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- Skin Imaging 1: Multiphoton Imaging
- Skin Imaging 2: Spectral Imaging
- Skin Imaging 3: OCT, Photoacoustic, and Confocal Imaging
- Skin Imaging and Spectroscopy: NIR and Fluorescence
- Skin Therapy
- Skin Therapy and Skin Diagnosis
- Poster Session
- Imaging and Miscellaneous Energy Applications
- Robotics
- Fibers and Prosthetics
- Applications of Light Energy
- Advanced Technology and Instrumentation in Otolaryngology: Lasers, Optics, Radio Frequency, and Related Technolgy
- Spectroscopy
- Myocardium: Diagnosis and Therapy
- Neuro Imaging I
- Tumor Diagnosis and Treatment
- Neuro Imaging II
- Poster Session
Skin Imaging 1: Multiphoton Imaging
Two-color excited-state absorption imaging of melanins
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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.
Noninvasive intravital cellular diagnosis of atopic dermatitis by using harmonic optical virtual biopsy
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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.
Quantitative multiphoton imaging for guiding basal-cell carcinoma removal
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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.
Multiphoton tomography of skin tumors after ALA application
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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.
Skin Imaging 2: Spectral Imaging
In vivo hyperspectral imaging of traumatic skin injuries in a porcine model
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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.
Biomechanical characterization of soft tissue injuries
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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.
Feasibility of multi-spectral imaging system to provide enhanced demarcation for skin tumor resection
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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.
Skin Imaging 3: OCT, Photoacoustic, and Confocal Imaging
Three-dimensional evaluation of in-vivo human skin by fiber-based polarization-sensitive spectral domain optical coherence tomography using B-scan-oriented polarization modulation
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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 of port-wine stains
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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.
Three-dimensional skin imaging using the combination of reflected confocal and multiphoton microscopy
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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.
Skin Imaging and Spectroscopy: NIR and Fluorescence
Digital fluorescent imaging system for quantitative analysis of facial sebum production
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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.
Oxygen saturation in arterial and venous occlusions for an animal flap model
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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.
Skin Therapy
Treatment of inflammatory facial acne vulgaris in Chinese patients with the 1450-nm diode laser: a pilot study
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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).
Ex-vivo investigations on endoluminal vein treatment procedures
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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.
Skin Therapy and Skin Diagnosis
Variable depth skin heating with lasers
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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.
Optimal spectral acquisition band for temperature profiling in human skin using pulsed photothermal radiometry
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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.
Poster Session
Skin topographical analysis by means of Talbot effect technique
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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.
Invariant high resolution optical skin imaging
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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.
Imaging and Miscellaneous Energy Applications
Optical coherence tomography of the rat cavernous nerves
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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.
Advanced imaging technique for automated classification of casts and crystals in urine
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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.
Thermal and histological effects of bipolar and monopolar electrosurgical resection of the prostate in a canine model
Show abstract
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.
Use of the 2-µm cw laser as addition and/or alternative for the Nd:YAG in urology
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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
Radio-frequency ablation of small renal tumors: minimum follow up of 1 year
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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.
Robotics
Peri-operative comparison between daVinci-assisted radical prostatectomy and open radical prostatectomy in obese patients
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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.
DaVinci-assisted laparoscopic radical prostatectomy: the learning curve
Show abstract
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 60th 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.
Is the learning curve endless? One surgeon's experience with robotic prostatectomy
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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.
Robot-assisted radical prostatectomy: histopathologic and biochemical recurrence data at one-year follow-up
Show abstract
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.
Robot-assisted laparoscopic pyeloplasty: minimum 1-year follow-up
Show abstract
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.
Fibers and Prosthetics
Transmission of high Erbium:YAG laser pulse energies through germanium oxide trunk fibers with sapphire tips
Show abstract
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.
Applications of Light Energy
Therapeutic efficacy and safety of photo-selective vaporization of prostate under local anaesthesia with light sedation
Carl-Jørgen Arum M.D.,
Paal Romundstad,
Jan Mjønes M.D.
Show abstract
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.
Vascular-targeted photodynamic of prostate cancer phase with Tookad for recurrent prostate cancer following radiation therapy: initial clinical studies
Show abstract
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.
Erbium:YAG laser incision of urethral strictures: early clinical results
Show abstract
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.
Advanced Technology and Instrumentation in Otolaryngology: Lasers, Optics, Radio Frequency, and Related Technolgy
Coblation for tonsillectomy in children: an evidence-based update on a maturing technology
Show abstract
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.
Effect of low level laser on ototoxicity prevention of FM1-43 in postnatal organotypic culture of rat utricles
Yong Won Chung M.D.,
Yong Saeng Kim M.D.,
Jin-Chul Ahn,
et al.
Show abstract
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 characterization of vocal folds by OCT-based laryngoscopy
Show abstract
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.
Spectroscopy
Study of normal, fibrous, and calcified aortic valve tissue by Raman and reflectance spectroscopy
Show abstract
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.
Broadband coherent anti-Stokes Raman scattering (CARS): a potential tool for atherosclerosis diagnostic imaging
Show abstract
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 of atheroslcerotic plaques
Show abstract
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.
Myocardium: Diagnosis and Therapy
Ho:YAG laser irradiation in blood vessel as a vasodilator: ex-vivo study
Show abstract
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.
Fiber photo-catheters with spatially modulated diffusers for laser treatment of atrial fibrillation
Show abstract
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.
Comparison of epicardial deformation in passive and active isolated rabbit hearts
Show abstract
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.
Development of novel short-term heating angioplasty: diameter and elasticity change of vascular wall ex vivo
Show abstract
In order to investigate the optimum operation parameters on novel short-term heating (<15s, approx. 70oC) 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=60-80oC, 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.
Neuro Imaging I
Noninvasive imaging of transgenic GFP expression in neonatal mouse brain
Show abstract
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.
In vivo optical characterization of pediatric epileptogenic lesions
Show abstract
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.
Toward forward-looking OCT needle tip vision of the spinal neuroforamen: animal studies
Show abstract
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.
Tumor Diagnosis and Treatment
Fluorescence of Pc 4 in U87 cells following photodynamic therapy
Show abstract
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.
Increased brain edema following 5-aminolevulinic acid mediated photodynamic in normal and tumor bearing rats
Show abstract
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 for the treatment of malignant gliomas
Show abstract
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.
Comparing the effects of repetitive and chronic ALA mediated PDT on human glioma spheroids
Show abstract
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.
Comparison of ALA and Photofrin in two rat glioma models
Sarah A. Ziegler,
Casey Hall,
Cherisse Loucks,
et al.
Show abstract
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
Neuro Imaging II
Analysis of 18F-fluorodeoxy-glucose PET imaging data captured before and after Pc 4-mediated photodynamic therapy of U87 tumors in the athymic nude rat
Show abstract
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.
Monitoring Pc 4-mediated photodynamic therapy of U87 tumors with 18F- fluorodeoxy-glucose PET imaging in the athymic nude rat
Show abstract
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.
Obstructive sleep apnea screening by NIRS imaging
Show abstract
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.
Mapping tissue chromophore changes in cerebral ischemia: a pilot study
Show abstract
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.
Poster Session
Hardware, software, and scanning issues encountered during small animal imaging of photodynamic therapy in the athymic nude rat
Show abstract
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.
Quantitative intracellular calcium sensing with two-photon fluorescence lifetime imaging microscopy
Show abstract
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+].