Search

Photodynamic therapy with methylene blue performed at the time of percutaneous CT- or US-guided abdominopelvic abscess drainage was safe and feasible, demonstrating a dose-dependent improvement in clinical response.

The non-invasive, in vivo measurement of microvascular blood flow has the potential to enhance breast cancer therapy monitoring. Here, longitudinal blood flow of 4T1 murine breast cancer (N=125) under chemotherapy was quantified with diffuse correlation spectroscopy based on layer models. Six different treatment regimens involving doxorubicin, cyclophosphamide, and paclitaxel at clinically relevant doses were investigated. Treatments with cyclophosphamide increased blood flow as early as 3 days after administration, whereas paclitaxel induced a transient blood flow decrease at 1 day after administration. Early blood flow changes correlated strongly with the treatment outcome and distinguished treated from untreated mice individually for effective treatments.

Using integrated Raman and angular scattering microscopy (IRAM), we follow the response of EMT6 cancer cells to photodynamic therapy (PDT) treatment. The study combines two non-labelling light scattering techniques to extract chemical information and organelle sizes from single cells. Each cell is measured repeatedly over several hours to follow changes in these parameters as the cell responds to the PDT treatment. An automated algorithm identifies which parameters are changing in time. Size parameters extracted from angular scattering measurements show a decrease in the size of 1-micron-diameter scatterers in treated cells. Treated cells also exhibit trends in several Raman peaks, denoting changes in chemical concentrations of proteins, nucleic acids, and lipids. Each of these parameters - acquired from both measurement modalities - can be monitored on a cell-by-cell basis. The ability to track these chemical and structural changes over time allows access to greater knowledge of biological processes.

A compact and efficient lightpipe device to deliver light to the human oral cavity for photodynamic therapy was designed and fabricated, having dimensions 6.8 mm×6.8 mm×46 mm. An average irradiance of 76 mW/cm^2 with an average deviation of 5% was measured on a square 25 mm^2 treatment field for an input power of 100 mW. The device limits irradiation of healthy tissue and offers potential for improvement over the current treatment procedure, which requires shielding of the whole cavity to avoid damage to healthy tissue.

The tumor extracellular matrix has been focused on by newer approaches to cancer therapy owing to its important functions in the process of drug delivery and cellular metastasis. This study aims to characterize tumor extracellular matrix structures in the presence and absence of therapy, as observed on second harmonic generation (SHG) images through both gray-level co-occurrence matrix (GLCM) derived texture features as well as Minkowski Functionals (MF) that focus on the underlying gray-level topology and geometry of the texture patterns. Thirteen GLCM texture features and three MF texture features were extracted from 119 regions of interest (ROI) annotated on SHG images of treated and control samples of tumor extracellular matrix. These texture features were then used in a machine learning task to classify ROIs as belonging to treated or control samples. A fuzzy k-nearest neighbor classifier was optimized using random sub-sampling cross-validation for each texture feature and the classification performance was calculated on an independent test set using the area under the ROC curve (AUC); AUC distributions of different features were compared using a Mann-Whitney U-test. Two GLCM features f3 and f13 exhibited a significantly higher classification performance when compared to other GLCM features (p < 0.05). The MF feature Area exhibited the best classification performance among the MF features while also being comparable to that obtained with the best GLCM features. These results show that both statistical and topological texture features can be used as quantitative measures is evaluating the effects of therapy on the tumor extracellular matrix.

Three compact and efficient designs are proposed to deliver an average irradiance of 50 mW/cm^2 with spatial uniformity well above 90% over a 25 mm^2 target area for photodynamic therapy of the oral cavity. The main goal is to produce uniform illumination on the target while limiting irradiation of healthy tissue, thus overcoming the need of shielding the whole oral cavity and greatly simplifying the treatment protocol. The first design proposed consists of a cylindrical diffusing fiber placed in a tailored reflector derived from the edge-ray theorem with dimensions 5.5 × 7.2 × 10 mm^3; the second device combines a fiber illuminator and a lightpipe with dimensions 6.8 × 6.8 × 50 mm^3; the third design, inspired by the tailored reflector, is based on a cylindrical diffusing fiber and a cylinder reflector with dimensions 5 × 10 × 11 mm^3. A prototype for the cylinder reflector was built that provided the required illumination for photodynamic therapy of the oral cavity, producing a spatial uniformity on the target above 94% and an average irradiance of 51 mW/cm^2 for an input power of 70 mW.

We evaluated intratumor (IT) versus intravenous (IV) administration of the photosensitizer Pc 4 with respect to tumor photosensitizer concentration, specificity, and responses to irradiation. BALB/c mice bearing intradermal EMT6 tumors were given 0.3 mg/kg Pc 4 injected IT or IV through the tail vein. Photosensitizer concentration was evaluated by chloroform extraction and localization assessed by fluorescence imaging and spectroscopy in vivo. Tumors were irradiated at 667 nm, 50 mW/cm2, and 100 J/cm2. Cures were defined as no palpable tumor 90 days after irradiation. Tumor Pc 4 concentrations 1 hour after IT administration were 35,000-fold higher than measured 24 hours after IV administration (0.112 vs0.317 x 10-5µg Pc 4/mg tumor). Exquisite tumor selectivity was observed 1 hour after IT injection. Fluorescence imaging of freshly sectioned tumors revealed no regions devoid of sensitizer at this time point, with pixel intensities in a midline section within a factor of 3 of the peak intensity. For identical photosensitizer doses, IT administration significantly improved tumor responses to irradiation, with more than 70% of tumors cured with IT-Pc 4-PDT. In this model, IT-Pc 4 administration provides improved tumor control, greater selectivity, and opportunity for a short drug-light interval.

Controlling metastases remains a critical problem in cancer biology. Within the peritoneal cavity, omental tissue is a common site for metastatic disease arising from intraperitoneal tumors; however, it is unknown why this tissue is so favorable for metastatic tumor growth. Using five different tumor cell lines in three different strains of mice, we found that the omentum was a major site of metastases growth for intraperitoneal tumors. Furthermore, initial attachment and subsequent growth were limited to specific sites within the omentum, consisting of organized aggregates of immune cells. These immune aggregates contained a complex network of capillaries exhibiting a high vascular density, which appear to contribute to the survival of metastatic cells. We found that the vasculature within these aggregates contained CD105+vessels and vascular sprouts, both indicators of active angiogenesis. A subset of mesothelial cells situated atop the immune aggregates was found to be hypoxic, and a similar proportion was observed to secrete vascular endothelial growth factor-A. These data provide a physiological mechanism by which metastatic tumor cells preferentially grow at sites rich in proangiogenic vessels, apparently stimulated by angiogenic factors produced by mesothelial cells. These sites provide metastatic cells with a microenvironment highly conducive to survival and subsequent growth.

We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 μm diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.

ABSTRACTTreatment of mucocutaneous and cutaneous Candida albicansinfections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicansto its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicansand examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicansbiofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicansbiofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment of C. albicans. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.

Efficient intratumor delivery of anticancer drugs and photo‐sensitizers is an important factor in the success of chemotherapy and photodynamic therapy, respectively. Unfortunately, their adequate and uniform intratumor distribution is impeded by several physiological barriers and by binding to tissue components. Measurement of gross tumor drug accumulation is a routine method of investigating the uptake and clearance of chemotherapy agents and photosensitizers but tells little about their extravascular spatial distribution. We use whole‐mount two‐color confocal fluorescence imaging and imaging spectroscopy of unprocessed excised murine tumor fragments to investigate the intratumor distribution of the photosensitizer meso‐tetrahydroxyphenyl chlorin (mTHPC) as a function of distance from blood vessels perfused with 0.2 μm diameter fluorescent microspheres. Significant mismatches between drug and perfused vasculature are caused by heterogeneities in tumor blood supply. We describe complex microscopic mTHPC gradients that reverse dramatically relative to the perfused vasculature with time after injection. This imaging technique can be applied to screen the dynamic intratumor distribution of other fluorescent photosensitizers and anticancer drugs.

Angularly resolved light scattering measurements were performed on suspensions of EMT6 cells and on mitochondria isolated from rabbit liver. Mie theory analysis of the scattering from intact cells indicated that mitochondrial-sized organelles dominated scattering in the range 5–90°. This interpretation was supported by the analysis of scattering from isolated mitochondria. Intact cells were subjected to oxidative stress by photodynamic insult. After 3h of incubation in the heme precursor aminolevulinic acid hexylester, EMT6 cells accumulated abundant protoporphyrin IX, an endogenous photosensitizer formed in mitochondria. Irradiation of aminolevulinic acid/protoporphyrin IX-sensitized cells with 10J cm−2 of 514nm light led to pronounced changes in angularly resolved light scattering consistent with mitochondrial swelling. Electron microscopy of similarly treated EMT6 cell monolayers showed significant changes in mitochondrial morphology, which included distension of the outer unit membrane and bloating of the internal mitochondrial compartment. Informed by these electron microscopy results, we implemented a coated sphere model to interpret the scattering from intact cells subjected to oxidative stress. The coated sphere interpretation was compatible with the scattering measurements from these cells, whereas simpler Mie theory models based on homogenous swelling were dramatically unsuccessful. Thus, in this system, angularly resolved light scattering reports oxidative-stress-induced changes in mitochondrial morphology.

We present two forward-adjoint models for recovering intrinsic fluorescence spectra and hemoglobin oxygen saturation of turbid samples. The first fits measured diffuse reflectance spectra to obtain the absorption and scattering spectra of the medium, and these are then used to correct distortions imposed on the fluorescence spectrum by absorption and scattering. The second fits only the measured fluorescence spectrum to determine simultaneously the amplitudes of absorption and fluorescence basis spectra and scattering parameters. Both methods are validated with Monte Carlo simulations and experimentally in scattering phantoms containing nicotinamide adenine dinucleotide and human erythrocytes. Preliminary measurements from murine tumors in vivo are presented.

We present a detailed investigation of Photofrin® photobleaching and photoproduct accumulation. Fisher rats were sensitized with 10 mg kg⁻¹ Photofrin and irradiated 24 h later with 514 nm light at 5 or 100 mW cm⁻². Fluorescence spectra were collected from the skin throughout treatment, and sensitizer bleaching and fluorescent photoproduct formation were quantified using spectral analysis. Photofrin bleaching was slightly more rapid at the higher irradiance under these conditions. However, accumulation of photoproduct was significantly enhanced at lower irradiance. To interpret these unexpected findings, we developed a new mathematical model in which reactions between singlet oxygen (¹O₂) and the photosensitizer and reactions between the sensitizer triplet and biological targets are both allowed to contribute to bleaching. Predictions of this model were tested in experiments performed on EMT6 spheroids sensitized with concentrations of 2.5, 10 and 30 µg mL⁻¹ Photofrin and subjected to PDT. Photofrin bleaching and photoproduct formation in these spheroids were measured using confocal fluorescence spectroscopy. In qualitative agreement with the mixed-mechanism model predictions, at the highest drug concentration Photofrin bleaching was more efficient via ¹O₂ reactions, while at the lowest concentration triplet reactions were more efficient. At all concentrations, photoproduct accumulation was greater under conditions of abundant oxygen.

ABSTRACTThe in vitro susceptibility of pathogenic Candidaspecies to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candidawas confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candidaand subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicansblastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabratatook up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicansdisplayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. kruseithat is relatively resistant to fluconazole compared to C. albicanswas equally sensitive to C. albicansat Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candidatherapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

We report results of experiments that evaluated the influence of oxygenation on the penetration of red light in tissue, with particular emphasis on 630 and 650 nm laser wavelengths commonly used in photodynamic therapy (PDT) of solid tumours. Direct measurements in tissue-simulating phantoms comprised of intact human erythrocytes suspended in a scattering emulsion demonstrated significant enhancements in fluence rate at depths of 0.5–2 cm from the irradiated surface when the cells were fully oxygenated versus fully deoxygenated. The 630 and 650 nm fluence rates at depth in the homogeneous phantoms continued to increase when examined over a continuous range of oxygen partial pressures from 0 to 160 Torr. When considered as a function of haemoglobin oxygen saturation, the largest increases in fluence rate were observed as the saturation increased beyond 70%. Dramatic increases in optical fluence rate were measured at the base of 1-cm-thick subcutaneous EMT6 mammary carcinomas in vivo when the tumour-bearing mouse was subjected to carbogen through a nose cone. These results indicate that improved tumour oxygenation is important in PDT not only for the maintenance of the oxygen-dependent photochemistry but, through the effects reported here, may also enable more efficient treatment of thicker lesions.

The Green’s function of the steady-state radiative-transport equation is derived in the P_3 approximation for isotropic sources. It is demonstrated that the P_3 approximation models the radiance in highly absorbing media or close to sources more accurately than does diffusion theory. Boundary conditions consistent with the P_3 approximation are also developed for semi-infinite media bounded by a nonscattering medium. Expressions for the reflectance remitted from media interrogated by a normally incident pencil beam are derived and fitted to simulated and experimental reflectance data from media having optical properties typical of biological tissue. The reconstructed optical properties are accurate to within ±10% in absorption and scattering for source–detector separations as small as 0.43 mm and albedos as low as 0.59. Methods for simplifying these expressions given some a priori knowledge of the scattering phase function are discussed.

Steady-state spatially resolved diffuse reflectance spectroscopy is used to reconstruct absolute visible absorption spectra (λ 450–610 nm) of tissue-simulating phantoms that contain intact mitochondria and red blood cells in physiologically relevant proportions. The spectra are analyzed to extract independent fitted concentrations of oxy- and deoxyhemoglobin and of oxidized and reduced mitochondria. The resulting hemoglobin oxygen-dissociation and mitochondrial oxidation-reduction curves exhibit p50parameters of 25 and 2.5 torr, respectively, in agreement with published values. These results demonstrate the potential for absolute quantitative cytochrome spectroscopy of tissue in the presence of hemoglobin in the visible wavelength region. Extension of these measurements to the in vivocase is discussed.

Phosphorescence quenching of certain metalloporphyrins is used to measure tissue and microvascular pO2. Oxygen quenching of metalloporphyrin triplet states creates singlet oxygen, which is highly reactive in biological systems, and these oxygen-consuming reactions are capable of perturbing tissue oxygenation. Kinetics of photochemical oxygen consumption were measured for a Pd-porphyrin in two model systems in vitro over a range of irradiances (1.34–134mWcm−2). For a given irradiance, and, after correction for differing porphyrin concentrations, rates of oxygen consumption were similar when the Pd-porphyrin was bound to bovine serum albumin and when it was taken up by tumor cells in spheroids. At irradiances comparable to those used in imaging superficial anatomy, rates of oxygen consumption were sufficiently low (2.5μMs−1) that tissue oxygenation would be reduced by a maximum of 6%. An irradiance of 20mWcm−2, however, initiated a rate of oxygen consumption capable of reducing tissue pO2 by at least 20–40%. These measured rates of consumption impose limitations on the use of phosphorescence quenching in thick tissues. The irreversible photobleaching of the Pd-porphyrin was also measured indirectly. The bleaching branching ratio, 23M−1, is significantly lower than that of porphyrin photodynamic agents.

We present a steady-state method for localizing a source of luminescence (i.e., fluorescence or phosphorescence) buried in a semi-infinite turbid medium with unknown optical properties. A diffusion theory expression describing the emittance of an isotropic point source is fit to spatially resolved surface measurements of the diffuse emittance from the luminescent source. The technique reports the location of the center of a 6.0-mm-diameter, fluorophore-containing spherical bulb embedded in a liquid phantom with an accuracy of 1.0 mm or better for source depths as great as 40.0 mm. Monte Carlo data are analyzed to investigate the range and the possible sources of error in the reconstructed source depth.

We present a steady-state radially resolved diffuse reflectance spectrometer capable of measuring the absorption and transport scattering spectra of tissue-simulating phantoms over an adjustable 170-nm wavelength interval in the visible and near infrared. Measurements in a variety of phantoms are demonstrated over the relevant range of tissue optical properties, and the accuracy of the instrument is found to be approximately 10% in both scattering and absorption. Monte Carlo simulations designed to test the accuracy of the instrument are presented that support the experimental findings.

The need for repositioning of surface coils and patients in MR examinations of the cervical and thoracic spine prolongs examination time. A new receiver design is proposed which overcomes this problem. The device is composed of two actively decoupled receiver coils mounted on the frame of a Philadelphia collar. These coils may be used separately to image either the thoracic or cervical spine or together to produce larger field-of-view images of the combined region. Signal-to-noise ratios of the separate cervical and thoracic spine images are not degraded as a result of mounting the receivers together. The full cervical and thoracic region is shown to be imaged at a signal-to-noise ratio significantly higher than that afforded by the body coil. A retrospective review of our case load suggests that a time saving could be achieved in approximately 13 of spine examinations by using this coil.

Light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology. Apoptosis, or programmed cell death, is associated with distinct morphological signatures such as mitochondrial swelling and nuclear condensation as well as characteristic biochemical signaling pathways, many of which are initiated by the release of cytochrome c from the mitochondria into the cytosol. In this Letter, we examine the time course of mitochondrial morphology changes as reported by light scattering and the subcellular location of cytochrome c measured by immunofluorescence microscopy in response to intracellular cell death signaling induced by photodynamic damage to lysosomes. We report that within this system, release of cytochrome c from the mitochondria into the cytosol occurs approximately simultaneously with mitochondrial-morphology-induced light scattering changes, providing further evidence that light scattering has the potential to play an important role in future studies of cell death biology.

We present a Mie theory-based analysis of angularly resolved light scattering from intact cells that involves the product of a particle size distribution ? and the scattering cross section ?. The analysis shows that the ability to size intracellular organelles depends strongly upon the assumed functional form of the particle size distribution; however, the product ?? extracts the dominant light scatterers regardless of this choice. We examine goniometer measurements from our own and another laboratory and find that this analysis reconciles seemingly conflicting results and confirms that mitochondrial-sized organelles dominate scattering from intact cells in the angular range 5°–90°.

We present the results of diffuse reflectance measurements made on the surface of a tissue-simulating phantom containing intact human erythrocytes. These measurements indicate that the absorption spectrum of hemoglobin in its natural environment is significantly different from that measured in homogeneous fluid solution, especially in the spectral regions of highest absorption. We show that this difference can be explained by the pigment packaging theory developed by Duysens [Biochim. Biophys. Acta19, 1 (1956)] and that the adoption of basis spectra that take this effect into account improves the accuracy of fitting diffuse reflectance spectra.

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.

Images of Vicia fabaL. root systems at 0.6-mm resolution, undisturbed and within the soil medium in which they were grown, have been obtained by using a 1.5-Tesla proton (1H) NMR medical imaging research system. Images of root systems in seven soil media (Cahaba soil, vermiculite, sand, perlite, fritted clay, potting soil, and peatlite) exhibited variable but useful quality owing to a diversity of magnetic properties of the soils. Root structure and pathology in the form of partial decay of hypogeal cotyledons were easily discernible. Water transport in roots with light-stressed foliage was demonstrated by using water doped with a paramagnetic NMR contrast agent, and the process of plant-wilt and recovery was monitored in situ. Images of germinated seeds within soil media indicated that dynamic observations of germination and growth are possible. The results suggest that NMR imaging can be an effective noninvasive tool for studying plants in situ.

To date, the lack of light delivery mechanisms to the oral cavity remains a barrier to the treatment of oral cancer with photodynamic therapy (PDT). The greatest impediment to medical practitioners is the current need to shield the normal tissues of the oral cavity, a costly and time-consuming procedure. In this research, we present the design of illumination devices to deliver light to the oral cavity for PDT, which will facilitate administration of PDT in the clinic. The goal for such an illumination device, as indicated by our clinical collaborators at Roswell Park Cancer Institute in Buffalo, NY, is to limit exposure of healthy tissue and produce an average irradiance of 100 mW/cm2over the treatment field, with spatial non-uniformities below 10%. Furthermore, the size of the device must be compact to allow use in the oral cavity. Our research led to the design and fabrication of two devices producing spatial non-uniformities below 6% over a treatment area of 0.25 cm2by design. One device consisted of an appropriately-sized reflector, inspired by solar concentrators, illuminated by a cylindrical diffusing fiber optimally located within the reflector; another was a solid lightpipe with a combination of optimized tapered and straight components.