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.

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.

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.

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.

Accounting for pharmacokinetic differences in dual-tracer receptor density imaging.

Dual-tracer molecular imaging is a powerful approach to quantify receptor expression in a wide range of tissues by using an untargeted tracer to account for any nonspecific uptake of a molecular-targeted tracer. This approach has previously required the pharmacokinetics of the receptor-targeted and untargeted tracers to be identical, requiring careful selection of an ideal untargeted tracer for any given targeted tracer. In this study, methodology capable of correcting for tracer differences in arterial input functions, as well as binding-independent delivery and retention, is derived and evaluated in a mouse U251 glioma xenograft model using an Affibody tracer targeted to epidermal growth factor receptor (EGFR), a cell membrane receptor overexpressed in many cancers. Simulations demonstrated that blood, and to a lesser extent vascular-permeability, pharmacokinetic differences between targeted and untargeted tracers could be quantified by deconvolving the uptakes of the two tracers in a region of interest devoid of targeted tracer binding, and therefore corrected for, by convolving the uptake of the untargeted tracer in all regions of interest by the product of the deconvolution. Using fluorescently labeled, EGFR-targeted and untargeted Affibodies (known to have different blood clearance rates), the average tumor concentration of EGFR in four mice was estimated using dual-tracer kinetic modeling to be 3.9 ± 2.4 nM compared to an expected concentration of 2.0 ± 0.4 nM. However, with deconvolution correction a more equivalent EGFR concentration of 2.0 ± 0.4 nM was measured.

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.

Stem-cell therapy for dilated cardiomyopathy: a pilot study evaluating retrograde coronary venous delivery.

OBJECTIVE: To evaluate retrograde coronary venous stem-cell delivery for Dobermanns with dilated cardiomyopathy. METHODS: Retrograde coronary venous delivery of adipose-derived mesenchymal stem cells transduced with tyrosine mutant adeno-associated virus 2 to express stromal-derived factor-1 was performed in Dobermanns with dilated cardiomyopathy. Cases were followed for 2 years and electrocardiograms (ECG), echocardiograms and Holter monitoring were performed. RESULTS: Delivery of cells was feasible in 15 of 15 dogs. One dog died following the development of ventricular fibrillation 24 hours after cell delivery. The remaining 14 dogs were discharged the following day without complications. Echocardiographic measurements of left ventricular size and function showed continued progression of disease. On the basis of Kaplan-Meier product limit estimates, median survival for dogs following stem-cell delivery was 620 days (range of 1-799 days). When including only the occult-dilated cardiomyopathy population and excluding those dogs already in congestive heart failure, median survival was 652 days (range of 46-799 days). CLINICAL SIGNIFICANCE: Retrograde venous delivery of tyrosine mutant adeno-associated virus 2-stromal-derived factor-1 adipose-derived mesenchymal stem cells appears safe. Stem-cell therapy in dogs with occult-dilated cardiomyopathy does not appear to offer advantage compared to recently published survival data in similarly affected Dobermanns.

Advantages of a dual-tracer model over reference tissue models for binding potential measurement in tumors.

The quantification of tumor molecular expression in vivo could have a significant impact for informing and monitoring emerging targeted therapies in oncology. Molecular imaging of targeted tracers can be used to quantify receptor expression in the form of a binding potential (BP) if the arterial input curve or a surrogate of it is also measured. However, the assumptions of the most common approaches (reference tissue models) may not be valid for use in tumors. In this study, the validity of reference tissue models is investigated for use in tumors experimentally and in simulations. Three different tumor lines were grown subcutaneously in athymic mice and the mice were injected with a mixture of an epidermal growth factor receptor-targeted fluorescent tracer and an untargeted fluorescent tracer. A one-compartment plasma input model demonstrated that the transport kinetics of both tracers was significantly different between tumors and all potential reference tissues, and using the reference tissue model resulted in a theoretical underestimation in BP of 50% ± 37%. On the other hand, the targeted and untargeted tracers demonstrated similar transport kinetics, allowing a dual-tracer approach to be employed to accurately estimate BP (with a theoretical error of 0.23% ± 9.07%). These findings highlight the potential for using a dual-tracer approach to quantify receptor expression in tumors with abnormal hemodynamics, possibly to inform the choice or progress of molecular cancer therapies.

Scattering phase function spectrum makes reflectance spectrum measured from Intralipid phantoms and tissue sensitive to the device detection geometry.

Reflectance spectra measured in Intralipid (IL) close to the source are sensitive to wavelength-dependent changes in reduced scattering coefficient ([Formula: see text]) and scattering phase function (PF). Experiments and simulations were performed using device designs with either single or separate optical fibers for delivery and collection of light in varying concentrations of IL. Spectral reflectance is not consistently linear with varying IL concentration, with PF-dependent effects observed for single fiber devices with diameters smaller than ten transport lengths and for separate source-detector devices that collected light at less than half of a transport length from the source. Similar effects are thought to be seen in tissue, limiting the ability to quantitatively compare spectra from different devices without compensation.

SU-E-T-11: LINAC Dose Profiling Using Cherenkov Emission Imaging.

PURPOSE: To demonstrate the potential for fast 3D dose profile imaging of a LINAC beam using images of the induced Cherenkov radiation in a water tank. A specialized time-gated imaging system was developed as a prototype to quantify and compare with Monte Carlo, to illustrate the concept. METHODS: Images were acquired from a water tank during irradiation from a 6 MV Varian-2100C linear accelerator beam using a time-gated CCD-based imaging system. The camera was placed normal to the tank wall to minimize parallax reflections, and resultant images were produced by evaluating the median of each pixel in a stack of 2000 images taken at a rate of 60 Hz with an exposure time of 10 ms. Experimental data was compared to images obtained from GEANT4 simulations of the optical setup. RESULTS: Examination of the scored quantities for dose and generated Cherenkov photons indicates that there is a strong similarity, which can be explained by considering the electron energy losses per unit path length. However, due to the complex convolution of the Cherenkov emission directionality and camera lens angular field of view, this relationship is distorted. These errors can be calibrated using the GEANT4 simulations to more accurately reflect the intrinsic dose in the water volume. CONCLUSIONS: This work demonstrates dose profiling using the induced Cherenkov radiation signal for the first time. These preliminary results serve as a proof of concept of imaging at one azimuthal angle. Analogous to SPECT, the technique could easily be translated to multiple angles yielding full dose reconstructions following filtered back projection. Further refinement of this technology could be the first step in a paradigm shift towards an alternative method for fast radiation field analysis. Advantages would include increased speed, as well as the ability to profile dynamic beam shapes within transparent solid anthropomorphic phantoms. This work has been financially supported by NIH grant R01CA109558.

SU-E-I-94: External Beam Radiation Cherenkov Emission in Tissue Used for Tissue Oxygen Sensing.

PURPOSE: To show that Cherenkov emission is generated by external radiotherapy beam in tissue, and could serve as optical source to excite an oxygen sensitive phosphor, Oxyphor G4, within tissue. The intensity and lifetime of the phosphorescence was measured with a time-gated system and reveals the oxygenation levels in the tissue phantom. METHODS: A tissue phantom made with PBS, 1% v/v Intralipid-20% (Sigma Aldrich), 1% v/v whole blood and Oxyphor G4 in 1 µM concentration is irradiated by 18MeV external radiotherapy electron beam at a dose rate of 4 Gy/min generated by a medical linear accelerator (Varian LINAC 2100C, Varian Medical Systems). On one side of the phantom, a fiber bundle is used to conduct optical signal to a spectrometer connected to a fast gating ICCD (PI-MAX3, Princeton Instruments). For each oxygenation level, a series of spectrum of phosphorescence at different time points is measured by the time domain gating technique. Lifetime of phosphorescence is analyzed by exponential fitting and is validated by comparison to an independent analysis by frequency domain phosphorimetry. Monte Carlo simulations using GEANT4, of the fiber optic collection of Cerenkov light were performed to decide the sensitivity of the optical system for a range of specified geometries and beam types. Simulation results identify the effective depth within the phantom that is sampled by the optical collection of the Cerenkov signal. RESULTS: Simulations show that we can detect the Cherenkov signals comes from an approximately 5 mm depth from within the tissue phantom. Lifetime of the phosphorescence and pO2 of the phantom could be measured and calculated correctly by the time domain gating system. CONCLUSIONS: This work indicates time domain gating techniques combined with an oxygen sensitive phosphor are capable of accurately monitoring tissue oxygenation from a reasonable sampling depth in tissue in vivo during external beam radiotherapy. NIH grant R01CA109558.

A three-dimensional finite element model and image reconstruction algorithm for time-domain fluorescence imaging in highly scattering media.

In this work, development and evaluation of a three-dimensional (3D) finite element model (FEM) based on the diffusion approximation of time-domain (TD) near-infrared fluorescence light transport in biological tissue is presented. This model allows both excitation and fluorescence temporal point-spread function (TPSF) data to be generated for heterogeneous scattering and absorbing media of arbitrary geometry. The TD FEM is evaluated via comparisons with analytical and Monte Carlo (MC) calculations and is shown to provide a quantitative accuracy which has less than 0.72% error in intensity and less than 37 ps error for mean time. The use of the Born-Ratio normalized data is demonstrated to reduce data mismatch between MC and FEM to less than 0.22% for intensity and less than 22 ps in mean time. An image reconstruction framework, based on a 3D FEM formulation, is outlined and simulation results based on a heterogeneous mouse model with a source of fluorescence in the pancreas is presented. It is shown that using early photons (i.e. the photons detected within the first 200 ps of the TPSF) improves the spatial resolution compared to using continuous-wave signals. It is also demonstrated, as expected, that the utilization of two time gates (early and latest photons) can improve the accuracy both in terms of spatial resolution and recovered contrast.

Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging.

Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters and the tissue is relatively homogeneous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins.

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.

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.

Comparison of fibroglandular tissue distributions for microwave tomographic breast images with complementary MR T2 weighted images.

We have recently demonstrated good correlation between the recovered permittivity from microwave imaging (MIS) and the recovered water content from near infrared imaging (NIR) for a common set of normal patients undergoing associated breast examinations. We have subsequently conducted a small sample of comparison breast examinations between microwave imaging and MR to assess possible correlation between the location and extent of the fibroglandular as seen on MR images with increased permittivity zones of the microwave images. From various physiological and MR breast studies, it has been shown that the fibroglandular regions are generally comprised of significantly higher levels of water than the more dominant adipose tissue. The initial results of this study are quite encouraging and demonstrate obvious correlations between the permittivity and MR-recovered fibroglandular regions for a set of patients with widely varying tissue type variations. In addition, they illustrate the value of extracting diagnostic information from multiple modalities especially where the amount of direct in vivo property measurements is limited or nonexistent.

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.

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.

Microwave image reconstruction utilizing log-magnitude and unwrapped phase to improve high-contrast object recovery.

Reconstructing images of large high-contrast objects with microwave methods has proved difficult. Successful images have generally been obtained by using a priori information to constrain the image reconstruction to recover the correct electromagnetic property distribution. In these situations, the measured electric field phases as a function of receiver position around the periphery of the imaging field-of-view vary rapidly often undergoing changes of greater than pi radians especially when the object contrast and illumination frequency increase. In this paper, we introduce a modified form of a Maxwell equation model-based image reconstruction algorithm which directly incorporates log-magnitude and phase of the measured electric field data. By doing so, measured phase variation can be unwrapped and distributed over more than one Rieman sheet in the complex plane. Simulation studies and microwave imaging experiments demonstrate that significant image quality enhancements occur with this approach for large high-contrast objects. Simple strategies for visualizing and unwrapping phase values as a function of the transmitter and receiver positions within our microwave imaging array are described. Metrics of the degree of phase variation expressed in terms of the amount and extent of phase wrapping are defined and found to be figures-of-merit which estimate when it is critical to deploy the new image reconstruction approach. In these cases, the new algorithm recovers high-quality images without resorting to the use of a priori information on object contrast and/or size as previously required.