostate cancer following RP. Methods: The patient elected to undergo MRI-guided LITT of the biopsy proven cancer recurrence using an FDAapproved MRI compatible, 980nm, 15-watt laser system with MR thermometry. Under T2-weighted MR(1.5T Siemens) imaging, guidance and targeting of the lesions with trans-perineal placement of laser applicators. Multiple cycles of laser energy were used to ablate the tumor. A MRI-compatible urethral cooling catheter was placed to prevent urethral thermal damage. Results: Intra-procedural temperature mapping allowed continuous monitoring of the ablation zone and permitted ablation control until tumor coverage was achieved. Additionally, the protective cooling effects of the urethral cooling catheter could also be seen with the temperature mapping. Post-ablation gadolinium and T2 weighted MR imaging demonstrated an ablation defect encompassing the recurrent tumor with no residual hyper-enhancing nodules. Three month follow-up shows no residual or recurrent tumor seen on MR imaging. Conclusion: This represents the first known, successful, MRI-guided, LITT procedures at 1.5T for locally recurrent prostate adenocarcinoma following RP.

Purpose: To evaluate the short term outcomes of 220 consecutive patients who underwent the 532 nm KTP photoselective vaporization of the prostate (PVP) procedure and to evaluate and categorize the complications of the procedure. Materials and Methods: A total of 220 patients with symptomatic benign prostatic obstruction were treated with KTP photoselective vaporization of the prostate. Evaluation measures included the AUA Symptom Score (AUASS)/Quality of Life Score (QOL), peak urinary flow rate (Qmax), post void residual urine (PVR) and adverse events. Results: Symptoms were evaluated at 3 months and adverse events at 1 and 3 months. 181 patients returned for their 1 month visit and 152 returned for their 3 month visit. The American Urological Association Symptom Score (AUASS) decreased from 21.8 to 6.7. The Quality of Life Score (QOL) decreased from 3.8 to 0.7. The peak urinary flow rate (Qmax) increased from 10.7 cc/sec to 22.7 cc/sec. And the post void residual urine (PVR) decreased from 262 cc to 105 cc. Most common adverse events lasting more than 10 days were mild hematuria in 45%, dysuria in 32%, and urgency/frequency in31%. Conclusion: These results confirm that photoselective vaporization of the prostate (PVP) is a safe and effective therapy for benign prostatic obstruction. However, there is frequent, but mild, hematuria and irritative voiding symptoms during the early postoperative period.

The cavernous nerves (CN) course along the prostate surface and are responsible for erectile function. Improved identification and preservation of the CN's is critical to maintaining sexual potency after prostate cancer surgery. Noncontact optical nerve stimulation (ONS) of the CN's was recently demonstrated in a rat model, in vivo, as a potential alternative to electrical nerve stimulation (ENS) for identification of the CN's during prostate surgery. However, the therapeutic window for ONS is narrow, so optimal design of the fiber optic delivery system is critical for safe, reproducible stimulation. This study describes modeling, assembly, and testing of an ONS probe for delivering a small, collimated, flat-top laser beam for uniform CN stimulation. A direct comparison of the magnitude and response time of the intracavernosal pressure (ICP) for both Gaussian and flat-top spatial beam profiles was performed. Thulium fiber laser radiation (λ=1870 nm) was delivered through a 200-μm fiber, with distal fiber tip chemically etched to convert a Gaussian to flat-top beam profile. The laser beam was collimated to a 1-mm-diameter spot using an aspheric lens. Computer simulations of light propagation were used to optimize the probe design. The 10-Fr (3.4-mm-OD) laparoscopic probe provided a constant radiant exposure at the nerve surface. The probe was tested in four rats, in vivo. ONS of the CN's was performed with a 1-mm-diameter spot, 5- ms pulse duration, and pulse rate of 20 Hz for a duration of 15-30 s. The flat-top laser beam profile consistently produced a faster and higher ICP response at a lower radiant exposure than the Gaussian beam profile due, in part, to easier alignment of the more uniform beam with nerve. With further development, ONS may be used as a diagnostic tool for identification of the CN's during laparoscopic and robotic nerve-sparing prostate cancer surgery.

Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early detection of prostate cancer. A trans-rectal probe adapted to fluorescence diffuse optical tomography measurements was developed by our team. This probe is based on a pulsed NIR laser source, an optical fiber network and a time-resolved detection system. A reconstruction algorithm was used to help locate and quantify fluorescent prostate tumors. In this study, two different kinds of time-resolved detectors are compared: High Rate Imaging system (HRI) and a photon counting system. The HRI is based on an intensified multichannel plate and a CCD Camera. The temporal resolution is obtained through a gating of the HRI. Despite a low temporal resolution (300ps), this system allows a simultaneous acquisition of the signal from a large number of detection fibers. In the photon counting setup, 4 photomultipliers are connected to a Time Correlated Single Photon Counting (TCSPC) board, providing a better temporal resolution (0.1 ps) at the expense of a limited number of detection fibers (4). At last, we show that the limited number of detection fibers of the photon counting setup is enough for a good localization and dramatically improves the overall acquisition time. The photon counting approach is then validated through the localization of fluorescent inclusions in a prostate-mimicking phantom.

Laser prostatectomy with various lasers has been shown to be effective in the treatment of benign prostate hyperplasia. However, the impact of laser parameters on tissue ablation is still in question. The aim of this study is to experimentally characterize laser-tissue interactions in terms of wavelength by comparing visible (λ= 532 nm) and infrared (λ= 2.01 μm) spectra. Porcine kidney tissue was used as it has thermal properties and glandular structure similar to human prostatic tissue. Q-switched 532 nm (GreenLight^TM HPS) and continuous-wave (CW) 2.01 μm (custom-made Tm:YAG) lasers were employed to remove soft tissue under various settings (power, working distance, and treatment speed). For both laser systems, ablation rate increased with power and was maximized at 4 mm/s. The 532 nm laser generated approximately 30% (p<0.005) higher ablation efficiency than the IR laser. A comparable ablation depth was found between the two wavelengths, but the 532nm laser generated relatively wider (up to 30%; p<0.005) craters. Owing to constant heating due the CW mode, the IR laser induced 20% thicker coagulation depth than the 532 nm (0.94 vs. 0.8 mm at 100 W; p<0.005). Histology also confirmed coagulation depth in response to each wavelength. Due to light absorption in aqueous environment, the IR laser exhibited a dramatic decrease in power transmission and ablation volume with increasing working distance whereas the 532 nm laser maintained relatively constant features. In conclusion, the characteristics of tissue ablation were contingent upon the applied wavelengths due to optical properties and laser parameters.

Development of a noninvasive vasectomy technique may eliminate male fear of complications and result in a more popular procedure. This study explores application of an optical clearing agent (OCA) to the scrotal skin to reduce both the laser power necessary for successful noninvasive laser vasectomy and the probability of scrotal skin burns. A mixture of DMSO/glycerol was noninvasively delivered into the scrotal skin using a Madajet. Near-infrared laser radiation with a range of average powers (7.0-11.7 W) was delivered in conjunction with a range of cryogen spray cooling rates (0.20-0.33 Hz) to the skin surface in a canine model, ex vivo and in vivo. Burst pressure (BP) measurements were conducted to quantify the strength of vas closure. A 30-min application of the OCA improved skin transparency by 26 ± 5 %, reducing the average power necessary for successful noninvasive laser vasectomy from 9.2 W without OCA (BP = 291 ± 31 mmHg) to 7.0 W with OCA (BP = 292 ± 19 mmHg). Control studies without OCA at 7.0 W failed to coagulate the vas with burst pressures (82 ± 28 mmHg) significantly below typical ejaculation pressures (136 ± 29 mmHg). Application of an optical clearing agent reduced the laser power necessary for successful noninvasive thermal coagulation of the vas by approximately 25%. This technique may result in the use of a less expensive laser system and eliminate the formation of scrotal skin burns during the procedure.

Development of a noninvasive vasectomy technique may eliminate male fear of complications (incision, bleeding, infection, and scrotal pain) and result in a more popular procedure. This study builds upon previously reported ex vivo tissue studies by exploring acute and short-term chronic in vivo canine studies. Isolation of the canine vas was achieved using a conventional vas ring clamp method. No perforation of the scrotal skin was necessary to occlude the vas. Laser radiation with a wavelength of 1075 nm, average power of 11.2 W, 500-ms pulse duration, 0.5 Hz pulse rate, and 3-mm-diameter spot was synchronized with cryogen spray cooling of the scrotal skin surface in a total of 8 dogs (n = 16 vasa) for a treatment time of 60 s. Burst pressure measurements were conducted at Days 0 and 21 (n = 8 vasa each day) to quantify the strength of vas closure. The vas was successfully thermally occluded in 15/16 (94%) procedures with 14/15 (93%) vas recording burst pressures above ejaculation pressure. One vas was not present, and another vas recorded a bursting pressure below ejaculation pressure. The coagulated vas bursting pressure averaged 283 ± 34 mm Hg at Day 0 and 260 ± 77 mm Hg at Day 21, significantly higher than reported vas ejaculation pressures of 136 ± 29 mm Hg. Minor scrotal skin burns were observed during the recovery period. Noninvasive thermal occlusion of the vas is feasible in an in vivo canine model. Elimination of minor skin burns and longer term chronic in vivo canine studies are needed to confirm azospermia after vas occlusion without recanalization.

The Thulium fiber laser has recently been tested as a potential alternative to the Holmium:YAG laser for lithotripsy. This study explores use of a short taper for expanding the Thulium fiber laser beam at the distal tip of a small-core fiber. Thulium fiber laser radiation with a wavelength of 1908 nm, 10 Hz pulse rate, 70 mJ pulse energy, and 1-ms pulse duration was delivered through a 2-m-length fiber with 150-μm-input-end, 300-μm-output-end, and 5-mmlength taper, in contact with human uric acid (UA) and calcium oxalate monohydrate (COM) stones, ex vivo (n=10 each). Stone mass loss, stone crater depths, fiber transmission losses, fiber burn-back, irrigation rates, and deflection through a flexible ureteroscope were measured for the above tapered fiber and compared with conventional fibers. After delivery of 1800 pulses through the tapered fiber, mass loss measured 12.7 ± 2.6 mg for UA and 7.2 ± 0.8 mg COM stones, comparable to conventional 100-μm-core fibers (12.6 ± 2.5 mg for UA and 6.8 ± 1.7 mg for COM stones). No transmission losses or burn-back occurred for the tapered fiber after 36,000 pulses, while a conventional 150-μm fiber experienced significant tip degradation after only 1800 pulses. High irrigation rates were measured with the tapered fiber inserted through the working port of an ureteroscope, without hindering ureteroscope deflection, mimicking that of conventional 150 ìm fibers. The short tapered distal fiber tip allows expansion of the laser beam, resulting in decreased fiber tip damage compared to conventional small-core fibers, without compromising fiber bending, stone vaporization efficiency, or irrigation rates.

The Holmium:YAG laser is currently the most common laser lithotripter. However, recent experimental studies have demonstrated that the Thulium fiber laser is also capable of vaporizing urinary stones. The high-temperature water absorption coefficient for the Thulium wavelength (μ_a = 160 cm^-1 at λ = 1908 nm) is significantly greater than for the Holmium wavelength (μ_a = 28 cm^-1 at λ = 2120 nm). We hypothesize that this should translate into more efficient laser lithotripsy using the Thulium fiber laser. This study directly compares stone vaporization rates for Holmium and Thulium fiber lasers. Holmium laser radiation pulsed at 3 Hz with 70 mJ pulse energy and 220 μs pulse duration was delivered through a 100-μm-core silica fiber to human uric acid (UA) and calcium oxalate monohydrate (COM) stones, ex vivo (n = 10 each). Thulium fiber laser radiation pulsed at 10 Hz with 70 mJ pulse energy and 1 ms pulse duration was also delivered through a 100-μm fiber for the same sets of 10 stones. For same number of pulses and total energy (126 J) delivered to each stone, mass loss averaged 2.4 ± 0.6 mg (UA) and 0.7 ± 0.2 mg (COM) for Holmium laser and 12.6 ± 2.5 mg (UA) and 6.8 ± 1.7 (COM) for Thulium fiber laser. UA and COM stone vaporization rates for Thulium fiber laser averaged 5-10 times higher than for Holmium laser at 70 mJ pulse energies. With further development, the Thulium fiber laser may represent an alternative to the conventional Holmium laser for more efficient laser lithotripsy.

The holmium:yttrium aluminum garnet (YAG) laser is the gold standard laser for intracorporeal lithotripsy.¹ Optical fibers are utilized to transmit laser energy to the surface of a stone for fragmentation via a predominant photothermal mechanism.² Previous work has demonstrated that performance characteristics of holmium:YAG optical fibers used for laser lithotripsy varies. Performance may difference not only between fibers made by different manufacturers but also between individual fibers produced by the same manufacturer.^3,4 Fiber failure with bending, such as during lower pole ureterorenoscopy, can lead to catastrophic endoscope damage resulting in costly repair. Manufacturers continue to develop new holmium:YAG optical fibers. In this study we evaluate a series of newly commercially available fibers using a previously designed testing protocol. This study was designed to determine the performance and threshold for failure of six newly available holmium:YAG laser fibers from Cook Medical and Fibertech Gmbh. We hypothesize that fiber performance will continue to vary amongst different holmium:YAG optical fibers.

Percutaneous nephrolithotomy has become the standard of care for large renal calculi requiring surgical removal. Ultrasonic intracorporeal lithotriptors are used to fragment and evacuate the calculi. Numerous commercially available systems exist. While previous study has demonstrated differing performance characteristics of the various systems, a standardized testing modality does not exist.^1-6 In addition, ultrasonic lithotriptor efficiency is thought to deteriorate over time, but an objective measurement technique has not been reported. Rather, subjective performance of the ultrasound during surgical procedures is used to gauge the effectiveness of the instrument. In this study we test the performance of two commercially available ultrasonic lithotriptors, the Lithoclast Select (Boston Scientific, Natick, MA) and Cyberwand (Gyrus ACMI, Southborough, MA). The Lithoclast Select combines pneumatic and ultrasonic lithotripsy in one handpiece but only the ultrasonic component was evaluated in this study. The Cyberwand combines two concentric ultrasound probes into a single handpiece. Several recent studies have shown the clinical effectiveness of the Lithoclast Ultra for PCNL.^1-5 The Cyberwand has been shown during in vitro testing to be an efficient lithotrite.⁶ G-force is the measurement of as objects acceleration relative to free-fall and is measured by the unit g. One g is equal to the standard of gravity which is 9.806 m/s2. In this study we measured the g-forces generated by the ultrasonic lithotriptors to determine if there was a difference in the forces generated by the devices.

During fluorescence cystoscopy, it is observed that the acquired images are sometimes blurred by a greenish background originating from the bladder washout fluid. Several fluorophores are involved in this overall liquid fluorescence, and their exact origin and relative contributions remain unknown. In this study, the bladder washout fluid is sampled at different times during fluorescence cystoscopy examinations. In total, 32 samples from 12 patients are analyzed with a spectrofluorimeter (excitation range: 350-445 nm, emission range 380-700 nm). This study shows clearly that the position of the fluorescence peaks (excitation/emission wavelengths: 450/525 nm, 405/625 nm) and shoulder (440/525 nm) is reproducible between different patients. It also suggests that an excitation at wavelengths higher than 400 nm helps to suppress this solution background fluorescence. Additionally, the pH of the solution seems to influence the position of the fluorescence peaks, and this suggests that changing the pH of the examination liquid could help in avoiding the greenish background.

OBJECTIVES: In this study, we evaluated histologically the effects of hexyl 5-aminolevulinateinduced photodynamic treatment in the AY-27 tumor cell induced rat bladder cancer model. MATERIAL & METHODS: The animals (fischer-344 female rats) were divided into 2 groups, half of which were orthotopically implanted with 400,000 syngeniec AY-27 urothelia1 rat bladder cancer cells and half sham implanted. 14 days post implantation 6 rats from each group were treated with hexyl 5-aminolevulinate-induced photodynamic treatment (8mM HAL and light fluence of 20 J/cm2). Additional groups of animals were only given HAL instillation, only light treatment, or no treatment. All animals were sacrificed 7 days after the PDT/only HAL/only light or no treatment. Each bladder was removed, embedded in paraffin and stained with hematoxylin, eosin, and saferin for histological evaluation at high magnification for features of tissue damage by a pathologist blinded to the sample source. RESULTS: In all animals that were AY-27 implanted and not given complete PDT treatment, viable tumors were found in the bladder mucosa and wall. In the animals treated with complete HAL-PDT only 3 of 6 animals had viable tumor. In the 3 animals with viable tumor it was significantly reduced in volume compared to the untreated animals. It was also noted that in the PDT treated animals there was a significantly increased inflammatory response (lymphocytic and mononuclear cell infiltration) in the peri-tumor area compared to implanted animals without complete HAL-PDT. CONCLUSION: Our results suggest that hexyl 5-aminolevulinate-induced photodynamic treatment in a rat bladder cancer model involves both direct effects on cell death (necrosis and apoptosis) and indirect effects to evoke the host immune-response, together contributing to tumor eradication.

Stereotactic navigational devices have been implemented in neurosurgery, orthopedics and ear-nose-throat to improve surgical accuracy. However, the feasibility of navigating inside the bladder has not yet been investigated. Occasionally, transurethral resections of bladder tumors (TURBTs) are impeded by bleeding and cloudiness inside the bladder and, consequently, the bladder lesions are not found back easily. In addition, small bladder lesions are often concealed when viewed with the camera some distance away from the bladder wall due to low contrast differences. The aim of the study is to investigate the feasibility of real-time bladder mapping using the Medtronic Stealthstation system, without the use of pre-operative images. Seven patients scheduled for a TURBT were included in the study. During the TURBT procedure, the spatial coordinates of the bladder lesions were recorded two times independently, after filling the bladder with a fixed volume of 390 ml. The distance between the spatial coordinates of two consecutive measurements, in millimeters, was calculated. We found that bladder lesions can be found back using the navigational system with an accuracy of less than 12 mm. Real-time bladder navigation is feasible without the necessity of pre-operative images or calibration. If the coordinates are directly superimposed on the video image this could facilitate the retrieval of bladder lesions during TURBT. This system could reduce the stress for the surgeon and decrease the operating time.

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.

The development of optical techniques for non-invasive diagnosis of cancer is an ongoing challenge to biomedical optics. For head and neck cancer we see two main fields of potential application 1) Screening for second primaries in patients with a history of oral cancer. This requires imaging techniques or an approach where a larger area can be scanned quickly. 2) Distinguishing potentially malignant visible primary lesions from benign ones. Here fiberoptic point measurements can be used as the location of the lesion is known. This presentation will focus on point measurement techniques. Various techniques for point measurements have been developed and investigated clinically for different applications. Differential Pathlength Spectroscopy is a recently developed fiberoptic point measurement technique that measures scattered light in a broad spectrum. Due to the specific fiberoptic geometry we measure only scattered photons that have travelled a predetermined pathlength. This allows us to analyse the spectrum mathematically and translate the measured curve into a set of parameters that are related to the microvasculature and to the intracellular morphology. DPS has been extensively evaluated on optical phantoms and tested clinically in various clinical applications. The first measurements in biopsy proven squamous cell carcinoma showed significant changes in both vascular and morphological parameters. Measurements on thick keratinized lesions however failed to generate any vascular signatures. This is related to the sampling depth of the standard optical fibers used. Recently we developed a fiberoptic probe with a ~1 mm sampling depth. Measurements on several leukoplakias showed that with this new probe we sample just below the keratin layer and can obtain vascular signatures. The results of a first set of clinical measurements will be presented and the significance for clinical diagnostics will be discussed.

The use of Raman spectroscopy in the detection and classification of malignancy within lymph nodes of the head and neck has been evaluated. Currently histopathology is considered the diagnostic gold standard. A consensus (majority) opinion from three expert histopathologists has been obtained and spectral diagnostic models developed by correlation with their opinions. Raman spectra have been measured at 830nm from 103 lymph nodes collected from patients undergoing surgery for a suspicious node. The pathologies covered reactive lymph nodes, primaries from Hodgkin's and non-Hodgkin's lymphomas and metastases from squamous cell carcinomas and adenocarcinomas. Spectral diagnostic models were constructed using PCA-fed-LDA and tested using leave-one-specimen-out cross validation. Models were constructed to distinguish between reactive and malignant nodes as well as a four group model to distinguish between the benign, metastatic and primary conditions. They achieved 89% and 84% correct prediction by node versus the gold standard, majority histopathology.

We developed a multiphoton microscope which integrates an all normal dispersion fiber laser, a double cladding photonic crystal fiber and a MEMS mirror scanner based hand-held probe. The fiber laser has a central wavelength of 1.06μm,a repetition rate of 76MHz and maximum average output power of more than 1W. The MEMS mirror based probe is compact and Second harmonic generation and two photon excited fluorescence images of biological sample were demonstrated.

Objective: Following tumour surgery in the head and neck region, skin flap transplants are usually required to cover the resection area. The purpose of the development was to provide a simple and reliable means to assess whether the transplanted flap is sufficiently perfused. Methods: Fluorescence of intravenously injected Indocyanine green (ICG) was detected with a slightly modified 3-chip CCD camera. Appropriately coated optical filters allow for excitation of ICG with NIR light and detection of NIR ICGfluorescence with the blue channel of the camera. In addition, low intensities of white light can be transmitted to allow for simultaneous display of a remission image in the green and red channels of the camera. Further processing was performed with a LabVIEW program. Results: A satisfactory white light image (red, green and blue display (RGB)) could be calculated from the remission images recorded with the green and red channels of the camera via a look-up table. The look-up table was programmed to provide an optimized blue intensity value for each combination of red and green values. This was generated using a reference image. Implementation of image tracking and intensity measurements in regions of interest (ROIs) in the images is useful to reliably monitor perfusion kinetics of flap and adjacent normal tissue.

Objectives: To describe the current and promising new applications of Optical Coherence Tomography (OCT) as a helpful tool when imaging the different sites in the head and neck. We used the OCT Niris system, which is the first commercially available OCT device for applications outside the field of ophthalmology. Methods: OCT images were obtained of normal, benign, premalignant and malignant lesions in different areas of the head and neck. The OCT imaging system has a tissue penetration depth of approximately 1-2mm, a scanning range of 2mm and a spatial depth resolution of approximately 10-20μm. Imaging was performed using a flexible probe in two different settings, the outpatient clinic and the operating room. Results: High-resolution cross-sectional images from the larynx were obtained with the patient awake, without the need for general anesthesia, under direct visualization with a flexible fiberoptic endoscope. The OCT probe was inserted through the nasal cavity and placed in slight contact with the laryngeal tissue. In the ears, cholesteatoma was differentiated from inflamed middle ear mucosa by the different hyperintensity. In the neck, normal as well as different pathologies of the thyroid were identified. Conclusions: This system is non invasive and easy to incorporate into the operating room setting as well as the outpatient clinic. It requires minimal set-up and only one person is required to operate the system. OCT has the distinctive capability to obtain highresolution images, and the microanatomy of different sites can be observed. OCT technology has the potential to offer a quick, efficient and reliable imaging method to help the surgeon not only in the operating room but also in the clinical setting to guide surgical biopsies and aid in clinical decision making of different head and neck pathologies, especially those arising form the larynx.

Optical coherence tomography (OCT) is a promising method in the early diagnosis of oral cavity cancer. The objective of the present study is to determine normal values of epithelial thickness in the oral cavity, as no such data are to be found in the literature. In healthy test persons, epithelial thickness of the oral mucosa was determined with the help of OCT separately for each side at nine different locations. Special attention was directed to those sites having the highest incidence for the development of dysplasias and carcinomas. Depending on the location within the oral cavity, the epithelium demonstrated a varying thickness. The highest values were found in the region of the tongue and the cheek, whereas the floor of the mouth showed the thinnest epithelium. Our data serve as reference values for detecting oral malignancy and determining the approximate grade of dysplasia. In this circumstance, a differentiated view of the different regions is important due to the variation in thickness of the epithelium within the normal oral cavity.

Laser septochondrocorrection (LSC) is a non-invasive, bloodless, painless procedure which takes only 10 minutes to complete and can be performed in outpatient settings. The efficacy and safety of this technology can be guarantied with the feed-back control system monitoring tissue characteristics in the course of laser treatment. Laser medical equipment for septochondrocorrection includes an Erbium doped glass fiber laser, special instrument, opto thermo mechanical contactor, and feed back control system measuring temperature on the nasal septum surface and switching the laser off when preset value of temperature is achieved. To the date LSC procedure has been applied for 380 patients, aging from 12 until 68 years. The new laser equipment LRC-701 manufactured by Arcuo Medical Inc. was tested for 106 patients in Moscow and for 64 patients in Crete. The positive results habe been obtained for 95 percent of the patients with two years follow-up. No age limitation, no complications and negative secondary effects were observed.

One common component of otolaryngological surgeries is the reshaping of cartilage. Previous studies have demonstrated the efficient achievement of this procedure through electromechanical reshaping (EMR), a technique that involves the direct application of voltage to cartilage that is mechanically deformed in a jig. Two main parameters, voltage and application time, may be regulated to achieve varying degrees of shape change. Although prior research has correlated these EMR parameters with degree of shape change, it remains necessary to correlate the same parameters with the degree of change in the mechanical properties of tissue. Once this is accomplished, an ideal balance may be determined, in which shape change is maximized while intrinsic tissue damage is minimized This study satisfies this need by providing comprehensive data on the pre- and post-EMR stiffness of both septal and auricular cartilage over a range of voltages (2-8V) with constant application time (2 min for septal, 3 min for auricular). EMR was applied using flat platinum electrodes to one of two 15 mm X 5 mm samples obtained from the same cartilage specimen, while the second sample was maintained as a control. Following a 15 min re-hydration period, the Young's modulus of the tissue was calculated for both the control and experimental sample from data obtained through a uniaxial tension test. A general reduction in stiffness was observed beginning at 3V, with the magnitude of reduction increasing at 6V.

Electromechanical reshaping (EMR) of cartilage is a promising noninvasive technique with potential for broad application in reconstructive surgery. EMR involves applying direct current electrical fields to localized stress regions and initiating a series of oxidation-reduction reactions, thus effecting a shape change. Previous EMR studies have focused on macroscopic structural measurements of the shape change effect or monitoring of electrical current flow. Only limited investigation of structural changes in the tissue at the histologic level have been performed, and not in real time. This study is the first to use optical coherence tomography (OCT) to examine structural changes in cartilage during EMR. Two platinum needle electrodes were inserted into fixed rectangular rabbit nasal septal cartilage specimens. The spectral domain OCT probe was then positioned above the section of cartilage in which the anode needle was inserted. A constant voltage of 6V was applied for 3 minutes, and images were obtained (8 frames/second). OCT was also performed in specimens undergoing dehydration under ambient conditions and during pH changes produced by the addition of HCl, as both processes accompany EMR. The OCT data identified distinct findings among the three conditions, suggesting that EMR causes a much greater degree of reshaping on a molecular level than dehydration or a change in pH alone. OCT provides a means to gauge structural changes in the tissue matrix during EMR. The application of OCT to image the EMR process will add to our understanding of the mechanisms of action involved and potentially facilitate optimization of this process.

Cryopreservation of cartilage has been investigated for decades and is currently an established protocol. However, the reliability and applicability of cartilage cryopreservation for the use in electromechanical reshaping (EMR) has not been studied exclusively. A system to cryopreserve large numbers of tissue specimens provides a steady source of cartilage of similar quality for experimentation at later dates. This will reduce error that may arise from different cartilage stock, and has the potential to maximize efficiency under time constraints. Our study utilizes a unique methodology to cryopreserve septal cartilage for use in EMR studies. Rabbit septal cartilage specimens were harvested and standardized to 20 x 8 x 1 mm, and placed in one of three solutions (normal saline, PBS, 10% DMSO in PBS) for four hours in a cold storage room at 4 degrees Celsius. Then, each cartilage specimen was vacuumed and sealed in an anti-frost plastic bag and stored in a freezer at -80 degrees Celsius for 1 to 3 weeks duration. EMR was performed using 2 and 6 volts for 2 minutes application time. Bend angle measurements of the cryopreserved cartilage specimens were compared to bend angles of fresh cartilage which underwent EMR using the same parameters. Results demonstrate that normal saline, phosphate buffered saline (PBS), and PBS with DMSO were effective in cryopreservation, and indicated no significant differences in bend angle measurements when compared to no cryopreservation. Our methodology to cryopreserve cartilage specimens provides a successful approach for use in conducting large-scale EMR studies.

Electro Mechanical Reshaping (EMR) with platinum needle electrodes has been recently developed to shape cartilage without conventional cut and suture surgery. This study investigates the relationship between the voltage applied, the electrical current measured during EMR, and the resulting shape. Monitoring the electrical current provides information to model the electro-chemistry, which will aid in determining the onset of shape stabilization. Porcine costal grafts, rabbit auricular, and porcine auricular tissue were bent into a 90° angle using a moulage. Platinum needle electrodes were then placed in contact with the cartilage and a constant voltage was applied for a set time. The electrical current was measured during the process and total charge transferred was calculated. The cartilage specimen was then removed from the jig and photographed after one minute in order to determine the resulting bend angle. Results show that a higher current in tissue is produced with increasing applied voltage. Each current trace is unique and is dependent on tissue thickness and inter-electrode distances. Understanding the electrical current process ultimately leads to optimizing EMR and feedback control. Voltage, for example, could be varied in real-time during EMR to produce a constant chemical reaction rate and potentially reduce total tissue dehydration in contact with electrodes. In conclusion, electric current traces provide information about chemical kinetics during EMR that depend on exposure settings, and monitoring these traces is an important step in optimizing the reshaping process.

Grafts obtained from peripheral regions of costal cartilage have an inherent tendency to warp over time. Laser irradiation provides a potential method to control the warping process, thus yielding stable grafts for facial reconstructive surgery. In our current study, we propose a simple and well-fitting model that numerically describes the degree of warping of laser irradiated costal cartilage grafts. Using a Nd:YAG laser (λ=1.32μm) at various exposure settings, grafts harvested from the peripheral regions of porcine costal cartilage were irradiated. The resulting graft geometry was objectively fitted to a curve using a quadratic regression model. The coefficient of determination (R²) demonstrated a very strong fit for all grafts modeled. A quadratic regression is simple to perform and results in a single numerical value that appropriately describes the degree of cartilage warping. Our proposed model is valuable in assessing the effect of laser irradiation on the warping process of costal cartilage.

A prototype device has been created to fuse septal tissue membranes as an alternative to sutures or staples through the controlled application of laser heating and pressure to induce protein denaturation and subsequent tissue fusion, through renaturation and intertwining, across the interface. Lasers have been used to close wounds in controlled laboratory tests over the last 15 years. Many encouraging results have been obtained; however, no commercial delivery systems are currently available. This is due primarily to two factors: requiring an inordinate amount of experience on the part of the operator, and attempting to achieve general applicability for multiple tissue systems. The present device overcomes these barriers as it is tailored for the particular application of septal laser fusion, namely for the coaptation of mucoperichondrial membranes. The important parameters involved in fusing biological tissues are identified. The development of the device followed from computational modeling based on Monte Carlo simulation of photon transport and on engineering firstprinciples. Experiments were designed and analyzed using orthogonal arrays, employing a subset of the relevant parameters, i.e., laser irradiance, dwell time and spot size, for a range of wavelengths. The in vitro fusion experiments employed 1cm by 1cm sections of equine nasal mucosa having a nominal thickness of 1mm.

Introduction: Titanium implants can be regarded as the current gold standard for restoration of sound transmission in the middle ear following destruction of the ossicular chain by chronic inflammation. Many efforts have been made to improve prosthesis design, while less attention had been given to the role of the interface. We present a study on chemical nanocoating on microstructured titanium contact surface with bioactive protein. Materials and Methods: Titanium samples of 5mm diameter and 0,25mm thickness were structured by means of a Ti:Sapphire femtosecond laser operating at 970nm with parallel lines of 5μm depth, 5μm width and 10μm inter-groove distance. In addition, various nanolayers were applied to titanium samples by aminosilanization, to which Star-Polyethylene glycole (Star-PEG) molecules plus biomarkers (e.g. RGD peptide sequence) were linked. Results: Chondrocytes could be cultured on microstructured surfaces without reduced rate of vital / dead cells compared to native surfaces. Chondrocytes also showed contact guidance by growing along ridges particularly on 5μm lines. On nanocoated titanium samples, first results showed a strong effect of Star-PEG suppressing unspecific protein absorption, while RGD peptide sequence did not promote chondrocyte cell growth. Discussion: According to these results, the idea of promoting cell growth on titanium prosthesis contact surfaces compared to non-contact surfaces (e.g. prosthesis shaft) by nanocoating is practicable. However, relative selectivity induced by microstructures for growth of chondrocytes compared to fibrocytes is subject to further evaluation.

Introduction: We performed laser myringoplasty to treat retraction pockets of the tympanic membrane using a CO₂ laser via flexible photonic band-gap fibers manufactured by Omni-Guide. Similar techniques to treat retraction pockets using laser myringoplasty have been described with both the CO₂ and KTP lasers. Objective: To report the efficacy of treating tympanic membrane retraction pockets using laser myringoplasty with a novel hand-held flexible photonic band gap fiber CO₂ laser. Methods: A hand-held flexible photonic band gap fiber CO₂ laser system (Omniguide) was used to treat tympanic membrane retraction pockets. Prior to use of the laser, attempts were made to release membrane adherent to the middle ear space structures. Tympanostomy tubes were placed in all patients. Results: Laser myringoplasty was performed on nineteen patients, for a total of 24 ears. Audiograms were performed on average three weeks after surgery. Air- bone gaps were calculated for pure tones at 0.5, 1, 2 and 3 kHz. The average preoperative ABG for the 25 ears was 13.9dB. The average postoperative ABG was 8.5dB. (P=0.02) The twenty ears with no effusion experienced an ABG closure from 12.1dB to 7.8dB whereas the four ears found to have an effusion had an average ABG closure from 22.7dB to 7.00dB. Conclusion: Laser myringoplasty using the Omni-Guide hand-held flexible fiber CO₂ laser independently produces hearing improvement. The handheld laser allows the surgeon to control the amount of energy delivered to the tympanic membrane and allows the surgeon to work outside of direct line of sight of the microscope resulting in improved accuracy and precision of the procedure. Ears found to have effusions experience the greatest hearing improvement. Patients with membrane adherence not amendable to valsalva may be at greater risk for sub-optimal hearing results.

Biofilm formation has been demonstrated for many mucosal pathogens such as Haemophilus influenzae. The presence of mucosal biofilms with chronic otitis media with effusion (COME) suggests that bacteria do not clear by antibiotics. Aim: To test the effect of photodynamic therapy (PDT) on H. influenzae induced biofilm in vivo. Methods: Gerbils were divided into control (C), HI group, Laser (L), PS, PDT A, and PDT B groups. The C group received no treatment. HI group was injected with 20μl (10⁸CFU/ml) of H. influenzae into the bullae and formation of biofilms in the bullae was obtained by 5 days. For L group, 120 J/cm² (100 mw × 20 min) of 632 nm LD laser was irradiated by a fiber inserted into the bullae 5 days after the H. influenzaeinjection. For PS group, photofrin 40μl (1mg/ml) were injected into the bullae 5 days after the H. influenzae injection. PDT A group received photofrin 1 mg/ml and LD laser 120 J/cm² that were administered into the bullae 5 days after the H. influenzae injection. PDT B group received photofrin 2 mg/ml and laser 150 J/cm² 5 days after the H. influenzae injection. The mucosal tissues in bullae were examined by H/E staining, and SEM. Results: The C group showed normal mucosa of bullae. The HI, L, and PS groups have shown well formed biofilm. Twenty five percent of the PDT A group and 50 % of the PDT B group have shown completely or partially resolved biofilm. Conclusion: The results of this study demonstrated that PDT appears to be effective to treat experimental H. influenzae induced biofilms in vivo. Clinical implication: PDT may be an alternative to antibiotic treatment on otitis media with biofilm formation.

Vocal fold scarring is one of the predominant causes of voice disorders yet lacks a reliable treatment method. The injection of soft biomaterials to improve mechanical compliance of the vocal folds has emerged as a promising treatment. Successful implementation of this method may benefit from improved localization of the injected material. Here, we create sub-surface vocal fold microsurgeries with a goal of eventually creating a plane in dense sub-epithelial scar tissue into which biomaterials can be injected. Specifically, we demonstrate the ablation of sub-epithelial voids in porcine vocal fold tissue within 100 μm below the surface such that a larger void in the active area of vocal fold mucosa (~3 × 10 mm²) can eventually be ablated in about 2 minutes. We use sub-μJ, 776 nm pulses from a compact, commercially available amplified femtosecond laser system operating at a 500 kHz repetition rate. The use of relatively high repetition rates, with a small number of overlapping pulses, is critical to achieving ablation in a very short period of time while still avoiding significant heat deposition. Additionally, we use the same laser for nonlinear optical imaging to provide visual feedback of tissue structure and confirm successful ablation. The ablation parameters, including pulse duration, pulse energy, spot size and scanning speed, are comparable to the specifications in our recently-developed miniaturized femtosecond laser surgery probe, illustrating the feasibility of developing an ultrafast laser surgical laryngoscope. We aim to further develop this clinical tool through demonstration of laryngeal microsurgery using a compact laser system in conjunction with a larynx-specific fiber-based surgery probe.

An optical coherence tomography study for imaging the round window niche and the promontorium tympani Tympanosclerosis may involve the tympanic membrane, the ossicles, and the oval and round window niche, respectively. The surgical treatment of the obliterated oval window niche is most challenging. Beside stapesplasty, vibroplasty coupling the floating mass transducer (FMT) onto the round window niche and into a new, so-called third window is indicated. In the latter situation, drilling a hole into the promontorium is necessary to couple the FMT close to the membranous endosteum. Damage of the membranous inner ear must be avoided. The question was whether OCT is useful to identify the endosteum and to provide microanatomical information of the round window niche. OCT was carried out on human temporal bone preparations, in which a third window was drilled leaving the membranous labyrinth and the fluid-filled inner ear intact and the overhang of the round window niche was removed. An especially equipped operating microscope with integrated OCT prototype (spectral-domain-OCT) was used. The OCT images and 3D reconstructions demonstrate the usefulness of OCT to measure the drilling cavity, to visualize the inner ear structures, and to obtain microanatomical information of the round and oval window niche. These findings may have an impact on stapes surgery, on cochlea implantation, and on vibroplasty coupling the FMT onto the round and third window. OCTguided drilling allows for more precise identification of the intact inner ear.

Advancements in implantable auditory prostheses now demand preservation of residual auditory function following the surgery. Atraumatic cochleostomy formation is essential to this goal. Clinically reported hearing outcomes in human implantation are still quite variable in this regard. The objective of the manuscript is to report on clinical indications and supportive laboratory evidence that the handheld CO₂ laser fiber (BeamPath® OTO-S, OmniGuide® Inc., Cambridge, MA, USA) is an effective tool for creating a soft cochleostomy.

The objective of this study was to develop an automated algorithm which uses fluorescence lifetime imaging microscopy (FLIM) images of human aortic atherosclerotic plaque to provide quantitative and spatial information regarding compositional features related to plaque vulnerability such as collagen degradation, lipid accumulation, and macrophage infiltration. Images were acquired through a flexible fiber imaging bundle with intravascular potential at two wavelength bands optimal to recognizing markers of vulnerability: F₃₇₇: 377/55 nm and F₄₆₀: 460/50 nm (center wavelength/bandwidth). A classification method implementing principal components analysis and linear discriminant analysis to correlate FLIM data sets with histopathology was validated on a training set and then used to classify a validation set of FLIM images. The output of this algorithm was a false-color image with each pixel color coded to represent the chemical composition of the sample. Surface areas occupied by elastin, collagen, and lipid components were then calculated and used to define the vulnerability of each imaged location. Four groups were defined: early lesion, stable, mildly vulnerable and extremely vulnerable. Each imaged location was categorized in one of the groups based on histopathology and classification results; sensitivities (SE) and specificities (SP) were calculated (SE %/SP %): early lesion: 95/96, stable: 71/97, mildly vulnerable: 75/94, and extremely vulnerable: 100/93. The capability of this algorithm to use FLIM images to quickly determine the chemical composition of atherosclerotic plaque, particularly related to vulnerability, further enhances the potential of this system for implementation as an intravascular diagnostic modality.

We have examined the possibility of non-thermal ablation technology for arrhythmia therapy with photosensitization reaction, in which photochemically generated singlet molecular oxygen may induce myocardial electrical conduction block. In the most popular energy source for arrhythmia catheter ablation; radiofrequency current, the thermal tissue injury causes electrophysiological disruption resulting in electrical isolation of ectopic beats. The temperature-mediated tissue disruption is difficult to control because the tissue temperature is determined by the heating and thermal conduction process, so that severe complications due to excessive heat generation have been the problem in this ablation. We demonstrated the electrical conduction block of surgically exposed porcine heart tissue in vivo with photosensitization reaction. The acute myocardial electrical conduction block was examined by the stimulation and propagation set-up consisting of a stimulation electrode and two bipolar measurement electrodes. Fifteen to thirty minutes after the injection of 5-10 mg/kg water-soluble chlorine photosensitizer, Talaporfin sodium (NPe6, LS11), the laser light at the wavelength of 663 nm with the total energy density of 50-200 J/cm² was irradiated several times with 3- 7 mm in spot-size to make electrical block line in myocardial tissue across the conduction pathway between the bipolar measurement electrodes. The propagation delay time of the potential waveform increased with increasing the irradiated line length. The observation of Azan-stained specimens in the irradiated area two weeks after the procedure showed that the normal tissue was replaced to the scar tissue, which might become to be permanent tissue insulation. These results demonstrated the possibility of non-thermal electrical conduction block for arrhythmia therapy by the photosensitization reaction.

Introduction: Quantitative optical coherence tomography (OCT) analysis is about to be relevant for the correct assessment of incomplete stent apposition which can result to late stent thrombosis. Nevertheless, the pseudo stent struts (PS), which show the strong signal same as the true strut, are sometimes seen at odd positions in the lumen and locate at the same distance from the image wire as a true strut. PS may be produced by the distorted beam and interfere accurate analysis. Our aim was to investigate the incidence of PS. Methods: We created a simple phantom model (2.5 mm-coronary artery stent apposed in 2.5-mm silicon tube). OCT pullback images at 1 mm/sec were obtained with an eccentric imagewire position for 5 times with different 5 pieces of imagewire. The strut location was recognized by the strong signal. Of these, PS was defined as: an irregularity of the alignment of strut locations; the difference in the strut-wire distance with one of the adjacent struts is within 20 micron. Strut contour with and without PS were delineated by semi-automated dedicated software with cubic spline interpolation, and symmetry index (= Min/Max diameter) was calculated. Results: In the phantom with Cypher stent and Tsunami stent, a pseudo strut reflection was observed in 71 of 7112 (1%) frames and 43 of 10302 (0.4%) frames, respectively. The PS incidence was the highest at fifth pull-back images. The symmetry index was significantly higher in strut contours without pseudo strut (0.95±0.02 versus 0.83±0.07, p<0.001). Conclusion: PS may mislead to the wrong data of clinical OCT analysis, in spite of the infrequency. When one starts to use the new image modality, one should be careful if there is some artifact which can affect on the data of the clinical investigations.

We previously presented a method to fabricate phantoms of normal coronary arteries. This method allows the deposition of multiple layers on a tubular structure, each layer replicating optical and mechanical properties of coronary artery layers. We now present an improved method to produce phantoms of arteries affected by atherosclerosis. The method now includes techniques to introduce structures that mimics the OCT signature of a calcification and of a lipid pool.

We report clinical study results of three-dimensional (3D) in vivo imaging of human coronary arteries using frequency domain optical coherence tomography (FD-OCT). At the time of this report, over 2000 patients in over 10 countries have been imaged using FD-OCT systems and disposable fiberoptic catheters developed by LightLab Imaging Inc. The first commercial versions of the systems were introduced in Europe in May 2009. The system operates at 50,000 axial lines/s, performing a 50 mm spiral pullback in 2.5 seconds with a rotational frame rate of 100 Hz. The commercial system employs a proprietary micro-cavity swept laser, allowing imaging of vessel diameters up to 10 mm. Data compiled from early studies indicate that FD-OCT is being used for post-intervention imaging of deployed coronary stents in over 40% of cases. High-resolution 3D imaging of stent geometry immediately following deployment enables detection of stent malapposition, which can increase the risk of thrombosis. Longer term follow-up imaging of stented vessels can detect thrombus formation, which can be treated pharmacologically, and excessive neointimal growth, which may require angioplasty or re-stenting. FD-OCT is also being used for pre-intervention imaging of stenotic lesions in about 60% of cases. Here FD-OCT is used to measure the minimum lumen area and to identify calcified deposits, side branches, or other vascular structures that could interfere with the stenting procedure. Overall, FD-OCT continues to be adopted at an increasing rate and has provided interventional cardiologists with a powerful tool for pre- and postintervention assessment of the coronary arteries.

The purpose of this study is to examine the peripheral tissue perfusion rates by time-series analysis of distribution and elimination kinetics of a clinically proven NIR fluorescence probe, indocyanine green (ICG). We developed a new method, dynamic ICG perfusion imaging technique to evaluate peripheral tissue perfusion that employs planar imaging with a CCD digital imaging system and time-series analysis of the spatiotemporal dynamics (150s) of intravenously injected ICG by using nonlinear regression and differential evolution methods. Six parameters (α, β, s, d, m; parameters which depend on an arterial input function (AIF) into a lower extremity and p; perfusion rates in the lower extremity) were estimated by the nonlinear regression modeling method. We have confirmed the validity of our new method by applying the method to a normal control and a patient with peripheral arterial occlusion disease (PAOD). PAOD patient showed a unique AIF curve pattern, which was caused by collateral blood flow bypassing the occluded major artery. The lower extremity tissue perfusion rate of the PAOD patient was estimated as about 35% of those of normal values. These results indicate that ICG perfusion imaging method is sensitive enough to diagnose PAOD and capable of diagnosing functional arterial diseases.

Cardiovascular disease is among the leading causes of death in the United States. Specifically, atherosclerosis is an increasingly devastating contributor to the tally and has been found to be a byproduct of arterial permeability irregularities in regards to lipoprotein penetration. To further explore arterial physiology and molecular transport, the imaging technique of Optical Coherence Tomography (OCT) was employed. With OCT, the permeation of glucose (MW = 180 Da), low density lipoprotein (LDL; MW = 2.1 × 10⁶ Da), and high density lipoprotein (HDL; MW = 2.5 × 10⁵ Da) in human carotid tissue was studied to determine the effect of different molecular characteristics on permeation in atherosclerotic tissues. The permeability rates calculated from the diffusion of the molecular agents into the abnormal carotid tissue samples is compared to those of normal, healthy tissue. The results show that in the abnormal tissue, the permeation of agents correlate to the size constraints. The larger molecules of LDL diffuse the slowest, while the smallest molecules of glucose diffuse the fastest. However, in normal tissue, LDL permeates at a faster rate than the other two agents, implying the existence of a transport mechanism that facilitates the passage of LDL molecules. These results highlight the capability of OCT as a sensitive and specific imaging technique as well as provide significant information to the understanding of atherosclerosis and its effect on tissue properties.

Optical methods have been widely used in basic neuroscience research to study the cerebral blood flow dynamics in order to overcome the low spatial resolution associated with magnetic resonance imaging and positron emission tomography. Although laser Doppler imaging and laser speckle imaging can map out en face cortical hemodynamics and columns, depth resolution is not available. Two-photon microscopy has been used for mapping cortical activity. However, flow measurement requires fluorescent dye injection, which can be problematic. The noninvasive and high resolution tomographic capabilities of optical coherence tomography make it a promising technique for mapping depth resolved cortical blood flow. Here, we present a functional Doppler optical coherence tomography (OCT) imaging modality for quantitative evaluation of cortical blood flow in a mouse model. Fast, repeated, Doppler OCT scans across a vessel of interest were performed to record flow dynamic information with a high temporal resolution of the cardiac cycles. Spectral Doppler analysis of continuous Doppler images demonstrates how the velocity components and longitudinally projected flow-volume-rate change over time, thereby providing complementary temporal flow information to the spatially distributed flow information of Doppler OCT. The proposed functional Doppler OCT imaging modality can be used to diagnose vessel stenosis/blockage or monitor blood flow changes due to pharmacological agents/neuronal activities. Non-invasive in-vivo mice experiments were performed to verify the capabilities of function Doppler OCT.

Measuring oxygen delivery in brain tissue is important for identifying the pathophysiological changes associated with brain injury and various diseases such as cancer, stroke, and Alzheimer's disease. We have developed a multi-modal imaging system for minimally invasive measurement of cerebral oxygenation and blood flow in small animals with high spatial resolution. The system allows for simultaneous measurement of blood flow using Fourier-domain optical coherence tomography, and oxygen partial pressure (pO₂) using either confocal or multiphoton phosphorescence lifetime imaging with exogenous porphyrin-based dyes sensitive to dissolved oxygen. Here we present the changes in pO₂ and blood flow in superficial cortical vessels of Sprague Dawley rats in response to conditions such as hypoxia, hyperoxia, and functional stimulation. pO₂ measurements display considerable heterogeneity over distances that cannot be resolved with more widely used oxygen-monitoring techniques such as BOLD-fMRI. Large increases in blood flow are observed in response to functional stimulation and hypoxia. Our system allows for quantification of cerebral metabolic rate of oxygen (CMRO2) with high spatial resolution, providing a better understanding of metabolic dynamics during functional stimulation and under various neuropathologies. Ultimately, better insight into the underlying mechanisms of neuropathologies will facilitate the development of improved therapeutic strategies to minimize damage to brain tissue.

We use the holographic method to project an arbitrary array of diffraction-limited focal spots suitable for multi-site twophoton excitation. The spot array can be projected arbitrarily within a three-dimensional (3D) volume, while the fourth dimension in time is attributed to high temporal resolution via high-speed non-iterative calculation of the hologram using a video graphics accelerator board. We show that the spots have sufficient energy and spatiotemporal photon density for localized two-photon excitation at individual spots in the array. The significance of this work points to 3D microscopy, non-linear micro-fabrication, volume holographic optical storage and biomedical instrumentation. In neuroscience, timecritical release of neurotransmitters at multiple sites within complex dendritic trees of neurons can lead to insights on the mechanisms of information processing in the brain.

In this report, we propose an efficient scheme to use High Definition Television (HDTV) in a console or notebook format as a computer terminal in addition to their role as TV display unit. In the proposed scheme, we assume that the main computer is situated at a remote location. The computer raster in the remote server is compressed using an HD E- >Mpeg2 encoder and transmitted to the terminal at home. The built-in E->Mpeg2 decoder in the terminal decompresses the compressed bit stream, and displays the raster. The terminal will be fitted with a mouse and keyboard, through which the interaction with the remote computer server can be performed via a communications back channel. The terminal in a notebook format can thus be used as a high resolution computer and multimedia device. We will consider developments such as the required HD enhanced Mpeg2 resolution (E->Mpeg2) and its medical ramifications due to improvements on compressed image quality with 2D to 3D conversion (Mpeg3) and using the compressed Discrete Cosine Transform coefficients in the reality compression of vision and control of medical robotic surgeons.

Introduction: Dynamic Contrast-Enhanced-Magnetic Resonance Imaging (DCE-MRI) may provide a means of tracking the outcome of Pc 4-sensitized photodynamic therapy (PDT) in deeply placed lesions (e.g., brain tumors). We previously determined that 150 μL of gadolinium (Gd-DTPA) produces optimal enhancement of U87-derived intracerebral tumors in an athymic nude rat glioma model. We wish to determine how consistently DCE-MRI enhancement will detect an increase in Gd-enhancement of these tumors following Pc 4-PDT. Methods: We injected 2.5 x 10⁵ U87 cells into the brains of 6 athymic nude rats. After 7-8 days pre-Pc 4 PDT peri-tumor DCE-MRI images were acquired on a 7.0T microMRI scanner before and after administration of 150 μL Gd. DCE-MRI scans were repeated on Days 11, 12, and 13 following Pc 4-PDT (Day 8 or 9). Results: Useful DCE-MRI data were obtained for these animals before and after Pc 4- PDT. In the pre-Pc 4-PDT DCE-MRI scans an average normalized peak Gd enhancement was observed in tumor tissue that was 1.297 times greater than baseline (0.035 Standard Error [SE]). The average normalized peak Gd enhancement in the tumor tissue in the scan following PDT (Day 11) was 1.537 times greater than baseline (0.036 SE), a statistically significant increase in enhancement (p = 0.00584) over the pre-PDT level. Discussion: A 150 μL Gd dose appears to provide an unambiguous increase in signal indicating Pc 4-PDT-induced necrosis of the U87-derived tumor. Our DCEMRI protocol may allow the development of a clinically robust, unambiguous, non-invasive technique for the assessment of PDT outcome.

The complete resection of the brain tumour is crucial to the patient life quality and prognosis. An autofluorescence probe aiming at helping the surgeon to improve the completeness of the removal is being developed. Autofluorescence spectroscopy is a promising approach to define whether the tissue is cancerous or not. First ex vivo measurements have been realised on an animal model. After tumorous cell injection in rat brain, autofluorescence intensity is revealed from the extracted brain. These autofluorescence data are compared to results from a histological analysis of same brains. First indicators are identified that may have the ability to differentiate tumorous and healthy tissues.

Introduction: Failure to eradicate infiltrating glioma cells using conventional treatment regimens results in tumor recurrence and is responsible for the dismal prognosis of patients with glioblastoma multiforme (GBM). This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB) and the blood brain tumor barrier (BBTB) which prevents the delivery of most anti-cancer agents. We have evaluated the ability of monocytes/macrophages (Mo/Ma) to cross the BBB in rats. This will permit access of anti-cancer agents such as nanoparticles to effectively target the infiltrating tumor cells, and potentially improve the treatment effectiveness for malignant gliomas. Materials and Methods: The infiltration of Mo/Ma into brain tumor spheroids in vitro was determined using fluorescent stained Mo/Ma. Tumors were also established in the brains of inbred rats and ALA-PDT was given 18 days following tumor induction. The degredation of the BBTB and quantification of the number of infiltrating Mo/Ma was examined on histological sections from removed brains. Results & Conclusion: PDT was highly effective in locally opening the BBTB and inducing macrophage migration into the irradiated portions of brain tumors.

In vitro studies were initiated to determine the suitability of murine and rat macrophages as delivery vehicles for gold nanoshells in the treatment of gliomas. Visualization of macrophage accumulation in and around gliomas may be accomplished using magnetic resonance imaging (MRI) and superparamagnetic iron oxide nanoparticles (SPIO). The optimal loading of both murine and rat macrophages with SPIO was determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES). Higher concentrations of SPIO were observed in rat macrophages and the optimal concentration in these cell lines was around 300 μg/ml. Higher concentrations resulted in significant cell toxicity. SPIO were visualized in fixed rat brains subjected to high field MRI using T₂*-weighted gradient echo pulse sequences. Macrophages were found to be very sensitive to near infra-red (NIR) laser irradiation.

Specific absorption rate (SAR) heating using radiofrequency (RF) waves is affected by the RF frequency and amplitude, and the conductivity of the tissue. Recently, conductive nanoparticles were demonstrated to induce hyperthermia in vitro and in vivo upon irradiation with an external 13.56 MHz RF field. The addition of conductive nanoparticles was assumed to increase the tissue conductivity and SAR. However, no quantitative studies have been performed that characterize the conductivities of biocompatible colloids or tissues containing nanoparticles, and relate the conductivity to SAR. The complex permittivities were measured for colloids containing single-wall carbon nanotubes (SWCNTs) in normal saline with 0.32% w/v Pluronic F108 nonionic surfactant. The carbon concentrations of the colloids ranged from 0 to 88 mM. The permittivities were measured using a dielectric probe and RF network analyzer for RF frequencies from 200 MHz to 3 GHz. The nonionic surfactant was added to the colloids to minimize flocculation of the nanotubes during the RF heating experiments. The results were compared with prior measurements of colloids containing 0.02% Pluronic F108. The dielectric and conductivity of the 0.02% Pluronic colloids rose linearly with carbon concentration but the 0.32% Pluronic colloids varied from linearity. Based on the permittivity results, selected colloid samples were placed inside a Bruker 7T/20 magnetic resonance (MR) imaging (MRI) system and irradiated at 300 MHz using a high duty cycle RF pulse sequence. The temperature changes were measured directly using fiber-optic thermometers and indirectly using MR thermometry and spectroscopy. Temperature changes were consistent with the colloid conductivities.

Autism is a socio-communication brain development disorder. It is marked by degeneration in the ability to respond to joint attention skill task, from as early as 12 to 18 months of age. This trait is used to distinguish autistic from nonautistic populations. In this study, diffuse optical imaging is being used to study brain connectivity for the first time in response to joint attention experience in normal adults. The prefrontal region of the brain was non-invasively imaged using a frequency-domain based optical imager. The imaging studies were performed on 11 normal right-handed adults and optical measurements were acquired in response to joint-attention based video clips. While the intensity-based optical data provides information about the hemodynamic response of the underlying neural process, the time-dependent phase-based optical data has the potential to explicate the directional information on the activation of the brain. Thus brain connectivity studies are performed by computing covariance/correlations between spatial units using this frequency-domain based optical measurements. The preliminary results indicate that the extent of synchrony and directional variation in the pattern of activation varies in the left and right frontal cortex. The results have significant implication for research in neural pathways associated with autism that can be mapped using diffuse optical imaging tools in the future.

High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural mechanisms, and for diagnosis of retinal disease and evaluation of treatment. Fast intrinsic optical signals (IOSs), which have the time courses comparable to retinal electrophysiological responses, hold the promise for high resolution imaging of retinal neural activities. However, application of fast IOS imaging has been hindered by contamination of slow, high magnitude, optical responses associated with transient hemodynamic and metabolic changes. We recently demonstrated the feasibility of separating fast IOSs from slow optical responses by combined dynamic differential imaging and high frequency flicker stimulation.

As part of our ongoing assessment of bone tissue composition and structure, we report the first experimental protocols of a prospective study to investigate the potential of using Raman spectroscopy to diagnose and predict skeletal fragility in postmenopausal osteoporosis patients. This multi-center study will assess several potential spectroscopic and X-ray based diagnostic techniques. One hundred and twenty participants will be enrolled in this five year study and the investigators will be blinded to information concerning patient history and status. Iliac crest bone biopsy specimens are provided with no identifying information except a patient study number. Our team will use micro-computed tomography (micro-CT) to identify regions of interest in both cortical and cancellous bone from specimens delivered to us. Raman mapping will be performed using a line-focused 785 nm laser in order to obtain local and averaged values on several spectroscopic metrics of bone quality. These metrics include carbonate/phosphate and phosphate/matrix ratios. Results from an initial set of biopsies will be presented. Protocols for obtaining measurements are discussed, with emphasis on the challenges presented by the use of fixed and polymer embedded specimens. These protocols are illustrated will data from a biopsy specimen.

In this study a whole field, non-contact optical method, Stereo Digital Image Correlation (SDIC), was used to determine the strain distribution and mechanical properties of fresh bone in Phosphate Buffered Saline (PBS) solution. Knowing the whole-surface strain distribution of bone is useful for understanding the effects of normal physiological loading, disease, drugs and aging. In addition, knowing the mechanical properties of bone will aid in the design of new biomaterials. Although there currently are methods for measuring the mechanical properties of bone, these methods have some limitations. Many miss areas of strain concentration, especially because of the inhomogeneous nature of bone. SDIC overcomes these limitations by being able to precisely measure whole-surface 3D contour and strain of samples in solution over a wide range of deformations. In this study, SDIC was used to measure the axial strain of fresh chicken tibia. A setup which has the capability to apply force axially was designed. This paper describes the methodology of SDIC for measuring fresh bone in a PBS solution. The effect of drying time on strain distribution was investigated. The usefulness of the SDIC system is demonstrated by examples of deformation and strain measurements for different chicken tibia in PBS solution.

Polarized Raman spectroscopy is widely used in the study of molecular composition and orientation in synthetic and natural polymer systems. Here, we describe the use of Raman spectroscopy to extract quantitative orientation information from bone tissue. Bone tissue poses special challenges to the use of polarized Raman spectroscopy for measurement of orientation distribution functions because the tissue is turbid and birefringent. Multiple scattering in turbid media depolarizes light and is potentially a source of error. Using a Raman microprobe, we show that repeating the measurements with a series of objectives of differing numerical apertures can be used to assess the contributions of sample turbidity and depth of field to the calculated orientation distribution functions. With this test, an optic can be chosen to minimize the systematic errors introduced by multiple scattering events. With adequate knowledge of the optical properties of these bone tissues, we can determine if elastic light scattering affects the polarized Raman measurements.

Bone is a highly specialized connective tissue comprised of cross-linked collagen fibers interspersed with apatitic mineral crystallites of various sizes, shapes, orientation, and composition. However, the nucleation, growth, and propagation of mineral crystallite into the collagenous matrix are not clearly understood. By using a research grade inverted microscope fitted with a line-shaped 830 nm laser and spectrograph, we show that the Raman scatter from mineralizing cell cultures in an incubation chamber can be collected and monitored directly through the bottom of the well-plates over a period of 24 hours. In our studies, murine-derived MC3T3 cells were incubated at 37°C in the presence of 5% CO₂ and 85% humidity. Results show a gradual shift in the phosphate ν₁ apatitic band center (955-957 cm^-1) during the first hour of mineralization. The phosphate ν₁ apatitic band width also narrowed during this time. To quantify the amount of crystal growth in vivo, we used a calibration curve derived from X-ray powder diffraction and Raman studies performed on a series of synthetic carbonated apatites and deproteinated mouse femoral specimens. Mineralization in neonatal mouse calvarial culture was observed along the lambdoid suture. Deposition proceeded in a stepwise fashion over the course of ~30 h.

This paper presents a visual servoing application with and without motion compensation and a fixed visual servoing configuration for CO2 laser osteotomy. A multi camera system from ART is used to track the position of the robot and a skull via marker spheres that are attached to both. A CT scan from the skull is performed and segmented to acquire a 3D model. Inside the model the position for the robot for the laser ablation is planned. The accuracy of the lightweight robot is increased with the additional supervision of an optical tracking system. Accuracy improvement was measured with an FARO measurement arm. A visual servoing control schema is presented. The demonstrator shows a working visual servoing application for laser osteotomy. To improve the error resulting mainly from the delay to acquire the data from the devices a motion compensation algorithm is introduced based on iterative learning and a normalized Least Mean Square (nLMS) filter. The results during the simulation and the experimental setup are shown. The system was then evaluated with the CO2 laser system OsteoLas X10 from Caesar - Bonn, Germany. Different cuts are performed with the robot and the CO2 laser system. For the breathing motion a robotic breathing simulator is used. The reached accuracy and the cutting results on bone are shown.

This work studies the possible variations of the properties of mineral and organic bone components with regard to the anatomical position and the patient's age. Autopsies of healthy human iliac crest have been analyzed within a wide range of ages (26-88), measuring different anatomical positions in trabecular bone by means of FT-IR spectroscopy. The study was focused on the analysis of ν1, ν3 phosphate, ν2 carbonate amida I and amida II bands. From the resulting spectra the cristallinity/maturity index, the collagen cross-links ratio and the carbonate/phosphate ratio were calculated. All of them provide information of bone mineral and collagen maturity. The results show a trend in the spatial distribution of mineral and collagen maturity in most of the samples. The most mature mineral and collagen of the bone were found to be located in the trabecular center, while the youngest were situated in the peripheral regions. However, this behavior has exceptions that seem to be related with the patient's age.

Although the mechanism of laser stimulation effect in bone has not completely understood, laser stimulation is recommended in the treatment of osteoporosis due to positive treatment efficacy. In this study, a minimal invasive laser needle system (MILNS) was developed using a fine hollow needle in order to stimulate directly bone site by guiding an optical fiber. In order to evaluate the MILNS as a treatment method, in-vivo animal experiment study was performed using osteopenic mice. Twelve virginal ICR mice were employed and divided two groups: SHAM-group and LASERgroup. SHARM-group was stimulated by only fine hollow needle and LASER-group by fine hollow needle combined with laser stimulation. All mice were served in-vivo micro-CT images before and after treatment. Three dimensional (3D) structural parameters and vBMD (volume bone mineral density, g/cm3) in the trabecular bone were measured. After 2 weeks of stimulation, the vBMD, BV/TV, Tb.Th and Tb.N in LASER-group were significantly higher than those in SHAM-group (p<0.05). Potentially, this study suggested that the MILNS might prevent the bone loss and maintains the bone mineral density of osteopenic mice.

Raman spectroscopic measurement of bone composition has shown promise as a medical diagnostic by measuring the molecular composition of the bone mineral and matrix. We previously demonstrated proof-of-principle transcutaneous Raman spectroscopy bone measurements in human cadavers. In this paper, we discuss further optimization of the instrumental configuration for efficient collection of bone signal using contact fiber-optic probe designs. To optimize collection of Raman signal through overlaying soft tissue, novel geometrically-accurate tissue phantoms were prepared. MRI and CT images of the human cadaveric specimens were used to create solid tissue phantoms with accurate geometric dimensions. In these tissue phantoms, optical properties can be varied systematically. Raman spectra of the prepared tissue phantoms were used to optimize the positions of the fibers in the fiber optic system, and the laser illumination sequence in the measurements. Three fiber optic probes were developed and tested with both novel tissue phantoms and human cadaveric specimens. The contact fiber optic probes were developed for arthroscopic measurements of joints, for transcutaneous measurements of bone in situ, and for contact measurements of exposed bone. By coupling the fiber optic probe to an imaging spectrograph, spectra were collected simultaneously at many positions on the tissue. Furthermore, spectra were collected with several different excitation laser patterns to enhance the effective spatial resolution of the measurements. Finally, a series of improvements were made in the data preprocessing to improve the recovered spectral signal. Together, these modifications improve signal-to-noise and spatial resolution.

The development of photothermal techniques to detect thermal waves in biological tissue has occurred with a concomitant advancement in the extraction of material thermophysical properties and knowledge regarding the internal structure of a medium. Human molars (n=37) were subjected to demineralization in acid gel (pH 4.5, 10 days), followed by incubation in different fluoride-containing remineralization solutions. PTR-LUM frequency scans (1 Hz - 1 kHz) were performed prior to and during demineralization and remineralization treatments. Transverse Micro-Radiography (TMR) analysis followed at treatment conclusion. A coupled diffuse-photon-density-wave and thermal-wave theoretical model was used to quantitatively evaluate changes in thermal and optical properties of sound, demineralized and remineralized enamel. Amplitude increase and phase lag decrease in demineralized samples were consistent with higher scatter of the diffuse-photon density field and thermal wave confinement to near-surface regions. A remineralized sample illustrates a complex interplay between surface and subsurface processes, confining the thermal-wave centroid toward the dominating layer. PTR-LUM sensitivity to changes in tooth mineralization coupled with optical and thermal property extraction illustrates the technique's potential for non-destructive evaluation of multi-layered turbid media.

This paper presents a pilot clinical study in detecting osteoarthritis (OA) in the hand using threedimensional (3-D) photoacoustic tomography (PAT). Distal interphalangeal (DIP) finger joints from OA patients and healthy volunteers were imaged with our 3-D PAT system in a spherical scanning configuration. Absorption coefficient images of the joint tissue were obtained using our finite element based photoacoustic image reconstruction algorithm coupled with the photon diffusion equation. The recovered quantitative photoacoustic images revealed significant differences in the absorption coefficient of the joint cavity (cartilage and synovial fluid) between the OA and healthy joints. Quantitative analysis of the joints also indicated an apparent difference in the recovered joint spacing between OA and healthy subjects, which is in agreement with the clinical observations. This study suggests that 3D PAT has the potential to become a useful tool for diagnosis of osteoarthritis.

An emerging clinical treatment option for articular cartilage injury includes bone marrow stimulation techniques, such as microfracture, which has grown increasingly popular among athletes. During the microfracture procedure, the surgeon penetrates the subchondral bone with an awl and creates "microholes" deep enough to ensure bleeding from the bone marrow. This procedure triggers a spontaneous repair response that results in the formation of fibrocartilaginous repair tissue. This preliminary study aimed to evaluate the potential use of femtosecond lasers and Erbium:YAG lasers as alternatives to microfracture surgery of the knee by assessing the effects of ablation on bovine femoral condyles. Bovine femoral condyles were obtained and 8mm cube blocks were extracted. The specimen were ablated with various laser dosimetry parameters and observed using a high power dissecting microscope to examine the effects of the lasers. Further imaging with conventional histology (hematoxylin and eosin staining) was done to provide more accurate information. Preliminary results show some carbonization but demonstrate little thermal damage to surrounding tissues. The femtosecond laser offers a more precise and efficient ablation than the Erbium:YAG laser, but both are demonstrated to be possible alternatives to the surgical-skill dependent microfracture procedure.

In our previous studies, near-infrared (NIR) diffuse optical tomography (DOT) had been successfully applied to imaging osteoarthritis (OA) in the finger joints where significant difference in optical properties of the joint tissues was evident between healthy and OA finger joints. Here we report for the first time that large joints such as the knee can also be optically imaged especially when DOT is combined with x-ray tomosynthesis where the 3D image of the bones from x-ray is incorporated into the DOT reconstruction as spatial a priori structural information. This study demonstrates that NIR light can image large joints such as the knee in addition to finger joints, which will drastically broaden the clinical utility of our x-ray guided DOT technique for OA diagnosis.

Biological hard tissues, such as those found in bone and teeth, are complex tissues that build a strong mineral structure over an organic matrix framework. The laser-matter interaction for bone hard tissues holds great interest to laser surgery and laser dentistry; the use of short/ultrashort pulses, in particular, shows interesting behaviors not seen in continuous wave lasers. High laser energy densities in ultrashort pulses can be focused on a small irradiated surface (spot diameter is 10-50 μm) leading to rapid temperature rise and thermal ablation of the bone tissue. Ultrashort pulses, specifically those in the picosecond and femtosecond ranges, impose several challenges in modeling bone tissue response. In the present paper we perform time-dependent thermal simulations of short and ultrashort pulse laser-bone interactions in singlepulse and multipulse (set of ultrashort pulses) modes of laser heating. A comparative analysis for both radiation modes is discussed for laser heating of different types of the solid bone on the nanosecond, picosecond and femtosecond time scales. It is shown that ultrashort laser pulses with high energy densities can ablate bone tissue without heating tissues bordering the ablation creator. This reaction is particularly desirable as heat accumulation and thermal damage are the main factors affecting tissue regrowth rates, and thus patient recovery times.

Prostate cancer is the most common primary tumor in men, with a high propensity to metastasize to bone. Bone metastases in prostate cancer are associated with active pathologic bone remodeling, leading to increased mortality and morbidity. Detailed characterization of bone metastases is important in the management of prostate cancer. Raman spectroscopy was applied in this study to investigate the structure and composition of metastatic bone in prostate cancer with the ultimate goal of identifying spectral features that are related to the alterations in bone quality as the bone metastases develop. Osteoblastic-, osteolytic- and tumor-absent bone specimens from prostate cancer patients were investigated using bench-top Raman microspectroscopy. Raman derived measurements of collagen mineralization, mineral crystallinity, and carbonate substitution were calculated. The osteolytic lesions demonstrated significantly lower collagen mineralization, determined by phosphate ν1/proline, and higher carbonate substitution than normal and osteoblastic bones. Mineral crystallinity was significantly lower in both blastic and lytic specimens. In addition, a significant increase in the ratio of hydroxyproine: proline was observed in the osteoblastic specimen, indicating an increase in the content of hydroxyproline at the blastic lesions. This study demonstrate that Raman spectroscopy shows promise in determining alterations in osteoblastic and osteolytic bone metastases as well as assessing the response of metastatic bone to therapies.

We have developed a spatial light modulator (SLM) based microscope that uses diffraction to shape the incoming two-photon laser source to any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision at high frame rates. Additionally, we have combined this microscope with a new class of two photon active neuromodulators with Ruthenium BiPyridine (RuBi) based cages that offer great flexibility for neuronal control.

Regional anesthetic blocks are performed on patients who will undergo surgery of the hand. In this study, thermal and oxygenation imaging techniques were applied to observe the region affected by the peripheral block as a fast objective, non-contact, method compared to the standard pinpricks or cold sensation tests. The temperature images were acquired with an IR thermal camera (FLIR ThermoCam SC640). The data were recorded and analyzed with the ThermaCam^TM Researcher software. Images at selected wavelengths were obtained with a CCD camera combined with a Liquid Crystal Tunable Filter (420-730 nm). The concentration changes of oxygenated and deoxygenated hemoglobin in the dermis of the skin were calculated using the modified Lambert Beer equation. In 10 patients an anesthetic block was placed by administering 20-30 ml Ropivacaine 7,5 mg/ml around the plexus brachialis. The anesthetic block of the axillary, ulnar, median and radial nerve causes dilatation of the blood vessels inducing an increase of blood flow and, consequently, an increase of the skin temperature and skin oxygenation in the lower arm. Both imaging methods showed distinct oxygenation and temperature differences at the surface of the skin of the hand with a good correlation with the areas with the nerve blocks. For oxygenation imaging a CCD camera with LED light source of selected wavelengths might be a relative inexpensive method to observe the effectiveness of regional blocks.

The cavernous nerves (CN) course along the prostate surface and are responsible for erectile function. Improved identification and preservation of the CN's is critical to maintaining sexual potency after prostate cancer surgery. Noncontact optical nerve stimulation (ONS) of the CN's was recently demonstrated in a rat model, in vivo, as a potential alternative to electrical nerve stimulation (ENS) for identification of the CN's during prostate surgery. However, the therapeutic window for ONS is narrow, so optimal design of the fiber optic delivery system is critical for safe, reproducible stimulation. This study describes modeling, assembly, and testing of an ONS probe for delivering a small, collimated, flat-top laser beam for uniform CN stimulation. A direct comparison of the magnitude and response time of the intracavernosal pressure (ICP) for both Gaussian and flat-top spatial beam profiles was performed. Thulium fiber laser radiation (λ=1870 nm) was delivered through a 200-μm fiber, with distal fiber tip chemically etched to convert a Gaussian to flat-top beam profile. The laser beam was collimated to a 1-mm-diameter spot using an aspheric lens. Computer simulations of light propagation were used to optimize the probe design. The 10-Fr (3.4-mm-OD) laparoscopic probe provided a constant radiant exposure at the CN surface. The probe was tested in four rats, in vivo. ONS of the CN's was performed with a 1-mm-diameter spot, 5-ms pulse duration, and pulse rate of 20 Hz for a duration of 15-30 s. The flat-top laser beam profile consistently produced a faster and higher ICP response at a lower radiant exposure than the Gaussian beam profile due, in part, to easier alignment of the more uniform beam with nerve. The threshold for ONS was approximately 0.14 J/cm², corresponding to a temperature increase of 6-8°C at the CN surface after a stimulation time of 15 s. With further development, ONS may be used as a diagnostic tool for identification of CN's during prostate cancer surgery.

The precise control of neural activity afforded by the use of light sensitive ion channels such as Channel Rhodopsin (ChR2) offers neuroscientists the means to devise new experiments. In this paper we present the Optogenetic Neural Stimulation (ONS) platform which enables complex in-vitro or ex-vivo investigation of neural activity. The platform is based on micro-meter sized Light Emitting Diodes (LEDs) integrated onto a single Gallium Nitrite chip. Mounted onto a microscope system, this system can be used to carry out experiments on networks of cells, or on sub-cellular regions of a neuron with millisecond timing and micrometer resolution.

In the adult nervous system, different population of neurons corresponds to different regenerative behavior. Although previous works show that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury¹, we have hitherto no details about the real dynamics of fiber regeneration. We coupled two photon imaging to laser-induced lesions to perform in vivo multiphoton nanosurgery in the CNS of living mice expressing fluorescent proteins to investigate the reparative properties of Climbing Fibers (CFs) in the adult CNS, following the time evolution of this plastic process in vivo. Here we show that a regenerative event may take place in a murine model in the days that follow a sub-micrometric lesion on the distal portion of the climbing fiber. Furthermore this unique model could allow, through manipulation of the viral vector, to explore in detail the biochemical mechanisms underlying the reparative process. The great potential of long-term two photon imaging, coupled to genetic manipulation, opens great opportunities to further investigate the dynamic properties of neurons and their rearrangement following an injury.