Computational Modeling and Imaging of the Intracellular Oxygen Gradient.

Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure (pO2). This study develops and evaluates a finite-element model of oxygen-consuming mitochondrial bioenergetics using the COMSOL Multiphysics program. The model derives steady-state oxygen (O2) distributions from Fickian diffusion and Michaelis-Menten consumption kinetics in the mitochondria and cytoplasm. Intrinsic model parameters such as diffusivity and maximum consumption rate were estimated from previously published values for isolated and intact mitochondria. The model was compared with experimental data collected for the intracellular and mitochondrial pO2 levels in human cervical cancer cells (HeLa) in different respiratory states and under different levels of imposed pO2. Experimental pO2 gradients were measured using lifetime imaging of a Förster resonance energy transfer (FRET)-based O2 sensor, Myoglobin-mCherry, which offers in situ real-time and noninvasive measurements of subcellular pO2 in living cells. On the basis of these results, the model qualitatively predicted (1) the integrated experimental data from mitochondria under diverse experimental conditions, and (2) the impact of changes in one or more mitochondrial processes on overall bioenergetics.

Mitochondria and oxygen homeostasis.

Molecular oxygen possesses a dual nature due to its highly reactive free radical property: it is capable of oxidizing metabolic substrates to generate cellular energy, but can also serve as a substrate for genotoxic reactive oxygen species generation. As a labile substance upon which aerobic life depends, the mechanisms for handling cellular oxygen have been fine-tuned and orchestrated in evolution. Protection from atmospheric oxygen toxicity as originally posited by the Endosymbiotic Theory of the Mitochondrion is likely to be one basic principle underlying oxygen homeostasis. We briefly review the literature on oxygen homeostasis both in vitro and in vivo with a focus on the role of the mitochondrion where the majority of cellular oxygen is consumed. The insights gleaned from these basic mechanisms are likely to be important for understanding disease pathogenesis and developing strategies for maintaining health.

Developing Analysis Protocols for Monitoring Intracellular Oxygenation Using Fluorescence Lifetime Imaging of Myoglobin-mCherry.

Oxygen (O2) is a critical metabolite for cellular function as it fuels aerobic cellular metabolism; further, it is a known regulator of gene expression. Monitoring oxygenation within cells and organelles can provide valuable insights into how O2, or lack thereof, both influences and responds to cell processes. In recent years, fluorescence lifetime imaging microscopy (FLIM) has been used to track several probe concentration independent intracellular phenomena, such as pH, viscosity, and, in conjunction with Förster resonance energy transfer (FRET), protein-protein interactions. Here, we describe methods for synthesizing and expressing the novel FLIM-FRET intracellular O2 probe Myoglobin-mCherry (Myo-mCherry) in cultured cell lines, as well as acquiring FLIM images using a laser scanning confocal microscope configured for two-photon excitation and a time-correlated single photon counting (TCSPC) module. Finally, we provide step-by-step protocols for FLIM analysis of Myo-mCherry using the commercial software SPCImage and conversion of fluorescence lifetime values in each pixel to apparent intracellular oxygen partial pressures (pO2).

1O2 determined from the measured PDT dose and 3O2 predicts long-term response to Photofrin-mediated PDT.

Photodynamic therapy (PDT) that employs the photochemical interaction of light, photosensitizer and oxygen is an established modality for the treatment of cancer. However, dosimetry for PDT is becoming increasingly complex due to the heterogeneous photosensitizer uptake by the tumor, and complicated relationship between the tissue oxygenation ([3O2]), interstitial light distribution, photosensitizer photobleaching and PDT effect. As a result, experts argue that the failure to realize PDT's true potential is, at least partly due to the complexity of the dosimetry problem. In this study, we examine the efficacy of singlet oxygen explicit dosimetry (SOED) based on the measurements of the interstitial light fluence rate distribution, changes of [3O2] and photosensitizer concentration during Photofrin-mediated PDT to predict long-term control rates of radiation-induced fibrosarcoma tumors. We further show how variation in tissue [3O2] between animals induces variation in the treatment response for the same PDT protocol. PDT was performed with 5 mg kg-1 Photofrin (a drug-light interval of 24 h), in-air fluence rates (ϕ air) of 50 and 75 mW cm-2 and in-air fluences from 225 to 540 J cm-2. The tumor regrowth was tracked for 90 d after the treatment and Kaplan-Meier analyses for local control rate were performed based on a tumor volume ⩽100 mm3 for the two dosimetry quantities of PDT dose and SOED. Based on the results, SOED allowed for reduced subject variation and improved treatment evaluation as compared to the PDT dose.

Genetically encoded FRET probes for direct mapping and quantification of intracellular oxygenation level via fluorescence lifetime imaging.

Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level and its concentration is used for the evaluation of many diseases (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration ([O2]) in living cells, tissues and organisms is challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor [O2] in the intracellular environment. We fused Myoglobin (Myo), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair, Myo-mCherry. The changes in the spectral properties of Myoglobin upon oxygen binding result in changes of the FRET-depleted emission intensity of mCherry, and this effect is detected by monitoring the fluorescence lifetime of the probe. We present here the preparation and characterization of a series of Myo-mCherry variants and mutants that show the versatility of our protein-based approach: the dynamic range of the sensor is tunable and adaptable to different [O2] ranges, as they occur in vitro in different cell lines, the probe is also easily targeted to subcellular compartments. The use of fluorescence overcomes the most common issues of data collection speed and spatial resolution encountered by currently available methods for O2-monitoring. By using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we can map the oxygenation level of cells in vitro, providing a quantitative assessment of [O2].

Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.

Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.

Three-dimensional finite-element based deformable image registration for evaluation of pleural cavity irradiation during photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is used after surgical resection to treat the microscopic disease for malignant pleural mesothelioma and to increase survival rates. As accurate light delivery is imperative to PDT efficacy, the deformation of the pleural volume during the surgery is studied on its impact on the delivered light fluence. In this study, a three-dimensional finite element-based (3D FEM) deformable image registration is proposed to directly match the volume of lung to the volume of pleural cavity obtained during PDT to have accurate representation of the light fluence accumulated in the lung, heart and liver (organs-at-risk) during treatment. METHODS: A wand, comprised of a modified endotrachial tube filled with Intralipid and an optical fiber inside the tube, is used to deliver the treatment light. The position of the treatment is tracked using an optical tracking system with an attachment comprised of nine reflective passive markers that are seen by an infrared camera-based navigation system. This information is used to obtain the surface contours of the plural cavity and the cumulative light fluence on every point of the cavity surface that is being treated. The lung, heart, and liver geometry are also reconstructed from a series of computed tomography (CT) scans of the organs acquired in the same patient before and after the surgery. The contours obtained with the optical tracking system and CTs are imported into COMSOL Multiphysics, where the 3D FEM-based deformable image registration is obtained. The delivered fluence values are assigned to the respective positions (x, y, and z) on the optical tracking contour. The optical tracking contour is considered as the reference, and the CT contours are used as the target, which will be deformed. The data from three patients formed the basis for this study. RESULTS: The physical correspondence between the CT and optical tracking geometries, taken at different times, from different imaging devices was established using the 3D FEM-based image deformable registration. The volume of lung was matched to the volume of pleural cavity and the distribution of light fluence on the surface of the heart, liver and deformed lung volumes was obtained. CONCLUSION: The method used is appropriate for analyzing problems over complicated domains, such as when the domain changes (as in a solid-state reaction with a moving boundary), when the desired precision varies over the entire domain, or when the solution lacks smoothness. Implementing this method in real-time for clinical applications and in situ monitoring of the under- or over- exposed regions to light during PDT can significantly improve the treatment for mesothelioma.

Evaluation of singlet oxygen explicit dosimetry for predicting treatment outcomes of benzoporphyrin derivative monoacid ring A-mediated photodynamic therapy.

Existing dosimetric quantities do not fully account for the dynamic interactions between the key components of photodynamic therapy (PDT) or the varying PDT oxygen consumption rates for different fluence rates. Using a macroscopic model, reacted singlet oxygen ( [ O 2 1 ] rx ) was calculated and evaluated for its effectiveness as a dosimetric metric for PDT outcome. Mice bearing radiation-induced fibrosarcoma tumors were treated with benzoporphyrin derivative monoacid ring A (BPD) at a drug-light interval of 3 h with various in-air fluences (30 to 350 ?? J / cm 2 ) and in-air fluence rates (50 to 150 ?? mW / cm 2 ). Explicit measurements of BPD concentration and tissue optical properties were performed and used to calculate [ O 2 1 ] rx , photobleaching ratio, and PDT dose. For four mice, in situ measurements of O 2

A Comparison of Dose Metrics to Predict Local Tumor Control for Photofrin-mediated Photodynamic Therapy.

This preclinical study examines light fluence, photodynamic therapy (PDT) dose and "apparent reacted singlet oxygen," [1 O2 ]rx , to predict local control rate (LCR) for Photofrin-mediated PDT of radiation-induced fibrosarcoma (RIF) tumors. Mice bearing RIF tumors were treated with in-air fluences (50-250 J cm-2 ) and in-air fluence rates (50-150 mW cm-2 ) at Photofrin dosages of 5 and 15 mg kg-1 and a drug-light interval of 24 h using a 630-nm, 1-cm-diameter collimated laser. A macroscopic model was used to calculate [1 O2 ]rx and PDT dose based on in vivo explicit dosimetry of the drug concentration, light fluence and tissue optical properties. PDT dose and [1 O2 ]rx were defined as a temporal integral of drug concentration and fluence rate, and singlet oxygen concentration consumed divided by the singlet oxygen lifetime, respectively. LCR was stratified for different dose metrics for 74 mice (66 + 8 control). Complete tumor control at 14 days was observed for [1 O2 ]rx ≥ 1.1 mm or PDT dose ≥1200 µm J cm-2 but cannot be predicted with fluence alone. LCR increases with increasing [1 O2 ]rx and PDT dose but is not well correlated with fluence. Comparing dosimetric quantities, [1 O2 ]rx outperformed both PDT dose and fluence in predicting tumor response and correlating with LCR.

A Comparison of Singlet Oxygen Explicit Dosimetry (SOED) and Singlet Oxygen Luminescence Dosimetry (SOLD) for Photofrin-Mediated Photodynamic Therapy.

Accurate photodynamic therapy (PDT) dosimetry is critical for the use of PDT in the treatment of malignant and nonmalignant localized diseases. A singlet oxygen explicit dosimetry (SOED) model has been developed for in vivo purposes. It involves the measurement of the key components in PDT-light fluence (rate), photosensitizer concentration, and ground-state oxygen concentration ([³O2])-to calculate the amount of reacted singlet oxygen ([¹O2]rx), the main cytotoxic component in type II PDT. Experiments were performed in phantoms with the photosensitizer Photofrin and in solution using phosphorescence-based singlet oxygen luminescence dosimetry (SOLD) to validate the SOED model. Oxygen concentration and photosensitizer photobleaching versus time were measured during PDT, along with direct SOLD measurements of singlet oxygen and triplet state lifetime (τΔ and τt), for various photosensitizer concentrations to determine necessary photophysical parameters. SOLD-determined cumulative [¹O2]rx was compared to SOED-calculated [¹O2]rx for various photosensitizer concentrations to show a clear correlation between the two methods. This illustrates that explicit dosimetry can be used when phosphorescence-based dosimetry is not feasible. Using SOED modeling, we have also shown evidence that SOLD-measured [¹O2]rx using a 523 nm pulsed laser can be used to correlate to singlet oxygen generated by a 630 nm laser during a clinical malignant pleural mesothelioma (MPM) PDT protocol by using a conversion formula.

Explicit macroscopic singlet oxygen modeling for benzoporphyrin derivative monoacid ring A (BPD)-mediated photodynamic therapy.

Photodynamic therapy (PDT) is an effective non-ionizing treatment modality that is currently being used for various malignant and non-malignant diseases. In type II PDT with photosensitizers such as benzoporphyrin monoacid ring A (BPD), cell death is based on the creation of singlet oxygen (1O2). With a previously proposed empirical five-parameter macroscopic model, the threshold dose of singlet oxygen ([1O2]rx,sh]) to cause tissue necrosis in tumors treated with PDT was determined along with a range of the magnitude of the relevant photochemical parameters: the photochemical oxygen consumption rate per light fluence rate and photosensitizer concentration (ξ), the probability ratio of 1O2 to react with ground state photosensitizer compared to a cellular target (σ), the ratio of the monomolecular decay rate of the triplet state photosensitizer (ß), the low photosensitizer concentration correction factor (δ), and the macroscopic maximum oxygen supply rate (g). Mice bearing radiation-induced fibrosarcoma (RIF) tumors were treated interstitially with a linear light source at 690nm with total energy released per unit length of 22.5-135J/cm and source power per unit length of 12-150mW/cm to induce different radii of necrosis. A fitting algorithm was developed to determine the photochemical parameters by minimizing the error function involving the range between the calculated reacted singlet oxygen ([1O2]rx) at necrosis radius and the [1O2]rx,sh. [1O2]rx was calculated based on explicit dosimetry of the light fluence distribution, the tissue optical properties, and the BPD concentration. The initial ground state oxygen concentration ([3O2]0) was set to be 40µM in this study. The photochemical parameters were found to be ξ=(55±40)×10-3cm2mW-1s-1, σ=(1.8±3)×10-5µM-1, and g=1.7±0.7µMs-1. We have taken the literature values for δ=33µM, and ß=11.9µM. [1O2]rx has shown promise to be a more effective dosimetry quantity for predicting necrosis than either light dose or PDT dose, where the latter is simplistically a temporal integral of the products of the photosensitizer concentration and light fluence rate.

Evaluation of the 2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) mediated photodynamic therapy by macroscopic singlet oxygen modeling.

Photodynamic therapy (PDT) is known as a non-invasive treatment modality that is based on photochemical reactions between oxygen, photosensitizer, and a special wavelength of light. However, a dosimetric predictor for PDT outcome is still elusive because current dosimetric quantities do not account for the differences in the PDT oxygen consumption rate for different fluence rates. In this study, we evaluate several dose metrics, total fluence, photobleaching ratio, PDT dose, and mean reacted singlet oxygen (mean [1 O2 ]rx ) for predicting the PDT outcome and a clinically relevant tumor re-growth endpoint. For this reason, radiation-induced fibrosarcoma (RIF) mice tumors are treated with 2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) and different in-air fluences (30 J/cm2 , 50 J/cm2 , 135 J/cm2 , 250 J/cm2 , and 350 J/cm2 ) and in-air fluence rates (20, 50, 75, 150 mW/cm2 ). Explicit measurements of HPPH and oxygen concentration as well as tissue optical properties are performed pre- and post-treatment. Then, this information is incorporated into a macroscopic model to calculate the photobleaching, PDT dose, and mean [1 O2 ]rx . Changes in tumor volume are tracked following the treatment and compared with the dose metrics. The correlation demonstrates that mean [1 O2 ]rx  serves as a better dosimetric quantity for predicting treatment outcome and a clinically relevant tumor re-growth endpoint.

Macroscopic singlet oxygen modeling for dosimetry of Photofrin-mediated photodynamic therapy: an in-vivo study.

Although photodynamic therapy (PDT) is an established modality for cancer treatment, current dosimetric quantities, such as light fluence and PDT dose, do not account for the differences in PDT oxygen consumption for different fluence rates ( ? ). A macroscopic model was adopted to evaluate using calculated reacted singlet oxygen concentration ( [ O 2 1 ] rx ) to predict Photofrin-PDT outcome in mice bearing radiation-induced fibrosarcoma tumors, as singlet oxygen is the primary cytotoxic species responsible for cell death in type II PDT. Using a combination of fluences (50, 135, 200, and 250 ?? J / cm 2 ) and ? (50, 75, and 150 ?? mW / cm 2 ), tumor regrowth rate, k , was determined for each condition. A tumor cure index, CI = 1 ? k / k control , was calculated based on the k between PDT-treated groups and that of the control, Available on the SPIE Digital Library.

Investigating the impact of oxygen concentration and blood flow variation on photodynamic therapy.

Type II photodynamic therapy (PDT) is used for cancer treatment based on the combined action of a photosensitizer, a special wavelength of light, oxygen (3O2) and generation of singlet oxygen (1O2). Intra-patient and inter-patient variability of oxygen concentration ([3O2]) before and after the treatment as well as photosensitizer concentration and hemodynamic parameters such as blood flow during PDT has been reported. Simulation of these variations is valuable, as it would be a means for the rapid assessment of treatment effect. A mathematical model has been previously developed to incorporate the diffusion equation for light transport in tissue and the macroscopic kinetic equations for simulation of [3O2], photosensitizers in ground and triplet states and concentration of the reacted singlet oxygen ([1O2]rx) during PDT. In this study, the finite-element based calculation of the macroscopic kinetic equations is done for 2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH)-mediated PDT by incorporating the information of the photosensitizer photochemical parameters as well as the tissue optical properties, photosensitizer concentration, initial oxygen concentration ([3O2]0), blood flow changes and ϕ that have been measured in mice bearing radiation-induced fibrosarcoma (RIF) tumors. Then, [1O2]rx calculated by using the measured [3O2] during the PDT is compared with [1O2]rx calculated based on the simulated [3O2]; both calculations showed a reasonably good agreement. Moreover, the impacts of the blood flow changes and [3O2]0 on [1O2]rx have been investigated, which showed no pronounced effect of the blood flow changes on the long-term 1O2 generation. When [3O2]0 becomes limiting, small changes in [3O2] have large effects on [1O2]rx.

Dosimetry study of PHOTOFRIN-mediated photodynamic therapy in a mouse tumor model.

It is well known in photodynamic therapy (PDT) that there is a large variability between PDT light dose and therapeutic outcomes. An explicit dosimetry model using apparent reacted 1O2 concentration ([1O2]rx) has been developed as a PDT dosimetric quantity to improve the accuracy of the predicted ability of therapeutic efficacy. In this study, this explicit macroscopic singlet oxygen model was adopted to establish the correlation between calculated reacted [1O2]rx and the tumor growth using Photofrin-mediated PDT in a mouse tumor model. Mice with radiation-induced fibrosarcoma (RIF) tumors were injected with Photofrin at a dose of 5 mg/kg. PDT was performed 24h later with different fluence rates (50, 75 and 150 mW/cm2) and different fluences (50 and 135 J/cm2) using a collimated light applicator coupled to a 630nm laser. The tumor volume was monitored daily after PDT and correlated with the total light fluence and [1O2]rx. Photophysical parameters as well as the singlet oxygen threshold dose for this sensitizer and the RIF tumor model were determined previously. The result showed that tumor growth rate varied greatly with light fluence for different fluence rates while [1O2]rx had a good correlation with the PDT-induced tumor growth rate. This preliminary study indicated that [1O2]rx could serve as a better dosimetric predictor for predicting PDT outcome than PDT light dose.

Deformable medical image registration of pleural cavity for photodynamic therapy by using finite-element based method.

When the pleural cavity is opened during the surgery portion of pleural photodynamic therapy (PDT) of malignant mesothelioma, the pleural volume will deform. This impacts the delivered dose when using highly conformal treatment techniques. To track the anatomical changes and contour the lung and chest cavity, an infrared camera-based navigation system (NDI) is used during PDT. In the same patient, a series of computed tomography (CT) scans of the lungs are also acquired before the surgery. The reconstructed three-dimensional contours from both NDI and CTs are imported into COMSOL Multiphysics software, where a finite element-based (FEM) deformable image registration is obtained. The CT contour is registered to the corresponding NDI contour by overlapping the center of masses and aligning their orientations. The NDI contour is considered as the reference contour, and the CT contour is used as the target one, which will be deformed. Deformed Geometry model is applied in COMSOL to obtain a deformed target contour. The distortion of the volume at X, Y and Z is mapped to illustrate the transformation of the target contour. The initial assessment shows that FEM-based image deformable registration can fuse images acquired by different modalities. It provides insights into the deformation of anatomical structures along X, Y and Z-axes. The deformed contour has good matches to the reference contour after the dynamic matching process. The resulting three-dimensional deformation map can be used to obtain the locations of other critical anatomic structures, e.g., heart, during surgery.

Intracellular localization and dynamics of Hypericin loaded PLLA nanocarriers by image correlation spectroscopy.

The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the first time, Hypericin containing NPs are prepared by combining the miniemulsion technique with the solvent evaporation method. This approach yields an efficient loading of the NPs with Hypericin and allows for additional cargo molecules. To monitor the release of Hypercin from the NPs, an additional fluorescent lipophilic dye Coumarin-6 is incorporated in the NPs. Temporal and spatiotemporal image correlation spectroscopy is used to determine the fate of the NPs carrying the potential cargo. Both directed and non-directed motions are detected. By using image cross-correlation spectroscopy and specific fluorescent labeling of endosomes, lysosomes and mitochondria, the dynamics of the cargo loaded NPs in association with the organelles is studied.

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.

In vivo outcome study of BPD-mediated PDT using a macroscopic singlet oxygen model.

Macroscopic modeling of the apparent reacted singlet oxygen concentration ([1O2]rx) for use with photodynamic therapy (PDT) has been developed and studied for benzoporphryin derivative monoacid ring A (BPD), a common photosensitizer. The four photophysical parameters (ξ, σ, ß, δ) and threshold singlet oxygen dose ([1O2]rx,sh) have been investigated and determined using the RIF model of murine fibrosarcomas and interstitial treatment delivery. These parameters are examined and verified further by monitoring tumor growth post-PDT. BPD was administered at 1 mg/kg, and mice were treated 3 hours later with fluence rates ranging between 75 - 150 mW/cm2 and total fluences of 100 - 350 J/cm2. Treatment was delivered superficially using a collimated beam. Changes in tumor volume were tracked following treatment. The tumor growth rate was fitted for each treatment condition group and compared using dose metrics including total light dose, PDT dose, and reacted singlet oxygen. Initial data showing the correlation between outcomes and various dose metrics indicate that reacted singlet oxygen serves as a good dosimetric quantity for predicting PDT outcome.

In-vivo outcome study of HPPH mediated PDT using singlet oxygen explicit dosimetry (SOED).

Type II photodynamic therapy (PDT) is based on the use of photochemical reactions mediated through an interaction between a tumor-selective photosensitizer, photoexcitation with a specific wavelength of light, and production of reactive singlet oxygen. However, the medical application of this technique has been limited due to inaccurate PDT dosimetric methods. The goal of this study is to examine the relationship between outcome (in terms of tumor growth rate) and calculated reacted singlet oxygen concentration ([1O2]rx) after HPPH-mediated PDT to compare with other PDT dose metrics, such as PDT dose or total light fluence. Mice with radiation-induced fibrosarcoma (RIF) tumors were treated with different light fluence and fluence rate conditions. Explicit measurements of photosensitizer drug concentration and tissue optical properties via fluorescence and absorption measurement with a contact probe before and after PDT were taken to then quantify total light fluence, PDT dose, and [1O2]rx based on a macroscopic model of singlet oxygen. In addition, photobleaching of photosenitizer were measured during PDT as a second check of the model. Changes in tumor volume were tracked following treatment and compared to the three calculated dose metrics. The correlations between total light fluence, PDT dose, reacted [1O2]rx and tumor growth demonstrate that [1O2]rx serves as a better dosimetric quantity for predicting treatment outcome and a clinically relevant tumor growth endpoint.