Imaging Cherenkov photon emissions in radiotherapy with a Geiger-mode gated quanta image sensor.

The emission of Cherenkov photons from human and animal tissue can be observed during clinical x-ray or particle beam irradiation. However, imaging this weak emission with the necessary single-photon sensitivity in the clinical room is challenging because of milliwatt-level ambient room lighting and the presence of stray high-energy radiation. In this Letter, we demonstrate, to the best of our knowledge, the first Cherenkov imaging with a time-gated quanta image sensor employing a large single-photon avalanche diode (SPAD) array. Detecting single Cherenkov photons was possible with high photon avalanche gain, fast temporal gating, and moderately high ∼7% photon detection probability. Single-bit digitization and active SPAD quenching enabled stray x-ray noise suppression and photon-noise-limited imaging in a clinical environment. This type of imaging allows the knowledge of location, shape, and surface dose of the therapeutic beam radiotherapy with the stability of solid state-based detection.

Phase I/II study of verteporfin photodynamic therapy in locally advanced pancreatic cancer.

BACKGROUND: Patients with pancreatic cancer have a poor prognosis apart from the few suitable for surgery. Photodynamic therapy (PDT) produces localised tissue necrosis but previous studies using the photosensitiser meso-tetrahydroxyphenylchlorin (mTHPC) caused prolonged skin photosensitivity. This study assessed a shorter acting photosensitiser, verteporfin. METHODS: Fifteen inoperable patients with locally advanced cancers were sensitised with 0.4 mg kg(-1) verteporfin. After 60-90 min, laser light (690 nm) was delivered via single (13 patients) or multiple (2 patients) fibres positioned percutaneously under computed tomography (CT) guidance, the light dose escalating (initially 5 J, doubling after each three patients) until 12 mm of necrosis was achieved consistently. RESULTS: In all, 12 mm lesions were seen consistently at 40 J, but with considerable variation in necrosis volume (mean volume 3.5 cm(3) at 40 J). Minor, self-limiting extrapancreatic effects were seen in multifibre patients. No adverse interactions were seen in patients given chemotherapy or radiotherapy before or after PDT. After PDT, one patient underwent an R0 Whipple's pancreaticoduodenectomy. CONCLUSIONS: Verteporfin PDT-induced tumour necrosis in locally advanced pancreatic cancer is feasible and safe. It can be delivered with a much shorter drug light interval and with less photosensitivity than with older compounds.

Singlet Molecular Oxygen: from COIL Lasers to Photodynamic Cancer Therapy.

Translation of experimental techniques from one scientific discipline to another is often difficult but rewarding. Knowledge gained from the new area can lead to long lasting and fruitful collaborations with concomitant development of new ideas and studies. In this Review Article, we describe how early work on the chemically pumped atomic iodine laser (COIL) led to the development of a key diagnostic for a promising cancer treatment known as photodynamic therapy (PDT). The highly metastable excited state of molecular oxygen, a1Δg, also known as singlet oxygen, is the link between these disparate fields. It powers the COIL laser and is the active species that kills cancer cells during PDT. We describe the fundamentals of both COIL and PDT and trace the development path of an ultrasensitive dosimeter for singlet oxygen. The path from COIL lasers to cancer research was relatively long and required medical and engineering expertise from numerous collaborations. As we show below, the knowledge gained in the COIL research, combined with these extensive collaborations, has resulted in our being able to show a strong correlation between cancer cell death and the singlet oxygen measured during PDT treatments of mice. This progress is a key step in the eventual development of a singlet oxygen dosimeter that could be used to guide PDT treatments and improve outcomes.

Monitoring of hemodynamic changes induced in the healthy breast through inspired gas stimuli with MR-guided diffuse optical imaging.

PURPOSE: The modulation of tissue hemodynamics has important clinical value in medicine for both tumor diagnosis and therapy. As an oncological tool, increasing tissue oxygenation via modulation of inspired gas has been proposed as a method to improve cancer therapy and determine radiation sensitivity. As a radiological tool, inducing changes in tissue total hemoglobin may provide a means to detect and characterize malignant tumors by providing information about tissue vascular function. The ability to change and measure tissue hemoglobin and oxygenation concentrations in the healthy breast during administration of three different types of modulated gas stimuli (oxygen/ carbogen, air/carbogen, and air/oxygen) was investigated. METHODS: Subjects breathed combinations of gases which were modulated in time. MR-guided diffuse optical tomography measured total hemoglobin and oxygen saturation in the breast every 30 s during the 16 min breathing stimulus. Metrics of maximum correlation and phase lag were calculated by cross correlating the measured hemodynamics with the stimulus. These results were compared to an air/air control to determine the hemodynamic changes compared to the baseline physiology. RESULTS: This study demonstrated that a gas stimulus consisting of alternating oxygen/carbogen induced the largest and most robust hemodynamic response in healthy breast parenchyma relative to the changes that occurred during the breathing of room air. This stimulus caused increases in total hemoglobin and oxygen saturation during the carbogen phase of gas inhalation, and decreases during the oxygen phase. These findings are consistent with the theory that oxygen acts as a vasoconstrictor, while carbogen acts as a vasodilator. However, difficulties in inducing a consistent change in tissue hemoglobin and oxygenation were observed because of variability in intersubject physiology, especially during the air/oxygen or air/carbogen modulated breathing protocols. CONCLUSIONS: MR-guided diffuse optical imaging is a unique tool that can measure tissue hemodynamics in the breast during modulated breathing. This technique may have utility in determining the therapeutic potential of pretreatment tissue oxygenation or in investigating vascular function. Future gas modulation studies in the breast should use a combination of oxygen and carbogen as the functional stimulus. Additionally, control measures of subject physiology during air breathing are critical for robust measurements.

Imaging radiation dose in breast radiotherapy by X-ray CT calibration of Cherenkov light.

Imaging Cherenkov emission during radiation therapy cancer treatments can provide a real-time, non-contact sampling of the entire dose field. The emitted Cherenkov signal generated is proportional to deposited dose, however, it is affected by attenuation from the intrinsic tissue optical properties of the patient, which in breast, ranges from primarily adipose to fibroglandular tissue. Patients being treated with whole-breast X-ray radiotherapy (n = 13) were imaged for 108 total fractions, to establish correction factors from the linear relationships between Cherenkov light and CT number (HU). This study elucidates this relationship in vivo, and a correction factor approach is used to scale each image to improve the linear correlation between Cherenkov emission intensity and dose ([Formula: see text]). This study provides a major step towards direct quantitative radiation dose imaging in humans by utilizing non-contact camera sensing of Cherenkov emission during the radiation therapy treatment.

Interstitial fluid pressure in soft tissue as a result of an externally applied contact pressure.

Manipulation of interstitial fluid pressure (IFP) has a clinical potential when used in conjunction with near-infrared spectroscopy for the detection of breast cancer. In order to better interpret how the applied pressure alters the vascular space and interstitial water volumes in breast tissue, a study on tissue-mimicking, gelatin phantoms was carried out to mimic the translation of external force into internal pressures. A complete set of three-dimensional (3D) pressure maps were obtained for the interior volumes of phantoms as an external force of 10 mmHg was applied, using mixtures of elastic moduli 19 and 33 kPa to simulate adipose and fibroglandular values of breast tissue. Corresponding linear elastic finite element analysis (FEA) cases were formulated. Shear stress, nonlinear mechanical properties, gravity and tissue geometry were all observed to contribute to internal pressure distribution, with surface shear stresses increasing internal pressures near the surface to greater than twice the applied external pressure. Average pressures by depth were predicted by the linear elastic FEA models. FEA models were run for cases mimicking a 93 kPa tumor inclusion within regions of adipose, fibroglandular tissue, and a composite of the two tissue types to illustrate the localized high fluid pressures caused by a tumor when an external force is applied. The conclusion was that external contact forces can generate potentially clinically useful fluid pressure magnitudes in regions of sharp effective elastic modulus gradients, such as tumor boundaries.

A photobiological and photophysical-based study of phototoxicity of two chlorins.

To understand the fundamental determinants of phototoxic efficacy and absorbed photodynamic dose, the triplet state and photobleaching quantum yields in living cells, cellular uptake, intracellular localization, and correlation with cell viability were studied for the two purpurins tin ethyl etiopurpurin 1 (SnET2) and tin octaethylbenzochlorin (SnOEBC) in ovarian cancer cells (OVCAR5). Although the triplet yields of these two photosensitizers were not significantly affected by cellular incorporation, the photobleaching yields were shown to be 3 orders of magnitude higher for cellular-bound sensitizer than for free or albumin-bound photosensitizer and higher for SnET2 than for SnOEBC for all of the cases. The intracellular concentration of SnOEBC was half that of SnET2 after 3 h- and 24 h-incubation times for both 0.1 microM and 1.0 microM incubation concentrations. Despite the lower concentrations of SnOEBC, the phototoxicity of the two photosensitizers was comparable at 1-microM incubation concentration and was up to 10-fold higher for SnOEBC at the lower concentration. The subcellular localization established using confocal microscopy and molecular probes showed that both photosensitizers were primarily lysosomally localized. SnOEBC, however, had an extra-lysosomal, mitochondrial localization component. The photophysical measurements allowed calculation of the intracellular singlet oxygen production, which indicated that the photosensitizer-light dose reciprocity was limited by photobleaching for SnET2 but only minimally for SnOEBC, and this was confirmed through cell-survival studies. Taken together, these data indicate that the critical determinant of differences in phototoxicity between the two molecules was their relative rates of photobleaching and their subcellular localization. The study points to the importance of combining photosensitizer uptake and localization with photophysical measurements in the relevant biological milieu to reasonably interpret and/or predict photosensitization efficacies.

Pre-treatment protoporphyrin IX concentration in actinic keratosis lesions may be a predictive biomarker of response to aminolevulinic-acid based photodynamic therapy.

BACKGROUND: Although aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy (PDT) is an effective FDA-approved therapy for actinic keratosis (AK), a substantial fraction of patients (up to 25%) do not respond to treatment. This study examined the feasibility of using pre-treatment measurements of PpIX concentration in AK lesions to predict response of ALA-PpIX PDT. METHODS: A non-invasive fiber-optic fluorescence spectroscopy system was used to measure PpIX concentration in patients undergoing standard-of-care ALA-PDT for AK. All patients provided assessments of pain at the time of treatment (n=70), and a subset reported pain and erythema 48-76 h after treatment (n=13). RESULTS: PpIX concentration was significantly higher in lesions of patients reporting high levels of pain (VAS score ≥5) immediately after treatment vs. patients reporting pain scores below VAS=5 (p<0.022) (n=70). However, pain was not an exclusive indicator of PpIX concentration as many patients with low PpIX concentration reported high pain. In a subpopulation of patients surveyed in the days after treatment (n=13), PpIX concentration measured on the day of treatment was uncorrelated with pain-reported immediately after treatment (r=0.17, p<0.57), but positive correlations were found between PpIX concentration and patient-reported pain (r=0.55, p<0.051) and erythema (r=0.58, p<0.039) in the 48-72 h following treatment. CONCLUSIONS: These data suggest that in vivo optical measurements of PpIX concentration acquired before light delivery may be an objective predictor of response to ALA-PpIX PDT. Identification of non-responding patients on the day of treatment could facilitate the use of interventions that may improve outcomes.

Introduction.

In the past decade, near-infrared sampling of tissue has become a large field of study, which has been called by many names (Photon migration, photon random walk, photon density waves, photon diffusion) encompassing work in spectroscopy and imaging of tissue. In this special issue, the focus is on diffuse optical tomography as it applies to medicine and biology. Diffuse optical tomography involves processing of near-infrared light measurements taken from tissue to create images of the interior.

Comparison of imaging geometries for diffuse optical tomography of tissue.

Images produced in six different geometries with diffuse optical tomography simulations of tissue have been compared using a finite element-based algorithm with iterative refinement provided by the Newton-Raphson approach. The source-detector arrangements studied include (i) fan-beam tomography, (ii) full reflectance and transmittance tomography, as well as (iii) sub-surface imaging, where each of these three were examined in a circular and a flat slab geometry. The algorithm can provide quantitatively accurate results for all of the tomographic geometries investigated under certain circumstances. For example, quantitatively accurate results occur with sub-surface imaging only when the object to be imaged is fully contained within the diffuse projections. In general the diffuse projections must sample all regions around the target to be characterized in order for the algorithm to recover quantitatively accurate results. Not only is it important to sample the whole space, but maximal angular sampling is required for optimal image reconstruction. Geometries which do not maximize the possible sampling angles cause more noise artifact in the reconstructed images. Preliminary simulations using a mesh of the human brain confirm that optimal images are produced from circularly symmetric source-detector distributions, but that quantitatively accurate images can be reconstructed even with a sub-surface imaging, although spatial resolution is modest.

Instrumentation and design of a frequency-domain diffuse optical tomography imager for breast cancer detection.

The instrument development and design of a prototype frequency-domain optical imaging device for breast cancer detection is described in detail. This device employs radio-frequency intensity modulated near-infrared light to image quantitatively both the scattering and absorption coefficients of tissue. The functioning components of the system include a laser diode and a photomultiplier tube, which are multiplexed automatically through 32 large core fiber optic bundles using high precision linear translation stages. Image reconstruction is based on a finite element solution of the diffusion equation. This tool for solving the forward problem of photon migration is coupled to an iterative optical property estimation algorithm, which uses a Levenberg-Marquardt routine with total variation minimization. The result of this development is an automated frequency-domain optical imager for computed tomography which produces quantitatively accurate images of the test phantoms used to date. This paper is a description and characterization of an automated frequency-domain computed tomography scanner, which is more quantitative than earlier systems used in diaphanography because of the combination of intensity modulated signal detection and iterative image reconstruction.

A theoretical study of light fractionation and dose-rate effects in photodynamic therapy.

The efficacy of photodynamic therapy is dependent upon the optical dose rate or upon the fractionation schedule on the light. These effects are thought to be limited by the time required for oxygen diffusion from the capillaries, since this therapy can consume oxygen faster than it can be supplied to tissues distant from the blood vessels. Oxygen diffusion and consumption by metabolic and photochemical mechanisms have been modeled here to compare theoretical predictions with experimental results of varying light fractionations and delivered dose rates. The mathematics of the problem have been described in the literature, and the present study extends these calculations to allow a more direct and quantitative comparison with fractionation experiments, using both analytical and numerical arguments. The optimum fraction time was found to depend only on the intercapillary spacing and not on the intensity of irradiation or the concentration of photosensitizer. The calculations indicate that experimentally observed optimum fractionation times of 30 and 60 s correspond to a distance from capillary to cell of approximately 1 mm. These results suggest that the fractionated light irradiation experiments need careful interpretation, and some possible reasons for longer optimum fractionation times are discussed.

Estimation of oxygen distribution in RIF-1 tumors by diffusion model-based interpretation of pimonidazole hypoxia and eppendorf measurements.

Numerical simulations of oxygen diffusion from the capillaries in tumor tissue were used to predict the capillary oxygen supply within and near hypoxic regions of the RIF-1 tumor. A finite element method to simulate the oxygen distribution from a histology section is presented, along with a method to iteratively estimate capillary oxygen concentrations. Pathological structural data for these simulations came from sections of the tumor stained with hematoxylin and eosin and were used to define the capillary positions and shapes, while overlapping regions of low oxygen concentration were defined by the hypoxia marker pimonidazole. These simulations were used to calculate spatial maps of the oxygen concentration and were tested for their ability to reproduce Eppendorf pO(2) histograms from the same tumor line. This simulation study predicted that capillary oxygen concentrations ranged from zero to above 20 microM, with a dominant peak in the hypoxic regions showing 78% of capillaries with less than 1 microM oxygen concentration, compared to only 12% in the non-hypoxic regions. The results were not highly sensitive to the metabolic oxygen consumption rate, within the range of 2 to 16 microM/s. This numerical method for oxygen capillary simulation is readily adaptable to histology sections and provides a method to examine the heterogeneity of oxygen within the capillaries and throughout the tumor tissue section being examined.

Image analysis for discrimination of cervical neoplasia.

Colposcopy involves visual imaging of the cervix for patients who have exhibited some prior indication of abnormality, and the major goals are to visually inspect for any malignancies and to guide biopsy sampling. Currently colposcopy equipment is being upgraded in many health care centers to incorporate digital image acquisition and archiving. These permanent images can be analyzed for characteristic features and color patterns which may enhance the specificity and objectivity of the routine exam. In this study a series of images from patients with biopsy confirmed cervical intraepithelia neoplasia stage 2/3 are compared with images from patients with biopsy confirmed immature squamous metaplasia, with the goal of determining optimal criteria for automated discrimination between them. All images were separated into their red, green, and blue channels, and comparisons were made between relative intensity, intensity variation, spatial frequencies, fractal dimension, and Euler number. This study indicates that computer-based processing of cervical images can provide some discrimination of the type of tissue features which are important for clinical evaluation, with the Euler number being the most clinically useful feature to discriminate metaplasia from neoplasia. Also there was a strong indication that morphology observed in the blue channel of the image provided more information about epithelial cell changes. Further research in this field can lead to advances in computer-aided diagnosis as well as the potential for online image enhancement in digital colposcopy.

Calibration of near-infrared frequency-domain tissue spectroscopy for absolute absorption coefficient quantitation in neonatal head-simulating phantoms.

Frequency-domain tissue spectroscopy is a method to measure the absolute absorption coefficient of bulk tissues, assuming that a representative model can be found to recover the optical properties from measurements. While reliable methods exist to calculate absorption coefficients from source-detector measurements less than a few centimeters apart along a flat tissue volume, it is less obvious what methods can be used for transmittance through the larger tissue volumes typically associated with neonatal cerebral monitoring. In this study we compare the use of multiple distance frequency-domain measurements processed with (i) a modified Beer-Lambert law method, (ii) an analytic infinite-medium diffusion theory expression, and (iii) a numerical finite element solution of the diffusion equation, with the goal of recovering the absolute absorption coefficient of the medium. Based upon our observations, the modified Beer-Lambert method provides accurate absolute changes in the absorption coefficient, while analytic infinite-medium diffusion theory solutions or finite element-based numerical solutions can be used to calculate the absolute absorption coefficient, assuming that the data can be measured at multiple source-detector distances. We recommend that the infinite-medium multi-distance method or the finite element method be used across large tissue regions for calculation of the absolute absorption coefficient using frequency-domain near-infrared measurements at multiple positions along the head.

Analysis of acetic acid-induced whitening of high-grade squamous intraepithelial lesions.

Immature and dysplastic cervical squamous epithelium whitens after the application of acetic acid during a colposcopic examination. The whitening process occurs visually over several minutes and subjectively discriminates between dysplastic and normal tissue. In this work, examples of the acetowhitening process are detailed in three ways: the color-imaged colposcopic appearance of the acetowhitening of high-grade cervical intraepithelial neoplasia (CIN 2/3), the kinetics of these reflectance patterns transformed to reduce noise in the signal, and a self-normalized green to red ratio measurement of the kinetics of these reflectance patterns. A total of six patients with biopsy confirmed CIN 2/3 were examined to obtain a set of timed images tracking the acetowhitening and the whitening-decay process over the course of 5-10 min. Regions of normal mature squamous epithelium within the same patients were also followed as an internal control. We determined that the temporal change over a 10 min time period in the ratio of green to red light intensities, taken from the respective color channels of the CCD, provides a reliable measure to clearly distinguish CIN 2/3 from normal cervical epithelium. This imaging and data normalization procedure may be applied to cervical lesions of different grades, to determine if a quantitative estimate provides predictive value during the colposcopic diagnosis.

Contrast-detail analysis for detection and characterization with near-infrared diffuse tomography.

Near-infrared (NIR) diffuse tomography is emerging as a medical imaging modality for obtaining information related to tissue hemoglobin concentration and oxygen saturation and may be used for characterizing diseased tissues such as breast cancer. The optimal methodology for NIR image reconstruction remains an ongoing research problem with several new approaches being demonstrated in recent years. However, a comparison of reconstruction methods is problematic because tools for the objective assessment of image quality have yet to be clearly defined for this type of nonlinear reconstruction problem. Contrast-detail analysis has become an accepted assessment tool to quantify x-ray mammography image quality, and in this study it has been applied to a prototype NIR diffuse tomography system that is being evaluated for breast cancer characterization. The minimum detectable levels of contrast have been defined for different sizes of objects, and the minimum contrasts which can be accurately reconstructed have also been determined for the same object sizes. In general, objects 8 mm and larger in diameter can be accurately reconstructed and detected for most absorption contrasts which are observed in human tissues (i.e., greater than 1% contrast in absorption). Objects as small as 2 mm can be detected with high contrast (i.e., near 100%), but cannot be accurately reconstructed. Within the size range of 2 mm to 8 mm, there is an inverse correlation between contrast and detail size which is characteristic of the total noise in the system. This analysis provides an objective method for assessing detection and characterization limits and can be applied to future improvements in hardware system architecture as well as reconstruction algorithms.

Photophysical properties of tin ethyl etiopurpurin I (SnET2) and tin octaethylbenzochlorin (SnOEBC) in solution and bound to albumin.

The photophysical characteristics of two second-generation PDT photosensitizers, tin ethyl etiopurpurin I (SnET2) and tin octaethylbenzochlorin (SnOEBC), have been measured in homogeneous solution and when bound to bovine serum albumin (BSA). The ground state and triplet state absorption spectra have been characterized, as have triplet lifetimes and quantum yields for intersystem crossing, singlet oxygen formation and photobleaching. In total, these parameters provide a complete set of data that can be used to quantitatively compare the photosensitizing efficiencies of these molecules. The photo-bleaching quantum yield of SnET2 is increased dramatically when it is bound to BSA, thus limiting the production of singlet oxygen at incident fluences above 1 J/cm2. In contrast, the quantum yield of photobleaching of SnOEBC is at least an order of magnitude lower than that of SnET2 under these conditions and does not significantly limit the photosensitization process for typical in vivo or in vitro fluences. This difference is expected to play a significant role in determining the relative photo-sensitizing ability of these compounds in vivo.

Analysis of the heterogeneity of pO2 dynamics during photodynamic therapy with verteporfin.

Photodynamic therapy (PDT) with verteporfin provides a reliable way to destroy malignant tissues. Changes in the blood flow and oxygen partial pressure (pO2) during verteporfin-PDT were studied here in the tumor tissue of the rat mammary R3230Ac carcinoma model. Oxygen microelectrodes (6-12 microns tip diameter) were used to measure the transients locally within tumors during intravenous injection of 1.0 mg/kg verteporfin followed by irradiation 15 min later with 690 nm light at 200 mW/cm2, for a cumulative dose of 144 J/cm2. The observed changes in pO2 were heterogeneous and there was a difference in the response of low-pO2 regions relative to higher-pO2 regions. The change in pO2 in hypoxic tissue regions (pO2 < 8 mmHg) had acute pO2 loss after treatment, whereas the response in regions of higher pO2 (> 8 mm Hg) was more heterogeneous with some areas maintaining their pO2 value after treatment was completed. Blood flow measurements taken on a subset of the animals indicated a significant loss in flow during the initial light delivery that remained low after treatment, indicating some vascular stasis. The results suggest that hypoxic or poorly perfused vessels may be more susceptible to acute stasis than normoxic vessels in this treatment protocol.

In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.