KEYWORDS: Monte Carlo methods, Photodynamic therapy, Tissue optics, Optical properties, Tissues, Safety, Resistance, Optical spectroscopy, In vivo imaging, Device simulation
Despite advances in image-guided percutaneous drainage, deep tissue abscesses remain a serious cause of morbidity, mortality, and hospital stay. We initiated a Phase 1 clinical trial exploring safety and feasibility of methylene blue (MB) photodynamic therapy during drainage. Five subjects have been treated, with no study-related adverse events and high technical success.
Monte Carlo simulations were used to examine effects of optical properties on delivered light dose. We found that light dose is highly dependent upon MB uptake and Intralipid concentration. These results motivated construction of an optical spectroscopy system for determination of abscess wall optical properties in vivo.
Intravenous administration of some photosensitizers, including the FDA-approved Photofrin, results in significant
systemic photosensitivity and a 2-3-day drug-light interval. Direct intratumor injection of photosensitizer could
potentially eliminate these negative aspects of photodynamic therapy (PDT), while requiring a lower photosensitizer
dose to achieve comparable drug concentration in the target tissue.
We performed PDT using intratumor injection of 3 photosensitizers, methylene blue (MB), Pc 4, and Photofrin, in
mouse tumor models. After a 0-15 minute drug-light interval, illumination was delivered by appropriate diode
lasers. For animals receiving MB or Pc 4, surface illumination was delivered using a microlens-terminated fiber. For
animals receiving Photofrin, interstitial illumination was delivered by a 1 cm diffuser.
In animals receiving MB or Pc 4, tumor dimensions were measured daily post-PDT, with a cure being defined as no
palpable tumor 90 days post-treatment. For Photofrin, animals were sacrificed 24 hours post-PDT and tumors were
excised, with samples HE stained to assess PDT-induced necrosis. 55% of tumors were cured with MB-PDT, and
significant tumor growth delay (p=0.002) was observed for Pc 4. For Photofrin PDT, the mean necrosis radius was
3.4±0.8 mm, compared to 2.9±1.3 mm for systemic administration, which was not a significant difference (p=0.58).
Intratumoral injection of the photosensitizers methylene blue, Pc 4, and Photofrin is feasible, and results in
appreciable tumor response. Further investigation is necessary to optimize treatment protocols and assess the
systemic photosensitivity induced by intratumor injection.
Interstitial photodynamic therapy (iPDT) describes the use of implanted optical fibers for delivery of treatment light to activate photosensitizer in regions that can be located deep within the body. Since sensitive healthy structures are often located nearby, this requires careful treatment planning that is dependent on tissue optical properties. Determination of these values usually involves the insertion of additional fibers into the volume, or the use of flat-cleaved optical fibers as both treatment sources and detectors. The insertion of additional fibers is undesirable, and cylindrical diffusers have been shown to offer superior treatment characteristics compared to flat-cleaved fibers. Using cylindrical diffusers as detectors for spectroscopic measurement is therefore attractive. We describe the determination of the detection profile for a particular cylindrical diffuser design and derive the scatterer concentration gradient within the diffuser core. This detection profile is compared to previously characterized diffusers, and is shown to be dependent on the diffuser design. For diffusers with a constant scatterer concentration and distal mirror, the detection profile is localized to the proximal end of the diffusing region. For diffusers with variable scattering concentration along their length and no distal mirror, the detection profile is shown to be more uniform along the diffusing region. We also present preliminary results showing the recovery of optical properties using arrays of cylindrical diffusing fibers as sources and detectors, with a mean error of 4.4% in the determination of μeff. The accuracy of these results is comparable to those obtained with other methods of optical property recovery.
Knowledge of optical properties is required to determine light dose in photodynamic therapy. We have designed an
interstitial optical probe, consisting of six helically arranged side-firing fibers enclosed in a 1.1 mm diameter
encapsulant, that can be used to determine these values. White light is delivered by one fiber and detected by the others.
Based on a Monte Carlo (MC) model of the probe, the absorption (μa) and reduced scattering (μs') coefficients of the
sample are determined. Recovery was verified in tissue-simulating phantoms containing MnTPPS or intact human
erythrocytes as absorbers and Intralipid as scatterer. Mean errors in recovery of μa and μs' were 9% and 19%,
respectively. In phantoms containing erythrocytes, hemoglobin oxygen saturation was recovered with mean error of
12%.
Using the MC model, we mapped the volumes sampled by particular spectroscopy fibers. For μa = 0.1 cm-1 and μs' = 20cm-1, 49% of photon packets detected at the fiber adjacent to the source sampled a radius further than 5 mm from the probe, while 24% of photon packets sampled further than 7.5 mm. When μs' was reduced to 10 cm-1, 54% of photon packets traversed a radius greater than 5 mm from the probe and 29% sampled further than 7.5 mm. Changing the value of μa to 0.2 cm-1 did not have an effect on the sampled volume.
We also provide a new probe design that aims to improve upon the accuracy of the current probe by incorporating a
wider range of source-detector separations.
We demonstrate interstitial recovery of absorption and scattering coefficients using a custom optical probe and a Monte Carlo (MC)–based recovery algorithm. The probe consists of six side-firing spectroscopy fibers contained in a 1.1-mm outer diameter cladding, with each fiber having a different axial and angular position on the probe. Broadband white light is delivered by one of the fibers and is detected steady-state by the remaining fibers. These spatially and spectrally resolved data are analyzed using a MC-based fitting algorithm in order to extract the local optical properties. The technique was verified in tissue-simulating phantoms consisting of Intralipid-20% as a scatterer and either manganese meso-tetra (4-sulfanatophenyl) porphine or intact human erythrocytes as an absorber. Absorption coefficients were recovered with a mean error of 9% and scattering coefficients were recovered with a mean error of 19%, whereas the hemoglobin oxygen saturation was recovered with a mean error of 12%. These results demonstrate the feasibility of optical property recovery for situations in which surface-contact spectroscopy is not a possibility, and where only a single probe can be inserted into the tissue.
We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1 + cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1 + cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1 + cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1 + cell population.
We measured the optical properties of freshly excised kidneys with renal parenchymal tumors to assess the feasibility of photodynamic therapy (PDT) in these patients. Kidneys were collected from 16 patients during surgical nephrectomies. Spatially resolved, white light, steady-state diffuse reflectance measurements were performed on normal and neoplastic tissue identified by a pathologist. Reflectance data were fit using a radiative transport model to obtain absorption (μ a ) and transport scattering coefficients (μ ′ s ), which define a characteristic light propagation distance, δ . Monte Carlo (MC) simulations of light propagation from cylindrical diffusing fibers were run using the optical properties extracted from each of the kidneys. Interpretable spectra were obtained from 14 kidneys. Optical properties of human renal cancers exhibit significant inter-lesion heterogeneity. For all diagnoses, however, there is a trend toward increased light penetration at longer wavelengths. For renal cell carcinomas (RCC), mean values of δ increase from 1.28 to 2.78 mm as the PDT treatment wavelength is increased from 630 to 780 nm. MC simulations of light propagation from interstitial optical fibers show that fluence distribution in tumors is significantly improved at 780 versus 630 nm. Our results support the feasibility of PDT in selected renal cancer patients, especially with photosensitizers activated at longer wavelengths.
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.
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/cm2 over 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 cm2 by 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.
We present a new Monte Carlo model of cylindrical diffusing fibers that is implemented with a graphics processing unit. Unlike previously published models that approximate the diffuser as a linear array of point sources, this model is based on the construction of these fibers. This allows for accurate determination of fluence distributions and modeling of fluorescence generation and collection. We demonstrate that our model generates fluence profiles similar to a linear array of point sources, but reveals axially heterogeneous fluorescence detection. With axially homogeneous excitation fluence, approximately 90% of detected fluorescence is collected by the proximal third of the diffuser for μs'/μa = 8 in the tissue and 70 to 88% is collected in this region for μs'/μa = 80. Increased fluorescence detection by the distal end of the diffuser relative to the center section is also demonstrated. Validation of these results was performed by creating phantoms consisting of layered fluorescent regions. Diffusers were inserted into these layered phantoms and fluorescence spectra were collected. Fits to these spectra show quantitative agreement between simulated fluorescence collection sensitivities and experimental results. These results will be applicable to the use of diffusers as detectors for dosimetry in interstitial photodynamic therapy.
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.
We present three designs for delivery of light in the oral cavity for photodynamic therapy (PDT) under the requirements
of average irradiance of 50 mW/cm2 and spatial non-uniformities well under 10% over a square area of 25 mm2. The
main goal is to design a device that avoids having to shield the oral cavity prior to irradiation for PDT. Illumination
theory is instrumental in identifying an effective geometry for the device. The designs proposed build upon the
technology that is already available for PDT and use illumination theory concepts to maximize the efficiency of the light
delivery. One design combines a cylindrical diffusing fiber with a reflector derived from the edge-ray theorem while a
second consists of a fiber illuminator coupled to a lightpipe device. Both designs are successful in delivering the light
reducing the need of shielding and in providing the desired irradiance and uniformity. The two approaches performed
comparably and provided a higher irradiance than needed, thus inspiring the design of a third, simpler design based on an
off-axis cylinder reflector.
Candida albicans is an opportunistic human fungal pathogen that requires an intact host immune response to prevent disease. Thus, studying host-pathogen interactions is critical to understanding and preventing this disease. We report a new model infection system in which ongoing C. albicans infections can be imaged at high spatial resolution in the ears of living mice. Intradermal inoculation into mouse ears with a C. albicans strain expressing green fluorescent protein results in systemic C. albicans infection that can be imaged in vivo using confocal microscopy. We observed filamentous growth of the organism in vivo as well as formation of microabscesses. This model system will allow us to gain significant new information about C. albicans pathogenesis through studies of host-C. albicans interactions in the native environment.
Motivated by recent successes in growing intradermal tumors in the ears of mice and establishing the
feasibility of in vivo confocal imaging of anatomic vessels in these tumors using fluorophore-conjugated
antibodies to CD31, we are exploring a number of applications of optical fluorescence imaging in
superficial murine tumor models in vivo. Immune responses induced by photodynamic therapy (PDT) are
dynamic processes that occur in a spatially and temporally specific manner. To visualize these processes
noninvasively, we have made progress in developing optical molecular imaging strategies that take
advantage of intradermal injection of fluorophore-conjugated-antibodies against surface antigens on
immune cells. This enables confocal imaging of the fluorescently labeled host cells to depths of at least
100 microns, and using this technique we have achieved in vivo imaging of granulocyte (GR-1)- and major
histocompatibility complex class II (MHC-II)-positive cell trafficking in tumors in response to PDT. The
latter include macrophages and dendritic cells. Data from tumors that were subjected to PDT with the
photosensitizer, HPPH, reveals a significantly enhanced level of
GR-1+ cell infiltration compared to
untreated control tumor. The temporal kinetics of GR-1+ and
MHC-II+ cells at different time intervals
post-PDT are being examined. The ability to image host responses in vivo without excising or perturbing the
tissue has opened up opportunities to explore means of optimizing them to therapeutic advantage.
Photodynamic therapy (PDT) using topical aminolevulinic acid (ALA) is currently used as a clinical treatment for
nonmelanoma skin cancers. In order to optimize PDT treatment, vascular shutdown early in treatment must be identified
and prevented. This is especially important for topical ALA PDT where vascular shutdown is only temporary and is not
a primary method of cell death. Shutdown in vasculature would limit the delivery of oxygen which is necessary for
effective PDT treatment. Diffuse correlation spectroscopy (DCS) was used to monitor relative blood flow changes in
Balb/C mice undergoing PDT at fluence rates of 10mW/cm2 and 75mW/cm2 for colon-26 tumors implanted
intradermally. DCS is a preferable method to monitor the blood flow during PDT of lesions due to its ability to be used
noninvasively throughout treatment, returning data from differing depths of tissue. Photobleaching of the photosensitizer
was also monitored during treatment as an indirect manner of monitoring singlet oxygen production. In this paper, we
show the conditions that cause vascular shutdown in our tumor model and its effects on the photobleaching rate.
We report an optical molecular imaging technique that exploits local administration of fluorophore-conjugated antibodies and confocal fluorescence microscopy to achieve high-contrast imaging of host cell populations in normal and tumor tissue in living mice. The method achieves micron-scale spatial resolution to depths greater than 100 µm. We illustrate the capabilities of this approach by imaging two dendritic cell populations in the skin and normal and tumor vasculature in vivo.
Silicon phthalocyanine Pc 4 photodynamic therapy (Pc 4-PDT) has emerged as a potentially effective treatment for cutaneous T-cell lymphoma (CTCL). Noninvasive reflectance and fluorescence spectroscopy before, during, and after PDT may provide useful dose metrics and enable therapy to be tailored to individual lesions. We present the design and implementation of a portable bedside spectroscopy system for initial clinical trials of Pc 4-PDT of CTCL. Reflectance and fluorescence spectra were obtained from an early stage CTCL patient throughout the course of the PDT treatment. Preliminary patient data show a significant effect of Pc 4 on the tissue absorption, modest Pc 4 photobleaching, and heterogeneity of Pc 4 within and between the lesions.
Angularly resolved light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology, with particular sensitivity to organelles the size of mitochondria and lysosomes. We have recently reported on a lysosome-staining-based method that provides scattering contrast between stained and unstained cells, and through the use of appropriate models, we extracted a size distribution and contribution to cellular light scattering that we attributed to lysosomes. We provide an independent measurement of the lysosomal size distribution and contribution to cellular light scattering by exploiting photodynamic ablation of lysosomes and observing its effect on angularly resolved light scattering measurements. From these measurements, we conclude that lysosomes scatter approximately 14% of the light from EMT6 cells at 633 nm and that their size distribution has a mean and standard deviation of 0.8 and 0.4 µm, respectively.
Motivated by recent successes in fluorescence imaging of whole mount tissue preparations and by rapid progress
in the fields of molecular imaging and molecular biology, we are exploring a number of applications of optical
fluorescence imaging in superficial murine tumor models in vivo. Imaging the PDT-induced expression of the
heat shock protein 70 (HSP70) in cells and in vivo is accomplished using stably transfected EMT6 cells in which
the gene for GFP is under the control of the HSP70 promoter. These cells readily form solid tumors in BALB/c
mice, enabling the direct imaging of the extent and time course of the activation of this promoter, with each
mouse serving as its own control. Imaging of similarly transfected EMT6 cells with a HIF-1&agr;/GFP fusion
protein vector enables visualization of HIF-1&agr; translocation to the nucleus. Recently, we have accomplished
fluorescent labeling of surface antigens in vivo using intratumor and intravenous injection of fluorophore-conjugated
antibodies. Injection of deep-red fluorophore-conjugated-anti-CD31 enables confocal fluorescence
imaging of the tumor vasculature to depths of at least 100 microns. With the vessels rendered fluorescent in this
way, a number of interesting studies become possible in the living mouse, including the direct visualization of
photosensitizer distribution from perfused vessels. Using the appropriate fluorophore-conjugated antibodies, we
have also been able to image infiltrating granulocytes in EMT6 tumors in response to PDT in vivo.
Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly
resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6
cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic
therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated
sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of
Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More
recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of
lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an
absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering
measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as
quantified by uptake of acridine orange, and can induce downstream effects including release of
mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell
Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the
induction of lysosomal morphology changes requires a fluence significantly higher than that reported for
cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial
swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes
remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of
scattering measurements at different wavelengths to different organelle populations.
Angularly resolved light scattering and wavelength-resolved darkfield scattering spectroscopy measurements were performed on intact, control EMT6 cells and cells stained with high-extinction lysosomal- or mitochondrial-localizing dyes. In the presence of the lysosomal-localizing dye NPe6, we observe changes in the details of light scattering from stained and unstained cells, which have both wavelength- and angular-dependent features. Analysis of measurements performed at several wavelengths reveals a reduced scattering cross section near the absorption maximum of the lysosomal-localizing dye. When identical measurements are made with cells loaded with a similar mitochondrial-localizing dye, HPPH, we find no evidence that staining mitochondria had any effect on the light scattering. Changes in the scattering properties of candidate populations of organelles induced by the addition of an absorber are modeled with Mie theory, and we find that any absorber-induced scattering response is very sensitive to the inherent refractive index of the organelle population. Our measurements and modeling are consistent with EMT6-cell-mitochondria having refractive indices close to those reported in the literature for organelles, approximately 1.4. The reduction in scattering cross section induced by NPe6 constrains the refractive index of lysosomes to be significantly higher. We estimate the refractive index of lysosomes in EMT6 cells to be approximately 1.6.
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 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.
Angularly-resolved light scattering is an established method of particle sizing. Scattering from intact cells provides information about the size distributions of intracellular scatterers. Mitochondria are important light scatterers, especially at forward angles. Nuclei play an important part in scattering light at extreme forward angles and in backscattering geometries. Because changes in mitochondrial morphology are among the early responses to photodynamic therapy (PDT) using mitochondrial-localizing sensitizers and because these changes may be important in determining the fate of the cell, it is interesting to consider light scattering as a means of assessing the response of cells and tissue to PDT. Simple transmission measurements in an absorption spectrophotometer report a rapid reduction in scattering in cells subjected to aminolevulinic acid (ALA)-PDT. ALA-PDT with a fluence of 5 J cm-2 induces a change in the angularly-resolved light scattering from EMT6 cells in suspension within approximately 45 minutes of irradiation. At earlier times following this fluence, the scattering differs only slightly from that observed with control cells. Analysis of the post-treatment scattering data at forward angles is consistent with mitochondrial swelling. Qualitatively similar changes in scattering are observed immediately after a fluence of 10 J cm-2 in cells sensitized with Pc 4.
The shift in optical absorption of hemoglobin upon binding of oxygen provides a basis for near-infrared monitoring of hemoglobin oxygen saturation, which is an important indicator of tissue oxygenation. Tumor oxygenation has long been studied, because hypoxic cells exhibit resistance to ionizing radiation therapy. The ability to measure noninvasively the oxygenation status of tumors and their response to oxygen modifiers is important in research and clinical settings. We have implemented a steady-state diffuse reflectance method of optical spectroscopy in scattering systems based on the theory of Farrell et al. (Med. Phys., 1992). In scattering phantoms containing erythrocytes, the method recovers the hemoglobin absorption spectrum (650 - 820 nm) and accurately monitors hemoglobin oxygen saturation. We have implemented a probe that individually positions several detection fibers normal to the surface of subcutaneous rodent tumors. Near-infrared absorption spectra reconstructed from diffuse reflectance measurements indicate a hemoglobin oxygen saturation of approximately 50% in R3230AC rat mammary adenocarcinomas when the anesthetized animal breathes room air. Administration of carbogen (95% oxygen, 5% carbon dioxide) via a nose cone produces a rapid and readily detectable increase in the saturation to 75% with no increase in tumor blood volume. Several methods of determining hemoglobin oxygen saturation from absorption spectra obtained by diffuse reflectance spectroscopy are compared, including singular value decomposition, which provides the ability to reconstruct the non-hemoglobin absorbing background without a priori knowledge of its structure or absolute magnitude.
We present two cw methods for localizing a source of fluorescence buried in a medium with optical properties similar to those of tissue in the near infrared region. The first approach is based on the fact that, for small excitation beam diameters, the absolute intensity at a given depth in the medium depends on the diameter of the incident beam. For a well-chosen pair of beam diameters, the ratio of these intensities in a scattering medium depends uniquely on the depth from the surface of incidence. Thus, the ratio of the fluorescence resulting from sequential excitation using two beam diameters can be used to determine the depth at which the fluorescence originated. The second method is based on spatially resolved surface measurements of the diffuse fluorescence from the buried source. Using a form of the diffusion theory analysis of Farrell et al. (Med. Phys., 1992) for the spatially resolved diffuse reflectance from a pencil beam incident on a scattering medium, it is possible to reconstruct the depth of the source from the shape of the surface fluorescence profile. Preliminary experimental results obtained using a 1.0 cm diameter sphere containing the tumor localizing fluorophore Nile Blue A show that the spatially resolved measurement reports the location of fluorescent sources as deep as 4.0 cm with an accuracy of 0.4 cm or better.
The oxygenation status of tissue is important in several medical applications. It has long been appreciated that hypoxic tumors are resistant to radiation therapy, while in photodynamic therapy (PDT), the dependence on oxygen is even more pronounced. Our laboratory studies several problems in PDT that originate with this 3O2-dependence. Use of 3O2-sensitive electrodes with small (<EQ 10 micrometers ) tip diameters enables accurate measurement of metabolic and photochemical 3O2 consumption in photosensitized tumor spheroids in vitro. The temporal response of these devices (< 1 s) is sufficient to capture rapid depletion of 3O2 during laser irradiation of individual spheroids. An important aspect of this research has been the development of appropriate mathematical models with which to interpret the microelectrode data and determine photophysical parameters. Recently, we have been establishing optical methods of measuring hemoglobin 3O2-saturation in order to monitor oxygenation in rodent tumors during PDT. We have adopted a steady-state diffuse reflectance technique, in which white light is injected into the tumor via an optical fiber, and the spatially-resolved diffuse reflectance is monitored using appropriately spaced detection fibers. The signals are spectrally dispersed onto a CCD camera by a grating spectrograph, providing simultaneous acquisition of spatially-resolved diffuse reflectance data for a 160 nm range of wavelengths. Absorption spectra reconstructed using a diffusion theory approximation are then fit to linear combinations of oxy- and deoxyhemoglobin spectra to obtain the 3O2-saturation.
We have previously reported on a microelectrode technique for mapping spatial and temporal distributions of oxygen in and around individual photosensitized multicell tumor spheroids. Steady-state and time-resolved measurements have been analyzed using diffusion theory ad have yielded estimates of the oxygen diffusion coefficient in the spheroid, the rate of metabolic oxygen consumption, and the fluence-rate-dependent rate of photochemical oxygen depletion. We have recently modified the theoretical treatment of the time-resolved measurements to include the oxygen dependence of the rate of therapy-induced oxygen consumption. The oxygen consumption term in the diffusion equation is now derived from kinetics of type II photochemistry. This expression contains the ration of two rate constants' the photosensitizer triplet decay rate (kp) and the bimolecular rate for collisional triplet-quenching interactions with oxygen (kot). From fits of numerical solutions of the diffusion equation to microelectrode measurements of PDT-induced oxygen transients, kp/kot, may be obtained for a photosensitizer in a multicell environment. The ration plays an important role in direct cell photosensitization by defining the concentration at which singlet oxygen formation is limited by the availability of oxygen. In a multicell environment where oxygen supply is diffusional, extremely low values of kp/kot exacerbate the oxygen depletion problem. Recent experiments and analysis indicate that in some cases photosensitizer bleaching rates may also be determined from microelectrode measurements in spheroids.
We have previously reported results of experiments in which EMT6/Ro spheroids were subjected to Photofrin-PDT consisting of a fixed incident fluence (60 J-cm-2) delivered at 200, 50, and 25 mW-cm-2. Surviving fractions from treated spheroids decreased as the incident fluence rate was lowered over this range. We have interpreted these data using a model wherein the cells compromising the surviving fractions are assumed to originate from within a therapy-induced anoxic volume resulting from Type-II photochemistry. In this paper, we demonstrate direct measurements of the phenomenon in individual photosensitized EMT6 spheroids. Steady-state measurements of 3O2 gradients in and around metabolizing spheroids allow determination of the 3O2 diffusion constant and the rate of metabolic 3O2 consumption within a spheroid. Time-dependent measurements obtained at a single spatial location during laser irradiation are fit to numerical solutions of a pair of time-dependent diffusion with consumption equations. Fits allow a determination of the rate of PDT-induced 3O2 consumption. Based on these fits, it is possible to calculate the spatial and temporal distributions of oxygen within a spheroid undergoing PDT.
Improved control of tumor growth has been accomplished by modification of the irradiation scheme applied to hematoporphyrin-induced sensitization. Significantly enhanced efficacy of PDT was seen with either reduction of fluence rate or with an intermittent light-dark protocol, together suggesting that consideration must be given to photochemical oxygen depletion as a rate-limiting component in the production of singlet oxygen, the mediator of cytotoxicity resulting from PDT. A model to explain the basis for the improved efficacy was developed and is being tested by study of homograft and xenograft tumors in vivo and in multicellular tumor spheroids in vitro.
Type II photo-oxidation depends on and consumes oxygen. Several factors, including the concentration of photosensitizer and the radiation fluence rate, determine the rate of oxygen consumption in tissue undergoing Photodynamic Therapy. If the tissue capillary density is sparse, as it is in many human tumors, our calculations indicate that for cells sufficiently distant from the nearest capillary, fluence rates commonly used in PDT (50 - 200 mW/cm2) deplete 3O2 levels below those necessary for 1O2 formation. The calculations suggest that under these conditions reduced fluence rates and radiation dose fractionation should be more effective than continuous radiation at high fluence rates in producing 1O2 throughout the treated tissue volume. These predictions are supported by results obtained in vivo. The data and their interpretation have implications for PDT dosimetry and offer the possibility of improved therapeutic ratio.
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