Photodynamic therapy leads to both direct and indirect tumor cell death. The latter also involves the consequences of vascular shut-down and immunologic effects. While these factors are a major factor in tumor eradication, there is usually an element of direct cell killing that can reduce the cell population by as much as 2-3 logs. Necrosis was initially believed to represent the predominant PDT death mechanism. An apoptotic response to PDT was first reported by Oleinick in 1991, using a sensitizer that targets the anti-apoptotic protein Bcl-2. Apoptosis leads to fragmentation of DNA and of cells into apoptotic bodies that are removed by phagocytosis. Inflammatory effects are minimized, and the auto- catalytic elements of the process can amplify the death signal. In this study, we examined consequences of Bcl-2 photodamage by a porphycene sensitizer that targets the ER and causes photodamage to the anti-apoptotic protein Bcl-2. Death patterns after Bcl-2 inactivation by a small-molecular antagonist were also assessed. In addition to apoptosis, we also characterized a hitherto undescribed PDT effect, the initiation of autophagy. Autophagy was initially identified as a cell survival pathway, allowing the recycling of components as nutrients become scarce. We propose that autophagy can also represent both a potential survival pathway after PDT damage to cellular organelles, as well as a cell-death pathway. Recent literature reports indicate that autophagy, as well as apoptosis, can be evoked after down-regulation of Bcl-2, a result consistent with results reported here.

We have previously described changes in angle-resolved light scattering measured from intact cells in suspension subjected to photodynamic therapy using photosensitizers that localize primarily to mitochondria. These changes were analyzed with a Mie theory-based model. For the sensitizers Pc 4 and ALA-induced protoporphyrin IX, the scattering data from PDT-treated cells was consistent with a coated sphere model, in which mitochondrial morphology changes were the predominant mechanism governing the scattering changes. This interpretation was supported by electron microscopy. Here we describe quite different changes in angle-resolved light scattering from cells sensitized with the lysosomal-localizing photosensitizer LS11. Unlike the case of the mitochondrial-localizing photosensitizers, analysis of these post-treatment scattering data reveals a shift toward a larger mean organelle diameter in the larger of the two particle size distributions identified from Mie-theory analysis of scattering from control cells. Further, the post-treatment scattering angular distributions are well interpreted in terms of homogeneous rather than coated spheres. On the basis of these results and results of fluorescence microscopy of LS11-PDT treated monolayers, we propose that the initial, pre-treatment scatterer population is comprised of lysosomes and mitochondria. LS11 PDT ablates a significant fraction of the lysosomes, leaving a relatively unperturbed population of mitochondria to dominate the scattering. These findings suggest that scattering measurements are capable of reporting a variety of PDT-induced changes to cell organelles. They further suggest that photodynamic action is a useful biophysical tool for understanding basic mechanisms of light scattering from intact cells.

Photodynamic therapy (PDT) involves the use of a photosensitizer, which, when activated by light becomes toxic to the cancer cells. Lasers provide light at a specific wavelength required to activate the photosensitizer while the monochromaticity of the lasers at specific wavelengths results in maximum effectiveness of the photosensitizer during treatment. An important property of photosensitizers is that they should absorb light at a long wavelength as the light has to be able to penetrate tissue, and low energy light is able to travel further through tissue than light which absorbs at a shorter wavelength. This study aimed at evaluating the effects of 2 different photosensitizers, Al (AlPcSmix), commercially known as Photosens^(R) and Ge (GePcSmix), both from the Phthalocyanine family of sensitizers, on oesophageal (SNO) and breast cancer (MCF-7) cells. Cells were irradiated at 660nm with a power output of 100 mW and a fluence of 10 J/cm². Cell viability and proliferation were assessed using adenosine triphosphate (ATP) luminescence and alamarBlue^TM staining. Lactate dehydrogenase (LDH) activity was used as a measure of cytotoxicity while the Comet assay was used to evaluate DNA damage. Heat shock protein 70 (Hsp70) induction acted as a measure of cellular stress. Both photosensitizers used during the course of this study are effective in targeting malignant cells, and have a cytotoxic effect on these cells when activated using laser irradiation. However, cytotoxic effects were also measured in the absence of laser irradiation, indicating the importance of photosensitizer concentration. Lower concentrations of photosensitizer in the presence of laser irradiation showed greater apoptotic inducing ability than with high concentrations. Morphologically, cells were affected to the detriment despite viability tests indicating the contrary.

HPPH (a chlorophyll-a analog) was linked with a cyanine dye and the resulting conjugate was found to be an efficient tumor imaging (fluorescence imaging) and photosensitizing agent (PDT). Our preliminary results suggest that tumor-avid porphyrin-based compounds can be used as vehicles for delivering the desired fluorophores to tumor for fluorescence imaging. In an early diagnosis of microscopic lesions in pre-clinical studies (C3H mice implanted with RIF tumors) the HPPH-cyanine dye conjugate showed tumor-imaging capability (λ_ex: 780 nm, λ_em: 860 nm) at the non- therapeutic doses that are 100 fold lower than those used therapeutically. Compared to the cyanine dye, the corresponding HPPH-conjugate showed enhanced long-term tumor imaging ability.

As the PDT effect may be enhanced by localized hyperthermia (HT), it would be logical to find a single agent that could bring about these two modalities at precisely the target site for synergism. Since highly localized HT can be induced by magnetic field excitation of superparamagnetic nanoparticles, we report here the design and synthesis of photosensitizer-decorated iron oxide nanoparticles and their tumoricidal effect. Thus, a porphyrin is covalently anchored on the iron oxide nanoparticle via dihydroxybenzene which binds tightly on the surface of the nanoparticle by M-O bond. The morphology of the resultant nanoparticle was studied to show that the crystallinality is not changed and the nanoparticle remains superparamagnetic at room temperature. The conjugate is also strongly fluorescent indicating that the iron oxide hardly affects the optical properties of the surface bound porphyrin moieties. The conjugate is readily taken by cancer cell (Hela cell line) and is able to trigger apoptosis after excitation by light.

The aminolevulinic acid (5-ALA) -based fluorescence diagnosis has been found to be promising for an early detection and demarcation of superficial oral squamous cell carcinomas (OSCC). This method has previously demonstrated high sensitivity, however this clinical trial showed a specificity of approximately 62 %. This specificity was mainly restricted by tumor detection in the oral cavity in the presence of bacteria. After topical ALA application in the mouth of patients with previously diagnosed OSSC, red fluorescent areas were observed which did not correlate to confirm histological findings. Swabs and plaque samples were taken from 44 patients and cultivated microbiologically. Fluorescence was investigated (OMA-system) from 32 different bacteria strains found naturally in the oral cavity. After ALA incubation, 30 of 32 strains were found to synthesize fluorescent porphyrins, mainly Protoporphyrin IX. Also multiple fluorescent spectra were obtained having peak wavelengths of 636 nm and around 618 nm - 620 nm indicating synthesis of different porphyrins, such as the lipophylic Protoporphyrin IX (PpIX) and hydrophylic porphyrins (water soluble porphyrins, wsp). Of the 32 fluorescent bacterial strains, 18 produced wsp, often in combination with PpIX, and 5 produced solely wsp. These results clarify that ALA-based fluorescence diagnosis without consideration or suppression of bacteria fluorescence may lead to false-positive findings. It is necessary to suppress bacteria fluorescence with suitable antiseptics before starting the procedure. In this study, when specific antiseptic pre-treatment was performed bacterial associated fluorescence was significantly reduced.

Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

Protoporphyrin IX (PpIX) is produced via the heme synthesis pathway by the cell following administration of aminolevulinic acid (ALA). ALA synthase, the enzyme that produces ALA in the cell from glycine and succinyl-coenzyme A, is inhibited in a feedback mechanism by heme and thus is the rate limiting enzyme in the heme synthesis pathway. Since ALA is administered systemically, the rate limiting step that naturally exists in the cells is bypassed, however it is currently unclear why cells have different rate limiting steps in the ALA-PpIX synthesis pathway, and more specifically which types of cancer cells are most productive. It has been determined that when the same amount of ALA is administered to a wide panel of cancer cells in vitro that vastly differing amounts of PpIX are produced. The steps for the ALA-PpIX pathway occur in and around the mitochondria of the cell, but interestingly no correlation is seen between PpIX production and mitochondrial content of the cell, following ALA administration. However, total cell area shows positive correlation with PpIX production. Administration of the iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed and thus increases the detectable amount of PpIX in each cell line. The cell lines that have the lowest PpIX production following administration of ALA alone show the largest increase in production following the combined administration of ALA and L1. PpIX fluorescence is thought to be a measure of cellular activity and the goal of the current study was to determine which cell lines would be the most promising targets for fluorescence detection or monitoring response to therapy. The results indicate that the cells with larger size and larger numbers of mitochondria may be good potential targets for this therapy. While this conclusion may appear obvious, it is not universally true, and cellular specific variations exist which are still not fully understood.

Photodynamic therapy (PDT) is a treatment modality which has been shown to be effective for both malignant and non-malignant diseases. New photosensitizers such as 5-aminolevulinic acid hexylester (hALA) may increase the efficiency of PDT. Monitoring of the tissue response provides important information for optimizing factors such as drug and light dose for this treatment modality. Optical spectroscopy may be suited for this task. To test the efficacy of hALA induced PDT, a study on rats with a superficial bladder cancer model, in which a bladder cancer cell line (AY-27) is instilled, will be performed. Preliminary studies have included a PDT feasibility study on rats, fluorescence spectroscopy on AY-27 cell suspensions, and optical reflection and fluorescence spectroscopy in rat bladders in vivo. The results from the preliminary studies are promising, and the study on hALA induced PDT treatment of bladder cancer will be continued.

The oxidative stress initiated by photodynamic therapy (PDT) results in accumulation of ceramide in various cell types undergoing apoptosis. Apoptotic responses to PDT are similar to those of the oxidative stress inducer and the Bcl-2 inhibitor HA 14-1 in murine leukemia L1210 cells. Here we tested whether de novo ceramide accumulation can be induced by HA 14-1 during apoptosis in Jurkat T human leukemia cells. Similar to photosensitization with Pc 4, treatment of Jurkat cells with HA14-1 leads to DEVDase activation and nuclear apoptosis. However, unlike Pc 4-PDT, HA 14-1 did not induce accumulation of de novo ceramide. The data suggest that, compared to HA 14-1, de novo ceramide accumulation is a distinct response to PDT in Jurkat cells.

m-THPC photodynamic therapy has been successfully studied in skin cancer, but no research effort concerning its topical application has been performed until now. Determination of the biodistribution of a special m-THPC thermogel formulation and its tumour selectivity was studied after topical application on hairless SKH-1 mice bearing non-melanoma skin carcinomas. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumour area and the concentration or demarcation of tumor by mTHPC fluorescence was measured at 2, 4 and 6 hours after drug application by three methods: 1. A fluorescence imaging system capturing images at two emission wavelengths (500 and 654 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 654 to 500 was calculated. 2. A fluorescence spectrometer with a fiber bundle for in vivo spectra recording after 420 nm excitation. 3 Each animal was euthanized and the photosensitizer was chemically extracted from liver, spleen, muscle, normal skin and tumour. The photosensitizer concentrations in the extracts and in plasma were determined by fluorescence spectroscopy. The in vivo methods showed a remarkable difference in the concentration of photosensitizer in normal skin and tumour. The highest concentration in tumour was observed 6h after drug application and the highest fluorescence intensity ratio of m-THPC in tumour to normal tissue was observed at 4 hours. Furthermore, no m-THPC was detected in normal tissues or plasma after drug topical application. In vivo and ex vivo results were consistent.

The study aims at developing a near infrared (NIR) tomography imaging system using a single rotating source/detector scanning device, which will be working for diffuse optical tomography (DOT) on medical applications. Some influential factors in terms of the design of this scanning device and test phantoms are investigated such as the temporal stability, air-absorption, container influence, and radiance normalization, etc. Then, a heterogeneous microsphere phantom with an off-center inclusion is investigated. It is observed that the radiance deviation between the heterogeneous and the homogenous is shifting with the inclusion position accordingly. Through the previously-mentioned system calibration, the back projection method as widely applied in the computed tomography (CT) technique is used to reconstruct optical images which indicate the distribution of optical property of the test phantom.

Photodynamic therapy (PDT) mediated with a novel vascular targeting photosensitizer pd-bacteriopheophorbide (Tookad) has been investigated as an alternative modality for the treatment of prostate cancer and other diseases. This study investigated, for the first time, the vascular photodynamic effects of Tookad-PDT on nerve tissues. We established an in situ canine model using the cutaneous branches of the saphenous nerve to evaluate the effect of Tookad-PDT secondary to vascular damage on compound-action potentials. With Tookad dose of 2 mg/kg, treatment with 50 J/cm2 induced little change in nerve conduction. However, treatment with 100 J/cm2 resulted in decreases in nerve conduction velocities, and treatment with 200 J/cm2 caused a total loss of nerve conduction. Vasculature surrounding the saphenous nerve appeared irritated. The nerve itself looked swollen and individual fibers were not as distinct as they were before PDT treatment. Epineurium had mild hemorrhage, leukocyte infiltration, fibroplasias and vascular hypertrophy. However, the nerve fascicles and nerve fibers were free of lesions. We also studied the effect of Tookad-PDT secondary to vascular damage on the pelvic nerve in the immediate vicinity of the prostate gland. The pelvic nerve and saphenous nerve showed different sensitivity and histopathological responses to Tookad-PDT. Degeneration nerve fibers and necrotic neurons were seen in the pelvic nerve at a dose level of 1 mg/kg and 50 J/cm2. Adjacent connective tissue showed areas of hemorrhage, fibrosis and inflammation. Our preliminary results suggest that possible side effects of interstitial PDT on prostate nerve tissues need to be further investigated.

We report results of in-vivo light dosimetry of light fluence (rate) in human prostate during photodynamic therapy (PDT). Measurements were made in-vivo at the treatment wavelength (732nm) in 15 patients in three to four quadrants using isotropic detectors placed inside catheters inserted into the prostate. The catheter positions are determined using a transrectal ultrasound (TRUS) unit attached to a rigid template with 0.5-cm resolution. Cylindrical diffusing fibers with various lengths are introduced into the catheters to cover the entire prostate gland. For the last four patients, distributions of light fluence rate along catheters were also measured using a computer controlled step motor system to move multiple detectors to different distances (with 0.1 mm resolution). To predict the light fluence rate distribution, a kernel-based model was used to calculate light fluence rate using either (a) the mean optical properties (assuming homogeneous optical properties) for all patients or (b) using distributions of optical properties measured for latter patients. Standard deviations observed between the calculations and measurements were 56% and 34% for (a) and (b), respectively. The study shows that due to heterogeneity of optical properties significant variations of light fluence rate were observed both intra and inter prostates. However, if one assume a mean optical properties (mua = 0.3 cm-1, mus' = 14 cm-1), one can predict the light fluence rate to within a maximum error 200% for 80% of the cases and a mean error of 105%. To improve the prediction of light fluence rate further would require determination of distribution of optical properties.

In photodynamic therapy (PDT), it is desirable to determine the light fluence distribution accurately for treatment planning. Earlier studies have shown heterogeneous distribution of optical properties in patients' prostates. Finite-element method (FEM) is suitable for dealing with heterogeneous media and irregular geometries. Cylindrical diffusing fibers (CDFs) were modeled as linear sources of finite lengths, using the same parameters as those used in the treatments. Meshes were generated in the three-dimensional (3D) prostate geometry, reconstructed using transrectal ultrasound images of the prostate. Heterogeneous optical properties measured in the prostate were applied in the calculation and the refractive-index mismatch boundary condition was studied. Compared with the measurements, the FEM calculations using heterogeneous optical properties show better agreements than those using homogeneous optical properties.

This paper summarizes ten approaches to quantifying fluorescence in tissues, and contrasts their strengths and weaknesses, relative to what their common applications are, and should be. The major issues involved in this analysis are to compare the accuracy of the method and the ability to quantify the active (i.e. non-aggregated) fraction of fluorophore in the tissue. In addition, issues of the depth of penetration and the availability of the method come into play when clinical applications are required. In general, tissue extraction and liquification methods are the 'gold standard' in this field, yet these are plagued by large variance in the values, raising questions about their ability to report on the true active fraction of drug in the tissue. Confocal and fiber optic microsampling methods allow direct quantification of the active fluorescence in vivo and are able to quantify the heterogeneity in the tissue. Yet both of these methods sample the most superficial layers of a tissue, unless invasive injection of the probe is done. Macroscopic sampling of the tissue is therefore the preferred choice for clinical use, yet there is truly no optimum method which can sample the drug concentration to arbitrary accuracy. Empirical bulk tissue sampling methods are the most commonly used, yet without model-based interpretation of the values it is generally not possible to be quantitative. Even relative uptake values can be distorted by the shape of the tissue, and so raster scanning or model-based assessment of the fluorescent yield is preferable, if available. Extending this concept further, tomographic methods can be implemented to quantify fluorescence, and can even be coupled into existing clinical imaging systems, but development and optimization of these methods will be required in the coming years. These are outlined, and case examples illustrated in this paper.

Optimal delivery of light in photodynamic therapy (PDT) requires not only optimal placement and power of light sources, but knowledge of the dynamics of light propagation in the tissue being treated and in the surrounding normal tissue, and of their respective accumulations of sensitizer. In an effort to quantify both tissue optical properties and sensitizer distribution, we have measured fluorescence emission and diffuse reflectance spectra at the surface of a variety of tissue types in the thoracic cavities of human patients. The patients studied here were enrolled in Phase II clinical trials of Photofrin-mediated PDT for the treatment of non-small cell lung cancer and cancers with pleural effusion. Patients were given Photofrin at dose of 2 mg per kg body weight 24 hours prior to treatment. Each patient received surgical resection of the affected lung and pleura. Patients received intracavity PDT at 630nm to a dose of 30 J/cm2, as determined by isotropic detectors sutured to the cavity walls. We measured the diffuse reflectance spectra before and after PDT in various positions within the cavity, including tumor, diaphragm, pericardium, skin, and chest wall muscle in 5 patients. The measurements we acquired using a specially designed fiber optic-based probe consisting of one fluorescence excitation fiber, one white light delivery fiber, and 9 detection fibers spaced at distances from 0.36 to 7.8 mm from the source, all of which are imaged via a spectrograph onto a CCD, allowing measurement of radially-resolved diffuse reflectance and fluorescence spectra. The light sources for these two measurements (a 403-nm diode laser and a halogen lamp, respectively) were blocked by computer-controlled shutters, allowing sequential fluorescence, reflectance, and background acquisition. The diffuse reflectance was analyzed to determine the absorption and scattering spectra of the tissue and from these, the concentration and oxygenation of hemoglobin and the local drug uptake. The total hemoglobin concentration in normal tissues varied from 50 to 300 µM, and the oxygen saturation was generally above 60%. One tumor measured exhibited higher hemoglobin concentration and lower saturation.

Effective Photodynamic therapy (PDT) treatment depends on the amount of active photosensitizer and the delivered light in the targeting tissue. For the same PDT treatment protocol, variation in photosensitizer uptake between animals induces variation in the treatment response between animals. This variation can be compensated via control of delivered light dose through photodynamic dose escalation based on online dosimetry of photosensitizer in the animal. The subcutaneous MAT-LyLu Dunning prostate tumor model was used in this study. Photosensitizer BPD-MA uptake was quantified by multiple fluorescence micro-probe measurements at 3 hours after verteporfin administration. PDT irradiation was carried out after photosensitizer uptake measurement with a total light dose of 75 J/cm2 and a light dose rate of 50 mW/cm2. Therapeutic response of PDT treatments was evaluated by the tumor regrowth assay. Verteporfin uptake varied considerably among tumors (inter-tumor variation 56% standard deviation) and within a tumor (largest intra-tumor variation 64%). An inverse correlation was found between mean photosensitizer intensity and PDT treatment effectiveness (R2 = 37.3%, p < 0.005). In order to compensate individual PDT treatments, photodynamic doses were calculated on an individual animal basis, by matching the light delivered to provide an equal photosensitizer dose multiplied by light dose. This was completed for the lower-quartile, mean and upper-quartile of the photosensitizer distribution. The coefficient of variance in the surviving fraction decreased from 24.9% in non-compensated PDT (NC-PDT) treatments to 16.0%, 14.0% and 15.9% in groups compensated to the lower-quartile (CL-PDT), the median (CM-PDT) and the upper-quartile (CU-PDT), respectively. In terms of treatment efficacy, the CL-PDT group was significantly less effective compared with NC-PDT, CM-PDT and CU-PDT treatments (p < 0.005). No significant difference in effectiveness was observed between NC-PDT, CM-PDT and CU-PDT. The results indicate that by measuring the mean photosensitizer concentration prior to light treatment, and then adjusting the light dose appropriately, a more uniform treatment can be applied to different animals thereby reducing the inter-individual variation in the treatment outcome.

In the research, the pseudo-model technique is proposed and implemented to simulate biological tissues under the framework of investigating near infrared (NIR) light propagating in diffusive media. The same optical characteristics inhere in the corresponding pseudo-models as in real tissues, where pseudo-models are constructed by using various volume densities of Intralipid. The pseudo-model technique proposed in the study has following advantages: 1.For the NIR tomography imaging system, the output signal from the real tissue may be too weak to be detected beyond the ability of current technologies. Thus, the pseudo-model is a viable alternative to cope with the limitation of the system in the measurement of real tissues. 2.Once the pseudo-model of a real tissue is decided, its optical properties can be investigated thoroughly. In addition, the initial estimates for the reconstruction of NIR optical property images can be selected adequately, and the image reconstruction algorithm can be modified accordingly with the information acquired from the pseudo-model. In the experiment, a pseudo-model of the background with an inclusion is performed for real tissues of pork inserted by a bone. It is observed that 1 % v.d. and 3 % v.d. of Intralipids can replace the pork and the bone, respectively, and the characteristics of the pseudo-model proposed here are consistent with those of the real tissues. As part of conclusion, the use of the pseudo-model technique is a promising approach to mimicking real tissues, especially for some parts of human body unable to be detected effectively.

Motivation: Photodynamic Therapy (PDT) with interstitial light delivery by multiple fibers for the treatment of large tissue volumes requires measurement of sensitizer distribution for dosimetric considerations. For stereotactic interstitial PDT of malignant glioma, for instance, a pre-irradiation comparison of the contrast enhancing tissue volume in MR-imaging with the photosensitized volume as assessed by fluorescence detection is desirable. For PDT of prostate cancer, the quantitative measurement of the selectivity of sensitizer uptake in cancer versus normal prostate parenchyma is important. Methods: It has previously been shown by others that the fluorescence intensity measured by a thin single optical fiber for excitation and detection is largely independent on optical parameters of the tissue that contains the fluorochrome. However, the investigators assumed similar values for excitation and emission wavelengths. This study concerned liquid phantom measurements (absorber: ink or hemoglobin, fluorochrome: Na-fluorescein) and Monte Carlo calculations, with extended conditions, where the absorption differs by a factor of 10 between excitation (426 nm) and emission (530 nm) wavelengths. The absorption coefficient (μ_a') was varied between 0.01 - 0.3 mm-1 (@ 426 nm), the effective scattering coefficient (μ_s') between 0.6 - 2.5 mm-1. A 200 μm and a 1000 μm core fiber were used. Results: Fluorescence intensity measured at 530 nm via a thin optical fiber (core diameter small compared to light penetration depth) depends minimally on optical tissue parameters. This result is valid for ink as absorber (μa identical at excitation and emission) as well as for hemoglobin (μa different). Fluorochrome concentration measurements seem possible with a 200 μm core fiber, but not with the 1000 μm core fiber.

Photodynamic therapy (PDT) is promising modality for cancer. Prostate cancer is the most common cancer in USA. We proposed transurethral prostate cancer treatment using the pulse-intensity-domain depth-controlled PDT to preserve urethra wall. We have found that photocytotoxicity has been suppressed under high-intensity pulsed excitation with the second generation photosensitizers. We aim to apply this effect to form intact portion on the surface of the irradiated field. Irradiation condition dependence of photocytotoxicity of rat prostate cancer cell line R3327-AT-1 was investigated with two clinical photosensitizers, Porfimer sodium and Talaporfin sodium. A pulsed laser was irradiated with the power energy density ranging from 1.25 to 10 mJ/cm2. Near-infrared luminescence from singlet oxygen in the solution of those two photosensitizers was measured transiently. We performed PDT against a rat subcutaneous prostate tumor mode with Talaporfin sodium (2mg/kg) injected intravenously 1 h prior to the irradiation. The laser was irradiated with the power energy density 2.5 or 10 mW/cm2, with the total fluence of 50 J/cm2. Photocytotoxicity in vitro and the singlet oxygen generation were both suppressed with the 10mJ/cm2 irradiation with Talaporfin sodium, while these with Porfimer sodium were kept relatively constant. The surface of the irradiated field of 1mm in thickness remained intact, while the tumor damaged layer of 1.3 mm in thickness was obtained in the case of 10mJ/cm2 irradiation. We think Talaporfin sodium has high sensitivity to the pulse energy density, which might be useful to realize urethra preserved PDT for prostate cancer.

Fluoresceinyl Cypridina Luciferin Analog (FCLA) is a chemiluminescence (CL) probe for detecting reactive oxygen species (ROS). Its detection efficiency of singlet oxygen can be significantly enhanced in the presence of human serum albumin (HSA). In the current study, the mechanism of the FCLA-HSA CL system is studied by means of direct CL measurement and spectroscopy techniques. Our results show that FCLA can combine with HSA via a single binding site to form a complex. The CL efficiency of the system is largely governed by an inter-system energy transfer between the two components upon interaction with singlet oxygen. The CL production reaches maximum in a synergetic manner when equal amount of FCLA and HSA are present simultaneously, but the production is less efficient at other ratios. This suggests that the FCLA-HSA system maybe used as a singlet oxygen detecting technique with higher sensitivity compared with that of conventional CL techniques. It may also provide a potential new technique for quantitatively analyze the presence of HSA in a sample.

We compared methylene blue (MB)-mediated photobactericidal efficacies against Pseudomonas aeruginosa when using nanosecond pulsed light and CW light. In the intensity range of 10-200 mW/cm2, there was no significant difference between two cases, while photobactericidal efficacy with nanosecond pulsed light was significantly lower than that with CW light at an intensity of 250 mW/cm2. This is attributable to the saturated absorption of MB molecules due to high peak intensity of nanosecond pulsed light. On the basis of these results, we estimated the depth dependence of bacterial killing, showing that in the skin tissue region deeper than 1.5 mm, photobactericidal efficacy with nanosecond pulsed light was higher than that with CW light. This suggests that the advantage of using high-peak-intensity pulsed light for deep tissue treatment.

Epilepsy is surgically curable if the seizure focus can be localized and does not include areas of eloquent cortex. Because epileptic cells are indistinct from surrounding brain, resection typically includes normal tissue. Using the rat kindling model of epilepsy, we evaluated Photodynamic Therapy (PDT) as a super-selective lesioning technique. We present a series of pilot studies to evaluate: 1) Protoporphyrin IX (PpIX) fluorescence, 2) the efficacy of PDT to raise seizure thresholds, 3) the safety of PDT using behavioral studies, and 4) histologic results. Bipolar electrodes were chronically implanted into the cortex and animals received successive low-level stimulation generating seizures of increasing severity. Following 5-aminolevulinic acid (ALA) administration, fully kindled rats received electrical stimulation to induce a generalized seizure. Animals were irradiated with laser light focused onto a temporal craniectomy. Our results show: 1) an increase in PpIX fluorescence in the seizure group, 2) PDT treated animals failed to demonstrate seizure activity following repeat stimulation, 3) no statistically significant difference between treated and control animals were observed on behavioral tests, 4) histology showed pyknotic hippocampal pyramidal cells in the CA3 region without areas of obvious necrosis. In conclusion, this is the first report of heightened PpIX-mediated fluorescence in epileptic brain. The selective accumulation of PpIX with laser PDT may provide a less invasive and more precise technique for obliteration of epileptic foci. PDT warrants additional research to determine if this technique may augment or replace existing procedures for the surgical management of epilepsy.

Photodynamic therapy (PDT) continues to evolve into a mature clinical treatment of a variety of cancer types as well as age-related macular degeneration of the eye. However, there are still aspects of PDT that need to be improved in order for greater clinical acceptance. While a number of new PDT photo-sensitizers, sometimes referred to as second- or third- generation therapeutic agents, are currently under clinical investigation, the direct treatment through the skin of subcutaneous tumors deeper than 5 mm remains problematic. Currently approved PDT porphyrin photo-sensitizers, as well as several modified porphyrins (e.g. chlorins, bacteriochlorins, etc.) that are under clinical investigation can be activated at 630-730 nm, but none above 800 nm. It would be highly desirable if new PDT paradigms could be developed that would allow photo-activation deep in the tissue transparency window in the Near-infrared (NIR) above 800 nm to reduce scattering and absorption phenomena that reduce deep tissue PDT efficacy. Rasiris and MPA Technologies have developed new porphyrins that have greatly enhanced two-photon absorption ( P A ) cross-sections and can be activated deep in the NIR (ca. 780-850 nm). These porphyrins can be incorporated into a therapeutic triad that also employs an small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR onephoton imaging agent that allows tracking the delivery of the triad to the tumor site, as well as clearance of excess triad from healthy tissue prior to the start of PDT treatment. We are currently using these new triads in efficacy studies with a breast cancer cell line that has been transfected with luciferase genes that allow implanted tumor growth and post- PDT treatment efficacy studies in SCID mouse models by following the rise and decay of the bioluminescence signal. We have also designed highly absorbing and scattering collagen breast cancer phantoms in which we have demonstrated dramatic cell kill to a depth of at least 4 cm. We have also demonstrated that at the wavelength and laser fluences used in the treatment of implanted tumors in the mouse mammary fat pads, there is little, if any, damage to the skin or internal mouse organs. In addition, we have also demonstrated that the implanted tumors can be treated to a depth of more than 1 cm by direct radiation through the dorsal side of the mouse.

A non contact all-optical method for surface photoacoustic is described. The surface acoustic waves were excited employing a ArF excimer laser in the 10 µm thin polyimide film and detected with a Michelson interferometer using 633 nm HeNe laser due to an active stabilization for interferometer a surface displacement of 0.2 A. could be detected used for the purpose of discriminating between normal and maligned tissue. The acoustic waves propagate several millimeters in the sample differentiating maligned areas from normal breast tissue samples which having different acoustic impedance.

Photodynamic therapy (PDT) has been widely studied in the last decades and it is becoming a promising tool in the treatment of tumors of many kinds. PDT is based on photoactivation of a sensitized drug that is restrained in the tumor cells, producing highly reactive species that can destroy tumoral cells with minimum collateral effect. This study aimed to evaluate the effect of the PDT in induced neoplasias of the colon by 1,2-dimetilhidrazine in rats, using as photosensitizing drug the chloroaluminum phthalocyanine incorporated to the liposomes and to compare the methods of irradiation using continuous or fractionated energy in PDT. Ten Wistar rats were distributed randomly in 3 groups (G1, G2 and C), anaesthetized and submitted to PDT with of fractionated (G1) or continuum (G2) irradiation energy using as a source of excitement an InGaAl laser. After 3 hours of the laser irradiation, 2 animals of the G1 group, 2 animals of the G2 group and 1 animal of C group were sacrificed and samples of tumoral tissue were collected for histological analysis; the same procedure was carried through 24 hours after irradiation. There were no significant differences between the extensions of the induced areas of necrosis for PDT in the groups under fractionated or continuous irradiation for the parameters used in this study. New studies must be carried through, using different parameters and intervals of laser irradiation, aiming to maximize the effect of the PDT for the treatment of colon tumors.

Photodynamic therapy (PDT) is the method of photosensitized tumor treatment. It is based on the photosensitizer (PS) selective accumulation in tumors, its subsequent activation under the light influence and oxygen active form formation that results in tumor destruction. Photodynamic action of some new water-soluble porphyrins was investigated in our laboratory. Dose-dependent effect of these porphyrins was shown on PC-12 murine pheochromocytoma cell line. The results revealed that the efficiency of the investigated porphyrins decreased in the following way: TOEPyP (meso-tetra-(4-N-oxyethylpyridyl)porphyrin) > Zn-TOEPyP > Ag-TOEPyP. It was shown that TOEPyP possessed nearly the same photodynamic activity (LD50) as well-known photosensitizer chlorin e6. These porphyrins have also demonstrated quite high photodynamic activity in vivo. The results were obtained in the experiments on white mice with engrafted C-180 (Croker's sarcoma). Antitumor activity of these porphyrins in the dark was 30-40%, whereas photodynamic activity was 45-60%.

The Monte Carlo (MC) method of calculating light distributions in turbid media such as tissue has become the gold standard, especially in complex geometries and heterogeneous tissue. The utility of the MC method, however, is limited by is computational intensity. In an effort to reduce the time needed for MC calculations, we have adapted a macro- Monte Carlo (MMC) method (Neuenschwander, et al. 1995, Phys. Med. Biol. 40, 543-574) to the solution of tissue optics problems. Traditional MC routines trace individual photons step-by-step through the tissue. Instead, the MMC approach relies on a data set consisting of spheres in which the light absorbed in each voxel is pre-calculated using a traditional MC routine. At each MMC step, the pre-calculated absorbed light dose in the appropriate sphere, aligned to the current position and direction of the sphere, is recorded in the dose matrix. The position and direction of the photon exiting the sphere are chosen from the exit distribution of the pre-calculated sphere, and the process is repeated. By choosing the size of the pre-calculated sphere appropriately, arbitrarily complex boundary geometries can be simulated. We compare the accuracy and calculation time of the MMC method with a traditional MC algorithm for a variety of tissue optical properties and geometries. We find that the MMC algorithm can increase the speed of calculation by as much as two orders of magnitude, depending on the optical properties being simulated, without a significant loss in accuracy.

Although phototriggered cleavage of chemical bonds induced by single-photon or two-photon-absorption provides attractive tools for controlled drug delivery, the choice of drugs is still limited by the linker system to which the therapeutic molecules need to be bound covalently. The use of a multifunctional linker system suitable for coupling a broad spectrum of drugs to the polymeric carrier will open a new field for drug delivery. We have developed a novel photocleavable multifunctional linker system based on coumarin dimers, whose unique photochemical behavior are well characterized. As a first example, an acrylic polymer-drug conjugate with antimetabolites is explored. The cleavage of the link between the drug and the polymer backbone is triggered by both single- as well as two-photon absorption. The release of the drug is investigated. It is possible to manufacture a polymeric drug delivery device with several drugs in different areas. In particular the two-photon-absorption induced process offers the possibility to address the drug of interest owing to the superior spatial resolution. The key to such devices is a versatile linker-system which can be adopted to work with various drug compounds.

Ten trans-A2B and A3-type corrole photosensitizers carrying functional groups were synthesized and screened for PDT activities. Photocytotoxicity was measured by the MTT cell reduction assay on a cultured human nasopharyngeal carcinoma (NPC) cell line (HONE-1). Experimental results indicated that corroles containing a single hydroxyphenyl substituent (3, 4 and 5) exhibit the highest activity among the corrole derivatives investigated. Confocal microscopy revealed that the site of cellular localization of the photosensitizers is predominantly at mitochondria. Also, nuclear staining detected apoptotic cell death.

Introduction: The external application of aminoleavulinic acid (ALA), which is a substrate of physiologic cell metabolism, represents a possible treatment option in superficial basal cell carcinomas (BCC). The development of new ALA-esters (mALA) with potential for higher penetration depths promises higher therapeutic success. This research aimed to prove the following hypothesis: The cytotoxic effect of the mALA- photodynamic therapy (mALA-PDT), when compared to the ALA-PDT, leads to a higher clinical success rate. Material and Methods: 24 patients with multiple facial tumors, after having received several local surgical excisions with known histology, were treated with either ALA- or mALA-PDT, during the past two years. In total, 89 basal cell carcinoma, 45 actinic keratoses, 6 keratoacanthoma, and 2 squamous cell carcinomas were treated. ALA-PDT: A thermo gel with 40 % mALA or ALA was applied from a cooled syringe. Three to five hours after gel application the skin was cleaned from any gel residues. Irradiation was done with a diode laser and was performed in two sessions, each 10 min long. After intervals of 2, 4 and 12 weeks, the patients were recalled to assess therapeutic efficacy. This was followed by photographic documentation. Results: More than 80% of the tumors treated primarily were resolved successfully. A recurrence rate of approximately 15% was observed. Three per cent of the tumors showed no reaction to therapy. There were no statistically significant differences between the two therapeutic groups. Discussion: The advantage of the use of ALA lies foremost in the fast metabolic use of the body's own photosensitizer PpIX. There are no known side effects of this therapy. Moreover, external application is superior to systemic application with regard to patient management. The method can be combined with other therapies. Although the mALA should have a better penetration in tumor tissue, the therapeutic outcome is similar to the use of ALA.

Photodynamic therapy using 5-aminolevulinic acid is an effective therapy for treating basal cell carcinoma, characterized by high lesion clearance and excellent cosmetic outcomes. Treatment optimization and lesion-tailored treatments making use of real-time treatment assessment promise still greater efficacy and improved comfort for patients. In order to monitor treatment parameters during therapy, instrumentation of our own design delivers a 633 nm treatment beam while simultaneously collecting fluorescence spectra. Fluorescence spectra from 650-800 nm are corrected for the effects of tissue optical properties and report protoporphyrin IX (PpIX) photobleaching as well as photoproduct dynamics in the lesion and in the perilesion margin during therapy. Brief treatment interruptions are made for acquisition of white light reflectance spectra from 420-800 nm that are used to generate corrections to fluorescence spectra and can be used to deduce blood volume and hemoglobin oxygen saturation. LabVIEW and Matlab scripts are used for real-time data analysis. Measurements have been made on 5 patients (7 BCC lesions) with a treatment fluence rate of 150 mW cm-2 and on 5 additional patients (5 BCC lesions) at 10 mW cm-2. Measurements are made for each lesion until greater than 90% photobleaching of PpIX is detected at which point the balance of the prescribed fluence is delivered at 150 mW cm-2 without interruption. PpIX bleaching rates between the two fluence rates varied significantly. These measurements were carried out during ALA-PDT treatment of BCC as part of a pilot study designed to guide treatment fluence and fluence rates in an anticipated clinical trial.

This work investigates the design of optimum distribution of photon density power among the source positions, and optimum modulation frequencies to maximize the detectability of heterogeneities embedded in turbid medium using near infrared light. The optimum waveforms are designed for the sources in near-infrared diffuse optical tomography which involves reconstruction of spatially varying optical properties of turbid medium as well as fluorophore lifetime and yield from boundary measurements. We start our analysis by first deriving the discrete source-to-detector map based on the finite-element formulation of the diffuse photon density wave equation and Robin boundary conditions. We determine statistical figures of merit to maximize the contrast of heterogeneities with respect to a given background. Next, we design optical waveforms that will maximize the figure of merit for the detectability of heterogeneities. When the figure of merit is derived based on optimal linear detection under the assumption of Gaussianity, the optimal source vector is given by the eigenvector corresponding to the maximum eigenvalue of the norm of the differences between the source-to-detector maps of homogeneous and heterogeneous domains. We extended our approach to investigate the optimum spatial positions and intensities of point sources to maximize the detectability of the heterogeneities. We explored the effect of tumor location with respect to the sources, tumor size, and the number of sources on detectability.

Background: Photodynamic therapy using porfimer sodium (Ps-PDT) is approved for use in patients with Barrett's highgrade dysplasia and esophageal carcinoma. Ps-PDT light dosimetry, however, is critically important to treatment outcomes since insufficient ablation results in residual dysplasia and carcinoma while excessive treatment results in stricture formation. Aim: The aim of this study was to model esophageal PDT with optical absorption and scattering coefficients derived from an ex-vivo porcine multilayer esophagus model. Methods: Optical coefficients were derived for the mucosal and muscle layers of normal pig esophagus. The mucosal layer (mucosa, muscularis mucosa and submucosa) was separated from the muscle layer. Diffuse reflectance and transmittance were measured with an integrating sphere spectrophotometer. Absorption and reduced scattering coefficients were determined with the inverse adding doubling method. (Table not available in abstract, see pdf of paper) Multilayer Monte Carlo simulation and single-layer mathematical dosimetry equations were employed to model esophageal PDT with the derived coefficients. Porfimer sodium addition was modeled with an increase in both absorption and scattering. Depth of injury, assumed to require a threshold light dose, was estimated for various light doses commonly used in clinical practice. Depth of injury was then compared to clinical outcomes reported in the literature for various light doses.

Photodynamic therapy is an effective, minimally invasive skin cancer treatment modality with few side effects. Improved therapeutic selectivity and efficacy is expected if treatment is optimized individually for each patient based on detailed measurements prior and during the treatment. The handheld system presented allows measuring optical properties of the skin, the rate of photosensitizer photobleaching during the ALA PDT and oxygen saturation in the tissue. The photobleaching rate is monitored using fluorescence spectroscopy, where protoporphyrin IX in tissue is exited by 410 nm (blue) or 532 nm (green) laser light, and fluorescence in the 580-800 nm range is monitored. The photobleaching rate is calculated by correlating the measured spectrum with known protoporphyrin IX, photoproduct and nonspecific tissue autofluorescence spectra using correlation analysis. Double-wavelength excitation allows a rough estimation of the depth of the fluorescence source due to the significant difference in penetration depth for blue and green light. Blood concentration and oxygenation in the tissue are found from the white light reflectance spectrum in the 460-800 nm range. Known spectra for the oxy- and deoxyhemoglobin, melanin, and tissue baseline absorption and tissue scattering are substituted in nonlinear equations to find the penetration depth and diffuse reflectance coefficient. The nonlinear equation for the diffuse reflectance coefficient is solved for blood and melanin concentrations and blood oxygenation values that provide the best fit to the measured spectrum. The optical properties of the tissue obtained from the reflectance spectroscopy are used to correct the fluorescence data. A noncontact probe with 5 fibers (3 excitation and 2 detection) focused to the same 5 mm diameter spot: 2 excitation lasers, a white light lamp and a two-channel spectrometer are used. A LabView program with custom nonlinear equation solvers written in C++ automatically performs the measurements and calculations, and writes data to a database. The system is currently used in a clinical trial to find the relationship between skin pigmentation, oxygen saturation in blood, photobleaching rate and optimal fluence rate for skin cancer treatment of actinic keratoses.

A non-contact fluorescence diffuse optical tomography (DOT) system capable of producing B-scan-type images of localized fluorescence regions up to depths of 15mm is presented. The B-Scan mode is analogous to ultrasound where the excitation and remission signals are delivered from the same surface of the tissue. This optical fluorescence system utilizes a 635 nm diode laser and two orthogonal galvanometers to raster scan the position of the source along the tissue surface. Using Protoporphyrin IX (PpIX) as a fluorescent agent, the amplitude of the remitted signal is separated by a 650 nm long pass filter and the fluorescence is then detected by a cooled CCD camera. Images are acquired for all source positions along the surface of the tissue, providing remission intensity images for each. This volume data set is then used in image reconstruction of the sub-surface volume, via a finite element method of modeling the fluorescence diffusion. The optimal remission imaging geometry, in terms of depth sensitivity, computation time, and image contrast-to-noise, was determined by performing sensitivity and singular-value decomposition analysis of the Jacobian for various source/detector combinations. The simulated results indicate that the fluorophore concentration and the inclusion size can not be recovered accurately in this mode; however, it is shown that the inclusion depth can accurately be predicted. Finally, by performing simulations on a mesh created from an MR image, we show that our system may prove useful in predicting the regions of local tissue fluorescence in the application of resection of residual brain tumor tissue under fluorescence guidance. This non-contact diagnostic system is being calibrated and finalized for potential use in this application of sub-surface imaging in the brain.

Mixed-metal supramolecular complexes coupling Ru and Os light absorber units to a central, reactive Rh site have been designed of the type [{(bpy)2M(dpp)}2RhCl2]5+. These complexes possess intense metal-to-ligand charge transfer transitions when excited at 500-700 nm making them good light absorbers. The presence of the Rh site introduces low lying metal-to-metal charge transfer states that are capable of visible light induced photocleavage of DNA via an oxygen independent pathway.1 We report here a study of the photodynamic action of supramolecular mixed-metal complexes showing that these systems inhibit cell replication after exposure to light while displaying no impact on cell replication in the dark. This photodynamic action has been studied using cultured Vero cells with a pre-incubation with the complexes, rinsing to remove complex from the media, followed by photo-activation and cell growth assay. The photodynamic action of this new series of complexes can be tuned as a function of components of the supramolecular assembly and complexes capable of coupling to targeting proteins and fluorescent reporter groups have been synthesized.