We have demonstrated a new method for imaging melanin with two-color excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes place in the medium. We can easily measure 10^-6 absorption changes caused by either instantaneous two-photon absorption or relatively long lived excited state absorption with a RF lock-in amplifier. Eumelanin and pheomelanin exhibit similar excited state dynamics. However, their difference in excited state absorption and ground state absorption leads to change in the phase of the transient absorption signal. Scanning microscopic imaging is performed with B16 cells, melanoma tissue to demonstrate the 3D high resolution imaging capability. Different melanosome samples are also imaged to illustrate the differences between eumelanin and pheomelanin signals. These differences could enable us to image their respective distribution in tissue samples and provide us with valuable information in diagnosing malignant transformation of melanocytes.

Atopic dermatitis (AD) is now very common in people who live in cities, especially for babies and children. Since the cause of AD is still not completely understood and each person may have his own mixed symptoms that can change over time, diagnosis of AD can not be done precisely. Unlike some skin diseases, physical biopsy is rarely used in diagnosing AD on account of its low urgency. Thus, only indirect diagnoses, like asking for a medical history to learn about the symptoms and to rule out other diseases can be carried out. To gain insight into cellular details of AD for long-term diagnosing without physical biopsy, a noninvasive in vivo tool with a sub-micron subsurface resolution and high penetrability has to be used. In this presentation, we show that harmonic optical virtual biopsy can provide the required noninvasive cellular imaging, and is ideal for future clinical diagnosis of AD. Harmonic optical microscopy has been demonstrated to have the capability to reveal cellular morphology of human skin from epidermis to dermis layer. Third harmonic generation (THG), which is sensitive to inhomogeneous interfaces, can show the structures of skins, and can be used to reveal the morphological changes, for example, the thicken cuticle which is a common symptom of AD. Second harmonic generation (SHG), which occurs in non-centrosymmetric structures, has excellent contrast in collagen fibers and can show the pathological changes of dermis layer. Utilizing both THG and SHG, useful information may be given to facilitate the diagnosis of AD.

For secure removal of the basal cell carcinoma tissue, the technique of Mohs' micrographic surgery is often used. However, Mohs' micrographic surgery is time-consuming. In this work, we evaluate the ability of multiphoton fluorescence (MF) and second harmonic generation (SHG) imaging to discriminate the borders of human basal cell carcinoma. Morphologically, basal cell carcinomas are featured by clumps of autofluorecent cells with relatively large nuclei and marked peripheral palisading in the dermis. In contrast, SHG from collagen contributes largely to the multiphoton signal in normal dermis. Within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals. The results suggest that normal collagen structures responsible for SHG have been altered in the cancer stroma and may reflect an up-regulated collagenolytic activity of cancer cells. To better delineate the cancer cells and cancer stroma from normal dermis, a quantitative MF to SHG index (MFSI) is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from normal dermis. Our work shows that MF and SHG imaging can be an alternative for the real-time guidance of the secure removal of basal cell carcinoma.

In skin, the most common form of cancer is the basal cell carcinoma, affecting about 800.000 people in US each year. One of the treatments is photodynamic therapy (PDT) with a topical application of 5-aminolevulinic acid (ALA) derivates. These photosensitizers accumulate mainly in cancerous cells and can be activated by light resulting in a destruction of the cells. We performed multiphoton tomography based on near-infrared (NIR) femtosecond laser pulses on skin biopsies in order to study the biosynthesis of protoporphyrine IX and the effect of treatment with submicron resolution. The fluorescence of the photosensitizer was excited using two-photon processes and monitored through the epidermis towards the basal layer. In comparison with normal skin the cancerous cells showed different morphology and fluorescence behavior. Multiphoton tomography might become a useful tool for high resolution imaging of cancerous tissue and to evaluate the therapeutical effects.

Studies of immediate skin reactions are important to understand the underlying biological mechanisms involved in traumatic or chemical damage to the skin. In this study the spatial and spectral information provided by hyperspectral images was used to identify and characterize non-penetrating skin injuries in a porcine model. A hyperspectral imaging system (Hyspex, Norsk Elektro Optikk AS) was used to monitor the temporal development of minor skin injuries in an anesthetized Norwegian domestic pig. Hyperspectral data were collected in the wavelength range 400-1000 nm (VNIR), with a spectral sampling interval of 3.7 nm. The measurements were initiated immediately after inflicting the injury, and were repeated at least five times at each site with irregular frequency. The last measurement was performed 4 hours after injury. Punch biopsies (5 mm), were collected from adjacent normal skin, and at the center and the margin of each injury. The study was approved by the national animal research authority. The hyperspectral data were analyzed with respect to oxy- and deoxyhemoglobin, and erythema index. The skin biopsies were examined to determine the extent of skin damage in the bruised zones. Preliminary results show that hyperspectral imaging allows discrimination between traumatized skin and normal skin in an early phase. The extent and location of the hemorrhages can be determined from hyperspectral images. These findings might contribute to a better understanding of immediate skin reactions to minor trauma, and thereby the development of a better diagnostic modality for non-penetrating skin injuries in forensic medicine.

Determining the cause of an injury and the force behind the impact may be of crucial importance in a court case. For non-penetrating soft tissue injuries there is a lack of information available in the literature. In this study controlled bruises were inflicted on an anesthetized pig by high speed, low-weight paintball projectiles (diameter 17.1 mm, weight 3.15 g). The speed of the object and the impact itself were monitored using a high speed camera. Punch biopsies (5 mm) were collected from the injury sites. A red and purple ring with a diameter of 1.5 cm appeared on the skin within 30 seconds after the paintball impact. The ring was visually fully established after 5-10 minutes. Numerical finite element simulations performed with ABAQUS\Explicit showed a build up of shear stresses in the skin where the ring formed. Biopsies demonstrated severe dermal vessel damage in the same area. It is concluded that considerable shear stresses during the impact will create dermal vessel damage and thereby cause a visible bruise. Larger forces are required for compressive stresses to inflict equivalent vascular damage.

Invading tumors like basal cell carcinoma have usually no distinct demarcation for the human eye. Therefore, during resection, an additional rim around the tumor is removed. However, extending sprouts can be missed since most lesions are not uniform. To improve the visualization of the tumor demarcation, we developed a multi-spectral imaging system especially adapted for dermatological applications based on tunable liquid crystal spectral tunable filter technology and LED illumination. Enhanced visualization of skin tumor demarcation was achieved using three strategies. The first strategy is based on creating false color images by combining narrow band spectral filtered images by placing them into the red, green and blue image components of a color image at three specific wavelengths. These specific wavelengths were determined using a trial on error tool to achieve the highest contrast between malignant and healthy tissue. The second strategy is to make ratio images of narrow band spectral filtered images at specific wavelengths. A trail on error tool was created which enables the user to try multiple wavelengths to obtain optimal contrast. This method could be applied in realtime. For the third strategy, on pixel spectral segmentation is applied by selecting the pixel spectra in the center of a tumor, surrounding tissue and healthy tissue far away from the tumor. The correlation between these specific spectra and all image pixels is calculated using a fast algorithm. The degree is correlation is graded by color coding and presented in a false color images showing a detailed demarcation of suspicious regions in the tissue. Although this strategy is expected to provide a higher specificity, it takes more time to calculate than the first strategy.

B-scan images of phase retardation provide the change of the phase-retardation slope in the cheek dermis with aging. Enface-based phase-retardation analysis of elder check suggests that the peripheral domain of infundibula have unique orientation of collagenous fibers.

Photoacoustic imaging is a hybrid imaging modality that is based on the detection of acoustic waves generated by absorption of pulsed light by tissue chromophores such as hemoglobin in blood. We show that photoacoustic imaging can be used to image blood concentrations in a port-wine stain.

Reflected confocal microscopy has been widely used in clinical application in dermatology. In recent years, multiphoton microscopy has also emerged as an important minimally invasive bioimaging technique for the skin. In this study, we combine reflected confocal microscopy and multiphoton microscopy for skin imaging. In the epidermis, reflected confocal signals are expected to help in delineating cell borders while multiphoton signals provide cytoplasmic morphologies. In the dermis, second harmonic generation signals provide the morphology of collagen fibers. When three-dimensional images are projected, the detailed distribution of cellular component and extracellular matrix in skin can be obtained. Properly developed, this technique is of great potential for in vivo clinical application.

Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifact due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system was developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consisted of a constant UV-A light source, digital color camera, and head-positioning device. We describe the system characterization for acquisition of a fluorescent facial image and the image analysis method. The imaging modality provided uniform light distribution on the facial mannequin model and presented a discernible color fluorescent image. Valuable parameters of sebum excretion were obtained after image analysis. The imaging system, which provides a non-contact method, was proven to be a useful tool to evaluate sebum excretion and to characterize the pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the method described herein is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time.

We studied ischemia and reperfusion in island groin flaps for nine Charles River rats. When the artery and/or vein are clamped, the oxygen saturation of hemoglobin (StO2) measured by an infra-red spectroscopic device (ViOptix, Inc., Fremont, CA) dropped significantly. The drop in arterial occlusion was usually faster than that in venous occlusion. When the clamp was released after more than 1 hour of vessel occlusion, the StO2 value came back, typically not to the pre-occlusive level but only about half of it.

The 1450-nm diode laser has been found to be effective for the treatment of inflammatory acne in USA, Europe and Japan. However, there is no report on its efficacy in Chinese acne vulgaris patients. We conduct this pilot study to evaluate the efficacy and safety of the 1450-nm diode laser in the treatment of inflammatory facial acne vulgaris in Chinese patients. Nineteen patients with inflammatory facial acne were treated with the 1450-nm diode laser at 4- to 6-week intervals. Clinical photographs and lesion counts were obtained at baseline and after each treatment. Subjective evaluation of response to treatment and pain was assessed using a questionnaire. In our study, clinical improvement was seen in all patients and was generally dramatic. Lesion counts decreased 34% after one treatment (p<0.01), 56% after two treatments (p<0.01), and 81% after three treatments (p<0.01). However, the treatment-related pain was comparatively hard to be tolerated in Chinese patitents, and the other main adverse effect was the hyper-pigmentation after treatments (36.84%, 7/19).

An ex-vivo model was developed for experimental evaluation of endoluminal thermal procedures for the occlusion of saphenous veins. The model consists of the subcutaneous foot vein from freshly slaughtered cows. Using this model primary and acute effects and initial mechanisms on vein vessel could be studied. In this study different energy sources (laser and radiofrequency generator), different energy application parameters (velocity, fluence, fluence rate, temperature) were compared. The dependency of using bare fibre and cylindrical diffusors could be investigated with respect to the induced effects on the vessels wall. Contraction of the vessels were measured and investigated macroscopically and microscopically as well as by means of optical coherence tomography. As a result an optimized treatment protocol could be developed and discussed with respect to the induced effects.

Laser based devices with ability to heat sub-surface zones at certain depths within skin have several potential applications. Methods to achieve such heating at different depths have been explored in this work. Monte Carlo modeling and heat transfer calculations were performed to calculate fluence distribution, temperature distribution, and thermal damage for various laser wavelengths in the range of 1,200-1,800 nm with various pulse durations. The treatment consisted of laser irradiation combined with contact cooling. Cooling leads to preservation of the top layer leading to a zone of thermally damaged tissue under the top layer. The results indicated that the thickness and mean depth of the thermally damaged sub-surface zone can be controlled by choice of laser wavelength and cooling and irradiation times. Thermally damaged zone was deeper with lower absorbing wavelengths and/or with longer pulse durations. Histological evaluation of ex vivo pig skin immediately after treatment was done to determine thermal damage band depth and thickness for various wavelengths and pulse durations. Histological evaluation supported the modeling results. Thus, variable depth heating can be achieved through selection of the wavelength and/or laser pulse duration. With a chosen wavelength device, a variable depth heating device can be constructed by varying the cooling and laser irradiation durations.

We studied pulsed photothermal radiometric temperature profiling in human skin in numerical simulations. We considered two detectors with different spectral responses (InSb, 3.0 - 5.3 &mgr;m; HgCdTe, 5.0 - 12.0 &mgr;m). By taking into account sensitivity of available radiation detectors, realistic noise, blackbody emission characteristics and spectral variation of human skin IR absorption coefficient for a certain acquisition band, we computed realistic PPTR signals for analytical temperature profiles (Hyper-Gaussian, 100 - 500 &mgr;m deep). For each spectral band we determined the optimal effective monochromatic absorption coefficient to be used in reconstructions. We reconstructed temperature profiles from the simulated signals using a custom code, based on the conjugate-gradient algorithm and including automatic adaptive regularization. Quantitative analysis of the reconstructed temperature profiles enables determination of optimal spectral acquisition band for each IR detector. In case of InSb detector, narrow acquisition bands yield lower reconstruction errors for shallow objects, but wide acquisition bands are preferred for deeper objects. The 4.1 - 5.6 &mgr;m spectral band offers a good compromise for objects placed at variable depths. For the HgCdTe detector the results indicate that optimal acquisition spectral band is 6.4 - 12.0 µm. The results also suggest that HgCdTe detector used at 6.4 - 12.0 µm performs better than InSb detector used at 4.1 - 5.6 &mgr;m.

Changes in the skin topography are early stage diagnosis tool for diseases and the skin response to medical and cosmetics treatment. The present work focuses on the applicability of the Talbot effect in the skin topography characterization in vivo. This work shows the design and the optical system mounting using Ronchy periodical grids for this characterization.

Optical Coherence Microscopy (OCM) is a bio-medical low coherence interferometric imaging technique that has become a topic of active research because of its ability to provide accurate, non-invasive cross-sectional images of biological tissue with much greater resolution than the current common technique ultrasound. OCM is a derivative of Optical Coherence Tomography (OCT) that enables greater resolution imposed by the implementation of an optical confocal design involving high numerical aperture (NA) focusing in the sample. The primary setback of OCM, however is the depth dependence of the lateral resolution obtained that arises from the smaller depth of focus of the high NA beam. We propose to overcome this limitation using a dynamic focusing lens design that can achieve quasi-invariant lateral resolution up to 1.5mm depth of skin tissue.

Improvements in identification, imaging, and visualization of the cavernous nerves during radical prostatectomy, which are responsible for erectile function, may improve nerve preservation and postoperative potency. Optical coherence tomography (OCT) is capable of real-time, high-resolution, cross-sectional, in vivo tissue imaging. The rat prostate serves as an excellent model for studying the use of OCT for imaging the cavernous nerves, as the rat cavernous nerve is a large, visible, and distinct bundle allowing for easy identification with OCT in addition to histologic confirmation. Imaging was performed with the Niris OCT system and a handheld 8 Fr probe, capable of acquiring real-time images with 11-&mgr;m axial and 25-&mgr;m lateral resolution in tissue. Open surgical exposure of the prostate was performed on a total of 6 male rats, and OCT images of the prostate, cavernous nerve, pelvic plexus ganglion, seminal vesicle, blood vessels, and periprostatic fat were acquired. Cavernous nerve electrical stimulation with simultaneous intracorporeal pressure measurements was performed to confirm proper identification of the cavernous nerves. The prostate and cavernous nerves were also processed for histologic analysis and further confirmation. Cross-sectional and longitudinal OCT images of the cavernous nerves were acquired and compared with histologic sections. The cavernous nerve and ganglion could be differentiated from the surrounding prostate gland, seminal vesicle, blood vessels, bladder, and fatty tissue. We report preliminary results of OCT images of the rat cavernous nerves with histologic correlation and erectile stimulation measurements, thus providing interpretation of prostate structures as they appear in OCT images.

We present the development and demonstration of a novel technique for microscopic analysis of urine particles. Casts and crystals in urine are indicative of clinically important abnormalities. Current urinalysis techniques using flow cytometry and image analysis are limited by their inability to detect, identify and classify crystals and casts. Casts, crystals and yeast cells reported by current automated urine particle analyzers must be confirmed by a second microscopic review involving a human operator to prevent false positives. Human examination of suspect urine samples is resource intensive and time consuming. We introduce a new imaging method to add functionality for recognition of casts and crystals in urine. Our approach uses a polarization microscopy technique to aid classification of crystals, casts and other urine particles. Crystals and casts in urine exhibit unique interference patterns when imaged using a fixed polarizer and analyzer in a crossed configuration. These interference patterns are a measure of birefringence (retardation angle) of the cast or crystal being imaged. Preliminary experiments indicate that uric acid shows a polarization color, and larger crystals exhibit a series of concentric black lines. We show that these unique 'signatures' when used in conjunction with a hierarchical pattern recognition technique can reliably classify the analytes with improved accuracy. The new imaging technique combined with the classification algorithm can address the shortcomings of current urinalysis techniques; and provide quicker and more accurate results.

Bipolar transurethral resection (TUR) is an alternative to monopolar TUR in the treatment of benign prostatic hyperplasia and offers the major advantage of utilizing 0.9% sodium chloride for irrigation. Claims have been made that bipolar electrosurgery in clinical use causes less thermal damage to adjacent tissues. We sought to assess tissue thermometry and histopathologic thermal effects of a bipolar system in comparison to standard monopolar TUR in an animal model. Eight male beagles were studied. A lower midline incision was used to access the prostate. Fiber-optic thermosensors were placed within the prostate. A midline cystotomy was used to perform antegrade resection of the prostate using a bipolar TUR system (VISTA CTR) with normal saline or a monopolar device using glycine. Resection of a 1cm cavity was performed in each lateral lobe. Animals were sacrificed acutely and the prostates excised for histopathological assessment of thermal damage. In both groups, prostatic temperature rises were transient and fell with increasing distance from the resection site. The greatest temperature increase occurred in the monopolar group (24.2 ± 3.9°C) compared to the bipolar group (6.8 ± 1.8°C, p<0.0001). The depth of thermal damage was greatest in the monopolar group (0.59 ± 0.27mm vs 0.15 ±0.02mm in the bipolar arm, p<0.0001). Bipolar TUR generated significantly less heat and produced less histopathological thermal damage compared to monopolar prostatic resection in a canine model. The clinical benefits of these findings remain to be determined.

Recently, 2 &mgr;m cw laser systems have been introduced for surgery. The 2 &mgr;m wavelength is predominantly absorbed by water and enables effective cutting and ablation of tissue similar to the cw CO₂ laser. In contrast to the CO₂ laser, the 2 &mgr;m wavelength is delivered through fiber optics and available for endoscopic procedures. After many years of experience with the 1.06 &mgr;m Nd:YAG laser, we started to use the 2 &mgr;m cw laser as alternative for various urological treatments. The treatments strategies and optimal settings were examined in the lab comparing the two 1.06 and 2 &mgr;m wavelengths performing thermal measurements. Consequently, the laser was applied for various urological cases. Penile tumors were resected with haemostatic effects and good aesthetic healing comparable with the Nd:YAG laser. Although the Nd:YAG has initially a deeper penetration, the blackening of the fiber during tissue cutting, provides a more superficial effect like the 2 &mgr;m laser. Bladder (pre)malignancies were ablated after biopsy. Only with higher stage tumors, coagulation depth of the Nd:YAG might be preferable for adequate treatment. Strictures in the urethra were incised and stents were effectively desobstructed: one patient with a stent implanted after a pelvic trauma, and one patient with catheterizable apedico stoma stenoses. The thermal damage during incision to deeper layers is minimal so recurrence due to scarring is not expected. Also hair grow in patients who underwent urethroplasty was effectively treated and scrotal atheromata cysts were effectively resected without recurrence. Laparoscopic nefrectomies are being considered using the 2 &mgr;m cw laser. The 2 &mgr;m cw laser has shown to be a versatile instrument for effective treatment of various urological indications. More patients and long term results are needed to prove the clinical significance compared to other treatment modalities

OBJECTIVE: With the increased utility of complex imaging modalities small renal tumors are being diagnosed with rising frequency. We performed radiofrequency ablation to treat tumors less than 4cm in size using a combination of temperature, impedance, ultrasound and laparoscopic guidance. In this article we reviewed the outcome of radiofrequency ablation of renal tumors at one year at our institution. MATERIALS AND METHODS: Over a three-year period 75 patients with a total of 93 renal tumors underwent radiofrequency ablation. Average patient age was 64.5 years with ASA of 2.9. Indications for nephron sparing were imperative in 33 (solitary kidney 21, renal insufficiency 12). Seventeen patients had significant co-morbidities with ASA score of 3 or more and were thought to be poor candidates for nephrectomy or partial nephrectomy. Five were Jehovah's Witness patients. Average tumor size was 3.2 cm (1.5-4.0). 60% of the tumors were exophytic and 40% deep. Radiofrequency ablation was performed via a transperitoneal approach using the single pronged 3cm Cool tip electrode (Radionics Inc). Tumor was isolated laparoscopically. Prior to ablation the lesions were biopsied. Ablation was performed using both laparoscopic and real-time ultrasound imaging of the boarders of the tumor. During ablation impedance and temperature monitoring was performed. For each tumor two separate ablations were performed at perpendicular angles, the first ablation was for 6 minutes and the second for 3 minutes. The center and periphery of the tumor was monitored to insure that the temperature rose above 70 degrees Celsius. Patients were followed at three-month intervals with triple phase CT scan or MRI to evaluate efficacy of the ablation. Our criteria for recurrent tumor were growth or enhancement of the lesion. RESULTS: Average operative time was 109 minutes with and average EBL of <25cc. Mean hospital stay was 1.4 days. At average follow up of 19.2 months (range 2-24), one lesion showed evidence of tumor recurrence which was corrected surgically. Two masses did show some mild enhancement on CT 6 months post operatively, biopsies showed no evidence of tumor with fibrosis. Of the patients with follow up of greater than 12 months 75% had decreasing size of the lesion, 25% had no change in size. No complications were seen. CONCLUSIONS: Radiofrequency ablation of renal tumors is a feasible alternative for patients that have imperative indication for nephron sparing surgery or those that have significant co-morbidities. The procedure is expedient, efficacious and carries minimal morbidity. It is of extreme importance to follow these patients closely with imaging of the lesion on a frequent basis.

Introduction: While the effects of increasing body mass index on prostate cancer epidemiology and surgical approach have recently been studied, its effects on surgical outcomes are less clear. We studied the perioperative outcomes of obese (BMI >= 30) men treated with daVinci-assisted laparoscopic radical prostatectomy (DLP) and compared them to those treated with open radical retropubic prostatectomy (RRP) in a contemporary time frame. Method: After Institutional Review Board approval, we used the Mayo Clinic Radical Prostatectomy database to identify patients who had undergone DLP by a single surgeon and those who had undergone open RRP by a single surgeon between December 2002 and March 2005. Baseline demographics, peri- and post-operative courses, and complications were collected by retrospective chart review, and variables from the two cohorts compared using chi-square method and least-squares method of linear regression where appropriate. Results: 59 patients who had DLP and 76 undergoing RRP were available for study. Baseline demographics were not statistically different between the two cohorts. Although DLP had a significantly lower clinical stage than RRP (p=0.02), pathological stage was not statistically different (p=0.10). Transfusion rates, hospital stay, overall complications, and pathological Gleason were also not significantly different, nor were PSA progression, positive margin rate, or continence at 1 year. After bilateral nerve-sparing, erections suitable for intercourse with or without therapy at 1 year was 88.5% (23/26) for DLP and 61.2% (30/49) for RRP (p=0.01). Follow-up time was similar. Conclusion: For obese patients, DLP appears to have similar perioperative, as well as short-term oncologic and functional outcomes when compared to open RRP.

Objective: To define the learning curve for daVinci-assisted laparoscopic radical prostatectomy (DLP) at our institution. Methods: The data from 170 patients who underwent DLP between August 2002 and December 2004 by a single surgeon (MTG) were reviewed. Operative time, hemoglobin decrease, conversion to open procedure, positive margin rates, complications, length of stay (LOS), length of catheterization, continence, and erectile function were analyzed. Results: Hemoglobin decrease (p=0.11), positive margin rates (p=0.80), and early urinary continence (p=0.17) did not significantly correlate with surgical experience. A trend towards lower complications (p=0.07) and an earlier return of erectile function (p=0.09) was noted with increased experience with DLP. Operative time, hospital stay, catheterization time, and open conversion showed significant association with patient sequence. Median operative time for the first 60 and the last 110 patients was 323.5 and 239.5 minutes (p=<0.0001), respectively. Median LOS for the aforementioned groups was 53 and 51 hours (p=0.009). Length of catheterization declined significantly between the first 60 and the remaining 110 patients, 14 as compared to 11.5 days (p=<0.0001). Eight open conversions occurred, six were in the first 30 patients (p=0.03). Conclusion: As an indicator of the learning curve, the operative time in our series showed no correlation with sequence after the 60^th patient. Thus, despite the advantages of robotics, the learning curve to efficient performance of daVinciassisted laparoscopic radical prostatectomy is long. Oncological and functional outcomes should not be affected during the learning curve.

Introduction: After performing 1,000 robotic prostatectomies we reflected back on our experience to determine what defined the learning curve and the essential elements that were the keys to surmounting it. Method: We retrospectively assessed our experience to attempt to define the learning curve(s), key elements of the procedure, technical refinements and changes in technology that facilitated our progress. Result: The initial learning curve to achieve basic competence and the ability to smoothly perform the procedure in less than 4 hours with acceptable outcomes was approximately 25 cases. A second learning curve was present between 75-100 cases as we approached more complicated patients. At 200 cases we were comfortably able to complete the procedure routinely in less than 2.5 hours with no specific step of the procedure hindering our progression. At 500 cases we had the introduction of new instrumentation (4th arm, biopolar Maryland, monopolar scissors) that changed our approach to the bladder neck and neurovascular bundle dissection. The most challenging part of the procedure was the bladder neck dissection. Conclusion: There is no single parameter that can be used to assess or define the learning curve. We used a combination of factors to make our subjective definition this included: operative time, smoothness of technical progression during the case along with clinical outcomes. The further our case experience progressed the more we expected of our outcomes, thus we continually modified our technique and hence embarked upon yet a new learning curve.

Introduction: Robotically assisted laparoscopic radical prostatectomy is a minimally invasive alternative for the treatment of prostate cancer. We report the histopathologic and short term PSA outcomes of 500 robotic prostatectomies. Materials and Methods: Five hundred patients underwent robotic radical prostatectomy. The procedure was performed via a six trocar transperitoneal technique. Prostatectomy specimens were analyzed for TNM Stage, Gleason's grade, tumor location, volume, specimen weight, seminal vesicle involvement and margin status. A positive margin was reported if cancer cells were found at the inked specimen margin. PSA data was collected every three months for the first year, then every six months for a year, then yearly. Results: Average pre-operative PSA was 6.9 (1-90) with Gleason's score of 5 (2%), 6 (52%), 7 (40%), 8 (4%), 9(2%). Post operatively histopathologic analysis showed Gleason's 6 (44%), 7(42%), 8(10%), 9(4%). 10%, 5%, 63%, 15%, 5% and 2% had pathologic stage T2a, T2b, T2c, T3a, T3b and T4 respectively. Positive margin rate was 9.4% for the entire series. The positive margin rate per 100 cases was: 13% (1-100), 8% (101-200), 13% (201-300), 5% (301-400) and 8% (401-500). By stage it was 2%, 4%, 2.5% for T2a, T2b, T2c tumors, 23% (T3a), 46% (T3b) and 53% (T4a). For organ confined disease (T2) the margin rate was 2.5% and 31% for non organ confined disease. There were a total of 47 positive margins, 26 (56%) posterolateral, 4 (8.5%) apical, 4 (8.5%) bladder neck, 2 (4%) seminal vesicle and 11 (23%) multifocally. Ninety five percent of patients (n=500) have undetectable PSA (<0.1) at average follow up of 9.7 months. Recurrence has only been seen with non organ confined tumors. Those patients with a minimum follow up of 1 year (average 15.7 months) 95% have undetectable PSA (<.1). Conclusion: Our initial experience with robotic radical prostatectomy is promising. Histopathologic outcomes are acceptable with a low overall margin positive rate. Short term biochemical recurrence free survival has also been good. We believe that the precise dissection allowed by the advantages of laparoscopic robotic surgery will translate into excellent long term oncologic outcomes.

Objectives: To evaluate the feasibility and efficacy of robotic-assisted laparoscopic pyeloplasty. Laparoscopic pyeloplasty has been shown to have a success rate comparable to that of the open surgical approach. However, the steep learning curve has hindered its acceptance into mainstream urologic practice. The introduction of robotic assistance provides advantages that have the potential to facilitate precise dissection and intracorporeal suturing. Methods: A total of 50 patients underwent robotic-assisted laparoscopic dismembered pyeloplasty. A four-trocar technique was used. Most patients were discharged home on day 1, with stent removal at 3 weeks. Patency of the ureteropelvic junction was assessed in all patients with mercaptotriglycylglycine Lasix renograms at 1, 3, 6, 9, and 12 months, then every 6 months for 1 year, and then yearly. Results: Each patient underwent a successful procedure without open conversion or transfusion. The average estimated blood loss was 40 ml. The operative time averaged 122 minutes (range 60 to 330) overall. Crossing vessels were present in 30% of the patients and were preserved in all cases. The time for the anastomosis averaged 20 minutes (range 10 to 100). Intraoperatively, no complications occurred. Postoperatively, the average hospital stay was 1.1 days. The stents were removed at an average of 20 days (range 14 to 28) postoperatively. The average follow-up was 11.7 months; at the last follow-up visit, each patient was doing well. Of the 50 patients, 48 underwent one or more renograms, demonstrating stable renal function, improved drainage, and no evidence of recurrent obstruction. Conclusions: Robotic-assisted laparoscopic pyeloplasty is a feasible technique for ureteropelvic junction reconstruction. The procedure provides a minimally invasive alternative with good short-term results.

Endoscopic applications of the Erbium:YAG laser have been limited due to the lack of a suitable optical fiber delivery system. This study compares the transmission of Er:YAG laser radiation through germanium oxide trunk fibers with silica or sapphire fiber tips for potential use in endoscopic tissue ablation. Er:YAG laser radiation with a wavelength of 2.94 &mgr;m, pulse length of 300 &mgr;s, pulse energies of 5-1360 mJ, and pulse rates of 3-10 Hz, was delivered through 1-m-long germanium oxide fibers with either 1-cm-long, 550-&mgr;m-diameter silica or sapphire tips. Transmission through the germanium oxide / sapphire fibers measured 65 + 5 % compared with 55 + 4 % for the germanium oxide / silica fibers (P < 0.05). The damage threshold for the hybrid fibers averaged 309 + 44 mJ and 126 + 43 mJ, respectively (n = 7 fibers each) (P < 0.05). Maximum pulse energies transmitted through the fibers were 700 mJ and 220 mJ, respectively. Improved index-matching of the trunk fiber and fiber tip at 2.94 &mgr;m resulted in higher transmission and damage thresholds for the germanium oxide / sapphire fibers. The germanium oxide / sapphire fiber may represent a promising mid-IR optical fiber delivery system for use in endoscopic applications of the Er:YAG laser requiring a flexible, biocompatible, and robust fiber delivery system for contact tissue ablation.

OBJECTIVES: We evaluated the therapeutic efficacy and safety of photo-selective vaporization of the prostate (PVP) under local anaesthesia in patients suffering from lower urinary tract symptoms (LUTS) secondary to prostatic obstruction. MATERIAL & METHODS: 150 patients at the average age of 73 (range 51-92) and a mean/median ASA-score of 2.4/2.0 were included. PVP was performed under either general or spinal anaesthesia in the first 67 patients and under local anaesthesia (peri-prostatic infiltration with 0.25% bupivacain-adrenalin 20 ml) and light sedation in the remaining 83 patients. Surgical variables including asa-score, operative-time, blood-pressure, oxygen saturation, pre- and post-op haemoglobin (Hgb) were recorded. Post operative need for pain medication, catheter-time, and time to pts. hospital discharge were also recorded. RESULTS: No patient with local anaesthesia required conversion to general anaesthesia. The median Hgb fall from pre-op. to post-op. was 0.55g/dl. The median requirement for post-op. catheterization was 2 hrs after local anaesthesia and 9 hrs after general or spinal anaesthesia. The median time from operation to hospital discharge was 12 hrs in local anaesthesia and 24 hrs for general or spinal anaesthesia (p<0.001). At 12 and 18 months postoperatively, the following factors 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. At 12 months urodynamic studies revealed significant improvement (p<0.001) for opening pressure, pressure @ flow-max, micturation resistance and bladder outlet obstruction index. CONCLUSION: PVP under local anaesthesia and light sedation provides excellent intraoperative safety, expedient post operative recovery, significant symptom relief as well as improvement in uro-dynamic outcomes.

We report on the first clinical application of vascular-targeted photodynamic therapy using a bacteriopheophorbide derivative, Tookad, in patients with localized prostate cancer following external beam radiation therapy. Patients received either escalating intravenous drug doses at a fixed light dose or escalated light doses at the highest photosensitizer dose. Two cylindrically diffusing fibers were placed transperineally in the prostate, along with light monitoring fibers in the prostate, urethra and rectum. Treatment response was assessed with 7-day gadolinium-enhanced T1-weighted MRI and 6-month biopsy. Lesion formation was strongly drug and light dose-dependent, with an apparent threshold response. Early biochemical and MRI responses support the clinical potential of TOOKAD-PDT to treat locally-recurrent prostate cancer.

Two cases involving Erbium:YAG laser incision of proximal bulbar urethral strictures are described. Erbium:YAG laser radiation with a wavelength of 2.94 &mgr;m, pulse energy of 10 mJ, and a pulse repetition rate of 15 Hz, was delivered through a 2-m-long, 250-&mgr;m-core sapphire optical fiber in contact with tissue. Total laser irradiation time was 5 min. The first patient suffering from a virgin urethral stricture was treated and is stricture-free. The second patient suffering from a recurrent urethral stricture required further treatment. This case report describes the first clinical application of the Er:YAG laser in urology.

Coblation technology (Arthrocare Corp., Sunnyvale, CA, USA) uses plasma-mediated ablation (PMA) for soft-tissue removal. The application of PMA for tonsillectomy has evolved since 1998 through changes in equipment, and surgical techniques. This paper reviews the evidence to date for Coblation tonsillectomy, and offers perspective on novel applications of this maturing technology.

Backgrounds and Objectives: The styryl pyridinium dye FM1-43 is nontoxic, fluorescent, cationic dye whose fluorescence markedly increases after partitioning into membrane. Rapid entry of FM1-43 is inhibited by drugs that block the mechanically gated transduction channels, suggesting the dye can itself act as a permanent blocker of the channels. In this study, the effects of low level laser (LLL) and FM1-43 on gentiamicin induced ototoxicity in postnatal organotypic culture of rat utricles were investigated. Materials and Methods: An organotypic culture of 2- 7-day-old rat utricular maculae was established. In a series of experiments utricles were exposed to either irradiation of low level laser(LG group)or 10 ?M FM1-43(FG group) or both(LFG group) followed by 1mM of gentamicin treatment for 12 hrs. The results of experimental groups were compared with the control group by confocal laser scanning and scanning electron microscopy. Results: LLL prevented vestibular hair cells ototoxicity. Rapid incubation with FM1-43 dye protected vestibular hair cell damage induced by gentamicin treatment. Substantial additive effect of LLL on ototoxicity prevention was noted in combination therapy with FM1-43. There were statistical significant differences among all groups but between control and LFG group by both confocal laser scanning and scanning electron microscopy. In addition, caspase-3 activity was hardly found in LFG group after double staining with Phalloidin-FITC by confocal laser scanning microscopy. Conclusion: These results suggest that there is an additive protection effect of LLL and FM1-43 against gentamicin ototoxicity in postnatal organotypic culture of rat utricles. LLL may have clinical preventive and therapeutic implications on ototoxicity.

Optical coherence tomography (OCT) is a new non-invasive method to investigate biological tissue. It is particularly suitable for examination of human vocal folds due to its optical penetration depth of around 1.5mm. We developed a modified laryngoscope with an integrated OCT beam path for non-contact imaging of human vocal folds. In vivo studies on awake patients show that synchronous OCT and conventional laryngoscopy works well. For enabling the choice of the appropriate OCT encoding technique for OCT-based laryngoscopy measurements on simulated vibrations were performed with time domain (TD) and fourier domain (FD) OCT devices. The results show that TD-OCT is much more suitable for non-contact imaging than FD-OCT. In studies on porcine and monkey vocal folds the performance of femtosecond laser tissue ablation was analyzed with OCT monitoring. Histopathological sections could be well correlated with the OCT images. In future applications a combined system that uses one femtosecond laser as light source for tissue ablation as well as for OCT imaging is conceivable. In conclusion, OCT can be used as a pre-, intra- and post-operative diagnostic instrument for analysing the vocal fold structure down to the vocalis muscle allowing a more precise indication for potential subsequent invasive procedures. We suggest that OCT should be used in addition to established examination methods for diagnostic evaluation of vocal fold malignancies and functional alterations.

Several studies have identified the degree of aortic valve calcification as a strong predictor both for the progression and outcome of aortic stenosis. In industrialized countries, aortic valve stenosis is most frequently caused by progressive calcification and degeneration of aortic cusps. However, there are no accurate methods to quantify the extent of aortic valve calcification. To provide a non-invasive alternative to biopsy, a range of optical methods have been investigated, including Raman and reflectance spectroscopy. A Raman spectrum can be used to access the molecular constitution of a particular tissue and classify it. Raman spectroscopy is largely used in the quantification and evaluation of human atherosclerosis, being a powerful technique for performing biochemical analysis without tissue removal. Nevertheless, increased thickness and disorganization of the collagen fibre network and extracellular matrix are known to affect the diffuse spectral reflectance of the tissue. A catheter with the "6 around 1" configuration, the central fiber transmit laser radiation to the sample and the scattered light is collected by the other six surrounding fibers, was used both for Raman and reflectance spectroscopy. A white light (krypton lamp, flashtube Model FX 1160 Perkin Elmer, USA) excitation was used for reflectance measurements. A Ti-sapphire (785nm, Spectra Physics, model 3900S, USA) laser, pumped by an argon laser (Spectra Physics, model Stabilite 2017, USA) was used as the near infrared Raman set up. Several ex-vivo spectra of aortic valve samples were analyzed. The results show a promising way to differentiate normal, fibrous and calcified tissue in aortic valve.

Nonlinear optical imaging technologies offer some intriguing medical diagnostic applications. Examples include fast imaging of elastin and collagen distributions in diseased tissues using two-photon fluorescence (TPF) and second harmonic generation (SHG), respectively. The 3D sectioning capabilities and biochemical specificity that enable fast imaging in highly scattering biological media lie at the heart of the appeal of these nonlinear approaches for medical applications. One of these promising nonlinear techniques relies on the resonance enhancement of the third order nonlinear susceptibility by a vibrational mode of a molecule. Coherent Anti-Stokes Raman Scattering (CARS) can provide similar vibrational information as a spontaneous Raman spectrum. The technique has been shown to be orders of magnitude more sensitive than spontaneous Raman, with video rate imaging demonstrated recently. In this work, we investigate the potential use of broadband CARS spectroscopy and CARS imaging for biochemical analysis of arterial tissue. Biochemical imaging data from broadband CARS is compared with spontaneous Raman microspectroscopy. The broadband CARS system comprised of a single femtosecond-laser is presented in detail. Issues related to data analysis, the advantages and current limitations of the CARS technique in biodiagnostics are discussed.

Multiphoton microscopy is a techniques that fascilitates three dimensional imaging of intact, unstained tissue. Especially connective tissue has a relatively strong nonlinear optical response and can easily be imaged. Atherosclerosis is a disease where lipids accumulate in the vessel wall and there is a thickening of the intima by growth of a cap of connective tissue. The mechanical strength of this fibrous cap is of clinically importance. If the cap ruptures a thrombosis forms which can block a coronary vessel and therby causing myocardial infarction. Multiphoton microscopy can be used to image the fibrous cap and thereby determine the thickness of the cap and the structure of the connective fibres. This could possibly be developed into a diagnostic and clincal tool to monitor the vulnerability of a plaque and also to better understand the development of a plaque and effects of treatment. We have collected multiphoton microscopy images from atherosclerotic plaque in human aorta, both two photon excited fluorescens and second harmonic generated signal. The feasability of using this technique to determine the state of the plaque is explored.

We studied Ho:YAG laser irradiation in blood vessel as a vasodilator ex vivo. We thought that the Ho:YAG laser-induced bubble expansion might be able to dilate the vessel because we found the vessel wall expansion after the Ho:YAG laser irradiation, that is steady deformation, in the vessel ex vivo. There have been many reports regarding to the Ho:YAG laser irradiation in the vessel. Most of studies concentrated on the interaction between Ho:YAG laser irradiation and vessel wall to investigate side effect on Ho:YAG laser angioplasty. We proposed to use the Ho:YAG laser-induced bubble expansion as a vasodilator. We studied vasodilation effect of the Ho:YAG laser-induced bubble ex vivo. The flash lamp excited Ho:YAG laser surgical unit (IH102, NIIC, Japan) (&lgr;=2.1&mgr;m) was used. The laser energy was delivered by a silica glass fiber (outer diameter: 1000&mgr;m, core diameter: 600&mgr;m). The laser-induced bubble was generated in the extracted fresh porcine carotid artery with the warmed saline perfusion. The laser energy at the fiber tip was ranging from 170-1300mJ per pulse. Number of the laser irradiation was ranged from 20pulses to 100pulses. The outer diameter of the vessel was observed. To examine the change in mechanical properties of the vessel wall, the stress-strain curve of the laser-irradiated vessel was measured. Birefringence observation and microscopic observation of staining specimen were performed. When the laser energy was set to 1300mJ per pulse, the outer diameter of the vessel after the laser irradiation was expanded by 1.4 times comparing with that of before the laser irradiation and the dilatation effect was kept even at 10minutes after the irradiation. The elasticity modulus of the artery by collagen was changed by the laser irradiation. In the polarized microscopic observation, the brightness of the intimal side of the vessel is increased comparing with that of the normal. We think this brightness increasing may be attributed to birefringence change by the arrangement of stretched collagen fiber. We suppose it is likely to be able to use the Ho:YAG laser irradiation as a temporary vasodilater tool in spite of further study should be performed.

A new generation of the optical fiber catheter has been developed for surgical treatment of atrial fibrillation with laser radiation. Atrial fibrillation (AF) is a heart rhythm abnormality that involves irregular and rapid heartbeats. The surgical maze procedure was the first treatment that offered a permanent solution for maintaining a normal sinus rhythm in patients with AF. This procedure involves the creation of a maze-like series of incisions in the left atrium, resulting in isolation of the pulmonary vein and removal of the left atrial appendage. To produce long continuous transmural lesions laser diodes operating in near IR range along with end-emitting fiber catheters have been used experimentally. The absence of side-emitting flexible catheters with the ability to produce long continuous lesions limits the further development of this technology. In this research, a new type of an optical catheter consisting of a flexible, spatially modulated fiber diffuser has been used to make continuous photocoagulation lesions for effective maze procedure treatments.

Mechanical deformation of isolated rabbit hearts through passive inflation techniques have been a viable form of replicating heart motion, but its relation to the heart's natural active contractions remain unclear. The mechanical properties of the myocardium may show diverse characteristics while in tension and compression. In this study, epicardial strain was measured with the assistance of computer-aided speckle interferometry (CASI)1. CASI tracks the movement of clusters of particles for measuring epicardial deformation. The heart was cannulated and perfused with Tyrode's solution. Silicon carbide particles were applied onto the myocardium to form random speckle pattern images while the heart was allowed to actively contract and stabilize. High resolution videos (1000x1000 pixels) of the left ventricle were taken with a complementary metal oxide semiconductor (CMOS) camera as the heart was actively contracting through electrical pacing at various cycle lengths between 250-800 ms. A latex balloon was then inserted into the left ventricle via left atrium and videos were taken as the balloon was repeatedly inflated and deflated at controlled volumes (1-3 ml/cycle). The videos were broken down into frames and analyzed through CASI. Active contractions resulted in non-uniform circular epicardial and uniaxial contractions at different stages of the motion. In contrast, the passive heart demonstrated very uniform expansion and contraction originating from the source of the latex balloon. The motion of the active heart caused variations in deformation, but in comparison to the passive heart, had a more enigmatic displacement field. The active heart demonstrated areas of large displacement and others with relatively no displacement. Application of CASI was able to successfully distinguish the motions between the active and passive hearts.

In order to investigate the optimum operation parameters on novel short-term heating (<15s, approx. 70^oC) balloon, named Photo-thermo dynamic balloon (PTDB), we studied diameter and elasticity change of vascular wall after dilatation ex vivo. We have been studying to develop the PTDB angioplasty in which we demonstrated sufficient vascular dilatation with lower pressure by heat- induced denaturation of arterial collagen. And we have also demonstrated the suppression of intimal hyperplasia in animal experiments. We need to understand the PTDB dilatation mechanism to determine the optimum operation parameters. The prototype PTDB with diameter of 3mm was used in our experiments. The internal diameters of extracted fresh porcine carotid arteries at pre- and post- PTDB dilatation were measured. Balloon parameters were follows; pressure P=2atm, peak temperature in balloon T=60-80^oC, and heating duration t=4-30s. Morphological change in the media of dilated artery with PTDB were microscopically examined with Weigert staining. Elastic properties were carried out by stress-strain measurements with calculation of young's modulus. We found that PTDB dilatation provided the effect to prevent elastic recoil. We explained that the reason of this effect might be arrangement of micro- structure in the media, i.e., heat-denatured collagen fibers sustained the elastic recoil due to rubbery elastin fibers. The arterial elasticity was not significant different after PTDB dilatation. It was suggested that there could be no compliance mismatch after PTDB dilatation in physiological range. We found that a part of PTDB dilatation mechanism, in which the vascular wall structure played an important role. The optimum operation parameters for PTDB might be determined in consideration of collagen denaturation progress and arterial composition.

Glial fibrillary acidic protein (GFAP) is a traditional biomarker for astrocytes of the central nervous system. In this study, non-invasive in vivo imaging of GFAP-GFP (green fluorescent protein) expression in the brain of neonatal transgenic mice is used as a novel method to investigate the relationship between the expression of the transgene at 0, 2, 4, 6 and 8 hr post-treatment in mice subjected to a single administration of 12 mg/kg of neurotoxin 1-methyl-4(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH₃-MPTP). The GFP elevation was found to peak at 6 hr and lasted to at least 8 hr after the toxin treatment. Histological examination of fixed brain sections using immunohistochemistry (IHC) shows an increase in GFP and GFAP signal from the substantia nigra pars compacta (SNpc) and the hippocampus. The results have provided quantitative fluorescence and qualitative histological evidence for the activation of the GFAP-GFP transgene in astrocytes following neurotoxin 2'-CH₃-MPTP administration, suggesting that the model described here could be used to study neuronal degeneration such as Parkinson's disease and in general, developmental neurotoxicity in live animals.

Epileptogenic lesions and their margins are often difficult to define intraoperatively. We hypothesize that optical spectroscopy can detect unique pathophysiological features of epileptogenic lesions in children and hence differentiate them from normal brain. This hypothesis was tested by comparing the in vivo optical and fluorescence characteristics of epileptogenic brain lesions (non-neoplastic) with those of normal brain. Patients were recruited from those receiving epilepsy surgeries at Miami Children's Hospital. Using a portable spectroscopic system, optical characterization of brain was performed intraoperatively. Fluorescence spectra were measured at 337 nm excitation, and diffuse reflectance spectra were measured between 400 and 850 nm. To date, seven epilepsy patients have been enrolled in the study. A couple interesting trends have been observed in the recorded optical spectra. First, sites within the resection zone, as defined by the intracranial electroencephalogram data, often show higher diffuse reflectance signals than normal sites do. This is especially prominent around 500 nm and between 650 and 850 nm. Secondly, several investigated sites with abnormal electroencephalogram and/or pathology show a unique blue shift in their fluorescence spectra, which is not seen in other cases.

Neurologic complications have been reported with spinal transforaminal injections. Causes include intraneural injection, plus embolization occlusion of the radicular artery with subsequent spinal cord infarction. ¹ Optical coherence tomography (OCT) is a non-invasive imaging modality, which is used to image tissue microstructure with very high resolution (less than 20 microns) in real-time. With a view toward needle tip OCT visualization of the spinal neuroforamen, we conducted animal studies to explore OCT imaging of paraspinal neurovascular structures. With institutional animal care committee approval, we performed ex-vivo and in situ OCT studies in a euthanized dog, pig, and rabbit. Image data was gathered on spinal nerve roots, dura, and brachial plexus. Two systems were used: frequency domain OCT imaging system developed at California Institute of Technology, and time domain Imalux NIRIS system with a 2.7 mm diameter probe. In a euthanized pig, excised dura was punctured with a 17-gauge Tuohy needle. FDOCT dural images of the puncture showed a subsurface cone-shaped defect. In a rabbit in situ study, puncture of the dura with a 26-gauge needle is imaged as a discontinuity. FDOCT imaging of both small artery and large arteries will be presented, along with H&E and OCT images of the brachial plexus.

Introduction: Given the length of procedures and the brightness of operating room lights, there is concern that photosensitizers used to locate brain tumors and treat them with photodynamic therapy (PDT) may photobleach before they can be fully utilized. The phthalocyanine photosensitizer Pc 4 is resistant to photobleaching. In this study, we tested the hypothesis that exposure of Pc 4-loaded glioma cells to photoactivating light will result in continuing fluorescence of Pc 4. Methods: U87 human glioma cells were cultured in MEM with 5% penicillin/streptomycin, 5% sodium pyruvate, 10% fetal bovine serum, and 25 mM HEPES. These cultures were given 0 or 125 nM Pc 4, followed 2 hours later by three separate exposures of 200 J/cm² of red light (&lgr;_max = 675 nm). Confocal fluorescence images were collected before and after each exposure. Results: Pc 4 fluorescence was localized to cytoplasmic membranes of the U87 glioma cells, as previously seen in other types of cells. After exposure to PDT, Pc 4 fluorescence was not reduced and even increased. Discussion: Pc 4 may be useful for the intra-operative detection of glioma by fluorescence and for PDT, since neither Pc 4 level nor its fluorescence is likely to decrease during exposure to operating room lights.

Introduction: Failure of treatment for high grade gliomas is usually due to local recurrence at the site of surgical resection indicating that a more aggressive form of local therapy, such as PDT, could be of benefit. PDT causes damage to both tumor cells as well as cerebral blood vessels leading to degradation of the blood brain barrier with subsequent increase of brain edema. The increase in brain edema following ALA-PDT was evaluated in terms of animal survival, histopatological changes in normal brain and tumor tissue and MRI scanning. The effect of steroid treatment, to reduce post-treatment PDT induced edema, was also examined. Methods:Tumors were established in the brains of inbred BD-IX and Fisher rats. At various times following tumor induction the animals were injected with ALA ip. and four hours later light treatment at escalating fluences and fluence rates were given. Nontumor bearing control animals were also exposed to ALA-PDT in a similar manner to evaluate damage to normal brain and degree of blood brain barrier (BBB) disruption. Results: Despite a very low level of PpIX production in normal brain, with a 200:1 tumor to normal tissue selectivity ratio measured at a distance of 2 mm from the tumor border, many animals succumbed shortly after treatment. A total radiant energy of 54 J to non-tumor bearing animals resulted in 50% mortality within 5 days of treatment. Treatment of tumor bearing animals with moderate fluence levels produced similar brain edema compared to higher fluence levels. ALA PDT in nontumor bearing animals produced edema that was light dose dependent. PDT appeared to open the BBB for a period of 24-48 hrs after which it was restored. The addition of post operative steroid treatment reduced the incident of post treatment morbidity and mortality. Conclusions: T2 and contrast enhanced T1 MRI scanning proved to be a highly effective and non-evasive modality in following the development of the edema reaction and the degree and time course of BBB dysfunction thus allowing the use of fewer animals.

Photochemical internalization (PCI) is a technique to improve the utilization of macromolecules (e.g. chemotherapeutic agents) in cancer therapy in a site-specific manner. The concept is based on the use of specially designed photosensitizers which localize preferentially in the membranes of endocytic vesicles. Upon exposure to light the photosensitizers induce the formation of reactive oxygen species such as singlet molecular oxygen. The photooxidation of the endocytic membranes leads to the release of the contents of the vesicles into the cytosol. In this way, macromolecules encapsulated by the vesicles will reach the cytosol and exert their biological activity instead of being degraded by lysosomal hydrolases. The utility of PCI for the treatment of malignant gliomas was investigated in vitro using an F98 rat glioma cell line. The cytotoxicity of 5-aminolevulinic acid (ALA) based PCI of bleomycin was compared to: (1) ALA-PDT, and (2) bleomycin. In all cases, monolayers were incubated in ALA, bleomycin, or ALA + bleomycin for 4 hours and were subsequently exposed to 635 nm light. Toxicity was evaluated using colony formation assays. F98 rat glioma cells in monolayer were found to be susceptible to the effects of both ALA-PDT and bleomycin. ALA-PDT was found to be particularly effective when light was delivered at a low irradiance of 5 mW cm^-2. In this case, a radiant exposure of 20 J cm^-2 resulted in only 4% survival. Bleomycin was found to be toxic at relatively low concentrations, incubation of F98 cells in 10 &mgr;g ml^-1 for 4 hours resulted in 1% survival. The PCI effect was found to be negligible for the parameters investigated in the F98 cell line suggesting that: (1) the incubation time was sub-optimal and/or (2) ALA was inappropriate for this application.

Following surgical removal of malignant brain tumors 80% of all cases develop tumor recurrence within 2 cm of the resected margin. The aim of postoperative therapy is therefore elimination of nests of tumor cells remaining in the margins of the resection cavity. However, it is unlikely that standard "one-shot" intraoperative PDT treatments can accomplish this goal. This is due mainly to the length of time required to deliver adequate light fluences to depths of 1-2 cm in the resection margin. Additionally, due to the short doubling time of malignant glioma cells, the kill rate per cell doubling indicates that it seems unlikely that a single relatively short treatment would be sufficient to prevent recurrence of the tumor. Multiple repetitive or chronic treatment protocols would therefore seem required. In repetitive PDT both phtosensitizer and light are given over relatively short treatment times (hours) with treatment repetition following relatively long intervals (weeks). In chronic PDT (also called metronomic), both the photosensitizer and light are delivered continuously at low rates for extended periods of time (days). The in vitro therapy response of human glioma spheroids to 5-aminolevulinic acid (ALA) mediated PDT in repetitive or chronic form were investigated. At 6J fluence, spheroid survival rates of 28 and 7% were observed for repetitive or chronic PDT protocols respectively. The results indicated that single chronic (24-48hrs) treatment) was more effective at inhibiting spheroid growth than PDT repeated at relatively long intervals (weeks) or daily fractionated PDT.

Photodynamic therapy (PDT) shows good promise as an adjuvant therapy for the treatment of gliomas. PDT requires the use of a photosensitizer but there have been few studies in which they have been directly compared. Two rat brain glioma cell lines, BT4C and F98 were used to study the effects of Photofrin (PF) and aminolevulinic acid (ALA) as photosensitizers. Both compounds were able to sensitize cells to red light (635nm) however, PF treated (2.5 &mgr;g/ml) cells displayed more cell death than ALA treated (1 mM) cells. ALA-PDT treated cells did not have a block in the cell cycle while PF-PDT treated cells had a fluence dependent G1 block 24 hrs after treatment. The BT4C cell line also displayed a G2 cell cycle block. Both photosensitizers were able to induce apoptosis as measured by flow cytometry but only PF-PDT was able to induce necrosis at the fluence rates investigated (1-20 J/cm²). These differences in cellular responses to ALA and

Introduction: Several workers have proposed the use of PET (Positron Emission Tomography) imaging for the outcome assessment of photodynamic therapy (PDT), especially for deep-seated tumors. We report on our study of 18Ffluorodeoxy- glucose (18F-FDG) PET imaging following brain tumor Pc4-PDT. Our working hypothesis was that the tumor's metabolic activity would decline dramatically following Pc 4-PDT owing to tumor necrosis. Methods: Seven days after intraparenchymal implantation of U87 cells, the brains of 12 athymic nude rats were imaged by micro-CT and/or micro-MR. These animals were also 18F-FDG micro-PET (&mgr;PET) scanned before and after Pc 4-PDT. 18F-FDG was used to trace metabolic activity that was monitored via &mgr;PET. Occurrence of PDT was confirmed on histology. The analysis of 18F-FDG dose and animal weight normalized &mgr;PET activity was studied over the 90 minute µPET scan. Results: Currently, &mgr;PET data have been studied for: (1) three of the animals that did not indicate tumor necrosis on histology and were assigned to a "Non-PDT" group, and (2) six animals that exhibited tumor necrosis on histology and were assigned to a "PDT" group. The &mgr;PET-detected 18F-FDG uptake activity in the tumor region before and after photoirradiation increased in the Non-PDT group an average of 2.28 times, and in the PDT group it increased an average of 1.15 times. Discussion: We are investigating the cause of the increase in 18F-FDG &mgr;PET activity that we observed in the PDT group. The methodology used in this study should be useful in determining whether this or other PET, SPECT, or MR functional imaging protocols will detect both the specificity and sensitivity of brain tumor necrosis following Pc 4-PDT.

Introduction: We have previously demonstrated the use of phthalocyanine Pc 4 for the photodynamic therapy (PDT) of ectopic human glial tumors in the athymic nude rat brain. We wish to determine whether 18F-fluorodeoxy-glucose (¹⁸F-FDG) Positron Emission Tomography (PET) imaging can detect the reduction in tumor metabolism that must occur after Pc 4-PDT-induced necrosis. Methods: 2.5 x 10⁵ U87 cells were injected into the brains of 12 athymic nude rats. After 7 days of tumor growth, all 12 animals were imaged functionally by ¹⁸F-FDG micro-PET (&mgr;PET) and structurally by micro-CT and/or micro-MR. These animals received 0.5 mg/kg b.w. Pc 4 via tail-vein injection. One day later the scalp was re-incised and the tumor illuminated with 30 J/cm² of 672-nm light from a diode laser. The next day these animals were again ¹⁸F-FDG &mgr;PET imaged. Next, the animals were euthanized and their brains were explanted for H&E histology. Results: Histology showed that tumors in the 6 Pc 4-PDT-treated animals demonstrated necrosis ranging from full to frank (severe). Preliminary analysis showed that 18F-FDG &mgr;PET activity in 3 of the 6 non-PDT group (i.e., no tumor necrosis observed) animals was seen to increase 2.28 times following tumor photoirradiation, whereas ¹⁸F-FDG &mgr;PET activity in 5 of the 6 PDT group (i.e., tumor necrosis observed) animals was seen to increase 1.15 times following tumor photoirradiation. Discussion: The increased ¹⁸F-FDG &mgr;PET activity in the PDT group was unexpected. We had expected this activity to decrease and are presently investigating the cause of this observation.

This study aimed at determining cerebral hemodynamic parameters in human subjects during breath holding using near infrared spectroscopy (NIRS). Breath holding serves as a method of simulation OSA (Obstructive Sleep Apnea). Data was acquired non-invasively from 40 subjects, twenty OSA sufferers (10 females, 10 males, age 20-70 years), and twenty normal volunteers (10 females, 10 males, age 20-65 years). Measurements were conducted using a LED Imager (LEDI) during breath holding. In comparing OSA subjects with controls during breath holding, a consistent increase or even a decrease in oxy- ([O₂Hb]), deoxy- ([HHb]), total hemoglobin ([tHb]) concentrations, and tissue hemoglobin oxygen saturation (SO₂) in the regional brain tissue were observed. The LEDI probe consists of 4 sources and 10 detectors serving as 4 sets of 1 source and 4 detectors each. A three wavelength (730, 805, and 850 nm) LED was used and the wavelengths were switched sequentially. The distance between sources and the source-detector separation were 2.5 cm. Data acquisition consisted of three segments, baseline for one minute, followed by a period of breath holding, and then 2 minutes of recovery time. The duration of the breath holding was subject-dependent. Our investigation proves that NIR spectroscopy could be used as a tool for detecting cerebral hemodynamics and also serves as a method of screening patients with OSA.

We describe the projection of spatially modulated light for quantitatively mapping changes in oxyhemoglobin, deoxyhemoglobin, and oxygen saturation in two pilot studies in the rat barrel cortex during both permanent and temporary cerebral ischemia. The approach is based on the projection of spatial modulation of white light onto the brain. The reflected light is captured on a CCD camera, which is then processed to obtain the concentration and distribution of chromophores over a wide field. Preliminary results confirm a measurable and quantifiable increase in tissue molecular concentration of deoxy-hemoglobin and decrease in hemoglobin oxygen concentration in both experimental settings. Our preliminary data from our pilot studies demonstrate that spatial modulation of light can provide quantitative chromophore mapping of the brain and has a potential role in monitoring the course and severity of cerebral ischemia in cerebrovascular disease patients.

Small animal imaging devices are now commonly used to study gene activation and model the effects of potential therapies. We are attempting to develop a protocol that non-invasively tracks the affect of Pc 4-mediated photodynamic therapy (PDT) in a human glioma model using structural image data from micro-CT and/or micro-MR scanning and functional data from 18F-fluorodeoxy-glucose (18F-FDG) micro-PET imaging. Methods: Athymic nude rat U87-derived glioma was imaged by micro-PET and either micro-CT or micro-MR prior to Pc 4-PDT. Difficulty insuring animal anesthesia and anatomic position during the micro-PET, micro-CT, and micro-MR scans required adaptation of the scanning bed hardware. Following Pc 4-PDT the animals were again 18F-FDG micro-PET scanned, euthanized one day later, and their brains were explanted and prepared for H&E histology. Histology provided the gold standard for tumor location and necrosis. The tumor and surrounding brain functional and structural image data were then isolated and coregistered. Results: Surprisingly, both the non-PDT and PDT groups showed an increase in tumor functional activity when we expected this signal to disappear in the group receiving PDT. Co-registration of the functional and structural image data was done manually. Discussion: As expected, micro-MR imaging provided better structural discrimination of the brain tumor than micro-CT. Contrary to expectations, in our preliminary analysis 18F-FDG micro-PET imaging does not readily discriminate the U87 tumors that received Pc 4-PDT. We continue to investigate the utility of micro-PET and other methods of functional imaging to remotely detect the specificity and sensitivity of Pc 4-PDT in deeply placed tumors.

In this work, we demonstrate the use of Two-Photon Fluorescence Lifetime Imaging Microscopy (TP-FLIM) for intracellular calcium ([Ca²⁺]i) measurement with a calcium sensitive fluorescent dye in a neuronal cell model and brain tissue. Calcium perturbations were induced via chemical stimulation and our results signify the potential of TP-FLIM for quantitative ([Ca²⁺]i) measurement within the physiological range of intracellular [Ca²⁺].