This PDF file contains the front matter associated with SPIE Proceedings Volume 8210, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Photodynamic therapy is known to evoke both autophagy and apoptosis. Apoptosis is an irreversible death pathway while autophagy can serve a cytoprotective function. In this study, we examined two photosensitizing agents that target lysosomes, although they differ in the reactive oxygen species (ROS) formed during irradiation. With both agents, the 'shoulder' on the PDT dose-response curve was substantially attenuated, consistent with loss of a cytoprotective pathway. In contrast, this 'shoulder' is commonly observed when PDT targets mitochondria or the ER. We propose that lysosomal targets may offer the possibility of promoting PDT efficacy by eliminating a potentially protective pathway.

Photosensitizers for photodynamic therapy (PDT) are most commonly delivered to patients or experimental animals via intravenous injection. After initial distribution throughout the body, there can be some preferential accumulation within tumors or other abnormal tissue in comparison to the surrounding normal tissue. In contrast, the photosensitizer precursor, 5-aminolevulinic acid (ALA) or one of its esters, is routinely administered topically, and more specifically, to target skin lesions. Following metabolic conversion to protoporphyrin IX, the target area is photoilluminated, limiting peripheral damage and targeting the effective agent to the desired region. However, not all skin lesions are responsive to ALA-PDT. Topical administration of fully formed photosensitizers is less common but is receiving increased attention, and some notable advances with selected approved and experimental photosensitizers have been published. Our team has examined topical administration of the phthalocyanine photosensitizer Pc 4 to mammalian (human, mouse, pig) skin. Pc 4 in a desired formulation and concentration was applied to the skin surface at a rate of 5-10 μL/cm² and kept under occlusion. After various times, skin biopsies were examined by confocal microscopy, and fluorescence within regions of interest was quantified. Early after application, images show the majority of the Pc 4 fluorescence within the stratum corneum and upper epidermis. As a function of time and concentration, penetration of Pc 4 across the stratum corneum and into the epidermis and dermis was observed. The data indicate that Pc 4 can be delivered to skin for photodynamic activation and treatment of skin pathologies.

Pancreatic ductal adenocarcinoma is a lethal disease that is often unresectable by the time of diagnosis and is typically non-responsive to chemo- and radiotherapy, resulting in a five year survival of only 3%. Tumors of the pancreas are characterized by a dense fibrous stroma rich in extracellular matrix proteins, which is implicated in poor therapeutic response, though its precise roles remain poorly understood. Indeed, while the use of therapeutics that target the stroma is an emerging paradigm in the clinical management of this disease, the primary focus of such efforts is to enhance drug penetration through dense fibrous stroma and it is unclear to what extent the characteristically rigid stroma of pancreatic tumors imparts drug resistance by acting as a complex signaling partner, or merely as a physical barrier for drug delivery. Here we use 3D in vitro co-cultures of pancreatic cancer cells and normal human fibroblasts as a model system to study heterotypic interactions between these populations. Leveraging this in vitro model along with image-based methods for quantification of growth and therapeutic endpoints, we characterize these co-cultures and examine the role of verteporfin-based photodynamic therapy (PDT) for targeting tumor-fibroblast interactions in pancreatic tumors.

Intrapleural photodynamic therapy (PDT) has been used as an adjuvant treatment with lung-sparing surgical treatment for mesothelioma. In the current intrapleural PDT protocol, a moving fiber-based point source is used to deliver the light and the light dose are monitored by 7 detectors placed in the pleural cavity. To improve the delivery of light dose uniformity, an infrared (IR) camera system is used to track the motion of the light sources. A treatment planning system uses feedback from the detectors as well as the IR camera to update light fluence distribution in real-time, which is used to guide the light source motion for uniform light dose distribution. We have reported previously the success of using IR camera to passively monitor the light fluence rate distribution. In this study, the real-time feedback has been implemented in the current system prototype, by transferring data from the IR camera to a computer at a rate of 20 Hz, and by calculation/displaying using Matlab. A dual-correction method is used in the feedback system, so that fluence calculation can match detector readings. Preliminary data from a phantom showed superior light uniformity using this method. Light fluence uniformity from patient treatments is also shown using the correction method dose model.

The ability to deliver uniform light dose in Photodynamic therapy (PDT) is critical to treatment efficacy. Current protocol in pleural photodynamic therapy uses 7 isotropic detectors placed at discrete locations within the pleural cavity to monitor light dose throughout treatment. While effort is made to place the detectors uniformly through the cavity, measurements do not provide an overall uniform measurement of delivered dose. A real-time infrared (IR) tracking camera is development to better deliver and monitor a more uniform light distribution during treatment. It has been shown previously that there is good agreement between fluence calculated using IR tracking data and isotropic detector measurements for direct light phantom experiments. This study presents the results of an extensive phantom study which uses variable, patient-like geometries and optical properties (both absorption and scattering). Position data of the treatment is collected from the IR navigation system while concurrently light distribution measurements are made using the aforementioned isotropic detectors. These measurements are compared to fluence calculations made using data from the IR navigation system to verify our light distribution theory is correct and applicable in patient-like settings. The verification of this treatment planning technique is an important step in bringing real-time fluence monitoring into the clinic for more effective treatment.

Photodynamic therapy (PDT) is an important treatment modality for localized diseases such as prostate cancer. In prostate PDT, light distribution is an important factor because it is directly related to treatment efficacy. During PDT, light distribution is determined by tissue optical property distributions (or heterogeneity). In this study, an interstitial diffuse optical tomography (iDOT) method was used to characterize optical properties in tissues. Optical properties (absorption and reduced scattering coefficients) of the prostate gland were reconstructed by solving the inverse problem using an adjoint model based on diffusion equation using a modified matlab public user code NIRFAST. In the modified NIRFAST method, linear sources were modeled for the reconstruction. Cross talking between absorption coefficients and reduced scattering coefficients were studied to have minimal effect, and a constrained optical property method (set either absorption coefficient or reduced scattering coefficient to be homogeneous) is also studied. A prostate phantom with optical anomalies was used to verify the iDOT method. The reconstructed results were compared with the known optical properties, and the spatial distribution of optical properties for this phantom was successfully reconstructed.

For efficient photodynamic treatment of wound infection, a photosensitizer must be distributed in the whole infected tissue region. To ensure this, depth profiling of a photosensitizer is necessary in vivo. In this study, we applied photoacoustic (PA) imaging to visualize the depth profile of an intravenously injected photosensitizer in rat burn models. In burned tissue, pharmacokinetics is complicated; vascular occlusion takes place in the injured tissue, while vascular permeability increases due to thermal invasion. In this study, we first used Evans Blue (EB) as a test drug to examine the feasibility of photosensitizer dosimetry based on PA imaging. On the basis of the results, an actual photosensitizer, talaporfin sodium was used. An EB solution was intravenously injected into a rat deep dermal burn model. PA imaging was performed on the wound with 532 nm and 610 nm nanosecond light pulses for visualizing vasculatures (blood) and EB, respectively. Two hours after injection, the distribution of EB-originated signal spatially coincided well with that of blood-originated signal measured after injury, indicating that EB molecules leaked out from the blood vessels due to increased permeability. Afterwards, the distribution of EB signal was broadened in the depth direction due to diffusion. At 12 hours after injection, clear EB signals were observed even in the zone of stasis, demonstrating that the leaked EB molecules were delivered to the injured tissue layer. The level and time course of talaporfin sodium-originated signals were different compared with those of EB-originated signals, showing animal-dependent and/or drug-dependent permeabilization and diffusion in the tissue. Thus, photosensitizer dosimetry should be needed before every treatment to achieve desirable outcome of photodynamic treatment, for which PA imaging can be concluded to be valid and useful.

Focusing a femto-second laser (1 mJ/pulse repetition 1 kHz) on a special tape, a strong radiation consisting of the electron beam of ~ 200 keV and X-rays of ~ 6.4 keV (5 %) has been generated. It has been verified that the radiation source is sufficient to kill the tumor cells and the DNA laddering structure in the in-vivo test is obtained. More test implanting the tumor under the skin of mouse and irradiating the laser-generated radiation, we have shown the radiation has a clear powerful therapeutic capability. For about 80 % of mice irradiated, their tumor disappeared. For further clinical test use, a compact laparoscope-type unit mounted on an articulated arm has been constructed and it can generate the necessary amount of the radiation dose.

Photodynamic Therapy (PDT) is an optical treatment modality used to destroy malignant tissues. Nowadays there are fixed clinical PDT protocols that make use of a particular optical dose, photosensitizer amount and drug-light interval. However the treatment response varies depending on the type of pathology and the patient. In order to adjust current dosimetry to get an optimal treatment outcome, the development of accurate predictive models has emerged as the ideal tool to achieve new personal protocols. Several attempts have been made in this way although the influence of the photosensitizer distribution on the optical parameters has not been taken into account until this moment. We present a first approach to predict the spatial-temporal variation of the absorption coefficient during the photodynamic process applied to a dermatological disease taking into account the photobleaching of a topical photosensitizer. The model presented also takes into account an inhomogeneous initial distribution of the photosensitizer, the propagation of light in the biological media and the evolution of the molecular concentrations of different components involved in the photochemical reactions. The obtained results permit us to investigate how the depletion of the photosensitizer during the photochemical reactions affects to the light absorption as it propagates within the target tissue.

Photodynamic therapy (PDT) is becoming a treatment of choice for cancer because of its low cost, high effectiveness and low damage to healthy tissue. Successful PDT outcome depends on accurate dosimetry, which is currently lacking, leading to variable and/or ineffective treatment outcome. We report on our research and developmental efforts towards an implicit dosimetric method for PDT that will provide an accurate assessment of treatment effectiveness by continuous monitoring of the in vivo drug concentration and the oxygen concentration in tissue. This approach uses the same tools presently available for PDT, making it attractive to the health professionals without increasing treatment cost.

Photodynamic therapy (PDT) is an important treatment modality for cancer and other localized diseases. In addition to PDT dose, singlet oxygen (¹O₂) concentration is used as an explicit PDT dosimetry quantity, because ¹O₂ is the major cytotoxic agent in photodynamic therapy, and the reaction between ¹O₂ and tumor tissues/cells determines the treatment efficacy. ¹O₂ concentration can be obtained by the PDT model, which includes diffusion equation for the light transport in tissue and macroscopic kinetic equations for the generation of the singlet oxygen. This model was implemented using finite-element method (FEM) by COMSOL. In the kinetic equations, 5 photo-physiological parameters were determined explicitly to predict the generation of ¹O₂. The singlet oxygen concentration profile was calculated iteratively by comparing the model with the measurements based on mice experiments, to obtain the apparent reacted ¹O₂concentration as an explicit PDT dosimetry quantity. Two photosensitizers including Photofrin and BPD Verteporfin, were tested using this model to determine their photo-physiological parameters and the reacted ¹O₂ concentrations.

Control of wound sepsis is an important challenge in traumatology. However, increase in the drug-resistant bacteria makes this challenge considerably difficult in recent years. In this study, we attempted to control burn wound sepsis in rats by photodynamic treatment, which has been reported to be effective against some drug-resistant bacteria. A 20% TBSA (total body surface area) full-thickness burn was made in rat dorsal skin, and five days after injury, a suspension of P. aeruginosa was applied to the wound surface. At 30 min after infection, a methylene blue (MB) solution was applied to the wound surface; 5 min afterwards, the wound was illuminated with a 665-nm light emitting diode (LED) array for 10 min. This treatment (application of MB and illumination) was repeated 3 times successively. The averaged light intensity on the wound surface was 3.3 mW/cm², the corresponding total light dose being 5.9 J/cm². One week after injury, the numbers of bacteria in the blood and liver were counted by colony forming assay. In the liver, the number of bacteria of the treated group was significantly lower than that of the sham control group without photodynamic treatment. In the blood, no bacteria were detected in the treated group, while a certain amount of bacteria was detected in the control group. These results demonstrate the efficacy of MB-mediated PDT with a red LED array to control burn wound sepsis.

Cancer is increasing fast nowadays through all over the world. Early diagnosis of cancer is a desirable subject as it can significantly improve the patient's chances of survival. In most cases the cancer is diagnosis using MRI, CT, PET. But, there are several disadvantages associated with high cost, low sensitivity and specificity, and health risks from radioactive. For that reason, significant efforts are being invested to improve the current imaging system. Thermography can offer some advantages. Chief among these are the contact free and low cost for detect cancer. But thermography has some disadvantages associated with low sensitivity for small tumors. In this research develops non contact, safe, high sensitivity, and low cost infrared imaging technique. Experiments were performed using lock in thermography with a small amount of magnetic nanoparticle (MNP) and radiofrequency generator. As a result, highly sensitive infrared thermography can a small amount of MNP be detected by the technique.

A light blanket is designed with a system of cylindrically diffusing optical fibers, which are spirally oriented. This 25x30 cm rectangular light blanket is capable of providing uniform illumination during intraoperative photodynamic therapy. The flexibility of the blanket proves to be extremely beneficial when conforming to the anatomical structures of the patient being treated. Previous tests of light distribution from the blanket have shown significant loss of intensity with the length of the fiber. This can be improved through the use of an optical adaptor which will be able to match the numerical aperture of the laser source to the numerical aperture of the blanket fiber; thus transmitting a higher percentage of light.

An ultrasound coupled handheld-probe-based optical fluorescence molecular tomography (FMT) system has been in development for the purpose of quantifying the production of Protoporphyrin IX (PPIX) in aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo. The design couples fiber-based spectral sampling of PPIX fluorescence emission with a high frequency ultrasound imaging system, allowing regionally localized fluorescence intensities to be quantified [1]. The optical data are obtained by sequential excitation of the tissue with a 633nm laser, at four source locations and five parallel detections at each of the five interspersed detection locations. This method of acquisition permits fluorescence detection for both superficial and deep locations in ultrasound field. The optical boundary data, tissue layers segmented from ultrasound image and diffusion theory are used to estimate the fluorescence in tissue layers. To improve the recovery of the fluorescence signal of PPIX, eliminating tissue autofluorescence is of great importance. Here the approach was to utilize measurements which straddled the steep Qband excitation peak of PPIX, via the integration of an additional laser source, exciting at 637 nm; a wavelength with a 2 fold lower PPIX excitation value than 633nm.The auto-fluorescence spectrum acquired from the 637 nm laser is then used to spectrally decouple the fluorescence data and produce an accurate fluorescence emission signal, because the two wavelengths have very similar auto-fluorescence but substantially different PPIX excitation levels. The accuracy of this method, using a single source detector pair setup, is verified through animal tumor model experiments, and the result is compared to different methods of fluorescence signal recovery.

The emergence of antibiotic resistant bacteria causes significant increase in deaths due to infections around the world. Nowadays, it could be impossible to find appropriate antibiotics to treat some bacterial strains, especially multidrug resistant types. Therefore, there is an urgent need to develop new and safe treatment techniques for multidrug resistant bacteria associated morbidity and mortality. In this study, Photodynamic Therapy was used to destroy these kinds of bacteria with near infrared light and Indocyanine Green. Different wavelengths of lasers mostly in the visible spectrum have been investigated for Photodynamic Therapy; however near infrared lasers have been used in very few studies. The main motivation to test photodynamic therapy with near infrared light and indocyanine green is that the near infrared laser (around 800-nm) has more penetration depth in the biological tissue than the other lasers have. Therefore it is supposed that it will show more antibacterial effect. And also indocyanine green has a very low toxicity and an FDAapproved drug. This study investigated optimum parameters for PDT with 809-nm laser and Indocyanine green (ICG) to kill P. aeruginosa in vitro. We were able to optimize the laser power and ICG concentration to non-toxic levels and achieved 99% decrease in bacterial load with 252 J/cm² laser light and 125 μg/ml ICG concentration. This study demonstrates that PDT with near-infrared light and ICG can be powerful and non-hazardous treatment strategy for untreatable pathogens.

Photodynamic therapy (PDT) that uses the second generation photosensitizer, verteporfin (VP), is a developing therapy for pancreatic cancer. The optimal timing of light delivery related to VP uptake and distribution in pancreatic tumors will be important information to obtain to improve treatment for this intractable disease. In this work we examined uptake and distribution of VP in two orthotopic pancreatic tumors with different histological structure. ASPC-1 (fast-growing) and Panc-1 (slower growing) tumors were implanted in SCID mice and studied when tumors were approximately 100mm³. In a pilot study, these tumors had been shown to differ in uptake of VP using lightinduced fluorescence spectroscopy (LIFS) in vivo and fluorescence imaging ex vivo and that work is extended here. In vivo fluorescence mean readings of tumor and liver increased rapidly up to 15 minutes after photosensitizer injection for both tumor types, and then continued to increase up to 60 minutes post injection to a higher level in ASPC-1 than in Panc-1. There was variability among animals with the same tumor type, in both liver and tumor uptake and no selectivity of tumor over liver. In this work we further examined VP uptake at multiple time points in relation to microvascular density and perfusion, using DiOC₇ (to mark blood vessels) and VP fluorescence in the same tissue slices. Analysis of DiOC7 fluorescence indicates that AsPC-1 and Panc-1 have different vascular densities but AsPC-1 vasculature is more perfusive. Analysis of colocalized DiOC7 and VP fluorescence showed ASPC-1 with higher accumulation of VP 3 hrs after injection and more VP at a distance from blood vessels compared to Panc-1. This work shows the need for techniques to analyze photosensitizer distribution in order to optimize photodynamic therapy as an effective treatment for pancreatic tumors.

Photodynamic therapy (PDT) mediated with verteporfin is currently being investigated to treat pancreatic cancer in patients who are not surgical candidates. Clinically, interstitial light delivery is administered through a fiber, via percutaneous needle implantation guided by ultrasound and/or verified by CT. Tumor response to PDT is based on photosensitizer (PS) dose, light dose, light dose rate and the timing of light application following PS injection. However, studies have shown that even when matching administered PDT treatment parameters such as drug dose and light level, there can be significant inter-patient variation in tissue damage post-PDT, and this has been primarily attributed to imprecise PS concentration at the target tissue site. In order to achieve optimal tumor response from PDT without causing major damage to surrounding tissue, it would be advantageous to measure the PS concentration in the target tissue just prior to light application. From these measurements, the clinician can adapt the light application dose to the measured target tissue PS concentration (i.e. insufficient target tissue PS concentrations compensated by higher light doses and vice versa.) in order to provide an optimal light dose for each patient. In animal studies, a spectrometer-based in-vivo fluorescence dosimetry system has been used to assess accumulated PS levels (verteporfin) in situ. Measurements are taken from skin, leg muscle, buccal mucosa and tumor tissue locations one hour after injection of the photosensitizer. Real-time spectral fitting, subtraction of background autofluorescence and ratiometric analysis is performed on the raw data to extract out only the photosensitizer fluorescence and therefore concentration. Using a pre-measured calibration data set of varying concentrations for verteporfin in tissue phantoms composed of intralipid and whole blood, it was possible detect concentrations of the photosensitizer below 0.5nM. In the clinical studies being performed at UCL Hospital in which verteporfin-PDT treatment is being given to patients with pancreatic cancer, the dosimetry system is being used to assess PS concentration the pancreatic tumor tissue prior to interstitial light dose treatment. The goal of the work here is to determine whether the dosimetry system can accurately and efficiently be used clinically by evaluating the measured local tissue PS concentration to treatment outcome (area of necrosis established). The results of this study will partially determine the need for fluorescence dosimetry to individualize PDT treatment for patients based on local tissue PS concentration.