Singular value decomposition metrics show limitations of detector design in diffuse fluorescence tomography

The spatial resolution and recovered contrast of images reconstructed from diffuse fluorescence tomography data are limited by the high scattering properties of light propagation in biological tissue. As a result, the image reconstruction process can be exceedingly vulnerable to inaccurate prior knowledge of tissue optical properties and stochastic noise. In light of these limitations, the optimal source-detector geometry for a fluorescence tomography system is non-trivial, requiring analytical methods to guide design. Analysis of the singular value decomposition of the matrix to be inverted for image reconstruction is one potential approach, providing key quantitative metrics, such as singular image mode spatial resolution and singular data mode frequency as a function of singular mode. In the present study, these metrics are used to analyze the effects of different sources of noise and model errors as related to image quality in the form of spatial resolution and contrast recovery. The image quality is demonstrated to be inherently noise-limited even when detection geometries were increased in complexity to allow maximal tissue sampling, suggesting that detection noise characteristics outweigh detection geometry for achieving optimal reconstructions

Bioenergetics and survival of chum (Oncorhynchus keta) and pink (O. gorbuscha) salmon in heated seawater

The use of heated seawater for enhancing the culture of Pacific salmon was investigated. Food consumption rate, gross food conversion efficiency, growth rate, and survival of chum (Oncorhynchus keta) and pink (O. gorbuscha} salmon fed to satiation were determined in relation to water temperature and body weight. Both species of salmon were raised at temperatures of 55°, 60°, 65°, and 70°F. The highest food consumption rate, gross food conversion efficiency, growth rate, and survival of chum salmon occurred at temperatures of 65°, 55°, 55°-65°, and 55°F, respectively, while those of pink salmon occurred at 65°, 55°-65°, 60°-65° and 55 -60°F, respectivety. The response to all conditions measured was poorest for chum and pink salmon at 70°F. Pink salmon generally had a higher food consumption rate, gross food conversion efficiency, and growth rate than chum salmon. Food consumption rate, gross food conversion efficiency, growth rate, and survival of chum and pink salmon decreased as body weight increased. As body weight increased, food consumption rate, gross food conversion efficiency, and growth rate generally decreased more at 60°-65°F than at 55°F for chum salmon, and more at 65°-70°F than at lower temperatures for pink salmon. Survival was influenced by behavior and disease in addition to temperature. Diseases encountered were bacterial kidney disease and vibriosis, of which the former was most prevalent. Chum and pink salmon can be cultured in heated seawater at temperatures not exceeding 65°F. Good growth (>5% gain in body weight/day) and survival (60-100%) can be expected

Automatic Exposure Control and Estimation of Effective System Noise in Diffuse Fluorescence Tomography

A diffuse fluorescence tomography system, based upon time-correlated single photon counting, is presented with an automated algorithm to allow dynamic range variation through exposure control. This automated exposure control allows the upper and lower detection levels of fluorophore to be extended by an order of magnitude beyond the previously published performance and benefits in a slight decrease in system effective noise. The effective noise level is used as a metric to characterize the system performance, integrating both model-mismatch and calibration bias errors into a single parameter. This effective error is near 7% of the reconstructed fluorescent yield value, when imaging in just few minutes. Quantifying protoporphyrin IX concentrations down to 50 ng/ml is possible, for tumor-sized regions. This fluorophore has very low fluorescence yield, but high biological relevance for tumor imaging, given that it is produced in the mitochondria, and upregulated in many tumor types

Fluorescence Tomography Characterization for Sub-Surface Imaging with Protoporphyrin IX

Optical imaging of fluorescent objects embedded in a tissue simulating medium was characterized using non-contact based approaches to fluorescence remittance imaging (FRI) and sub-surface fluorescence diffuse optical tomography (FDOT). Using Protoporphyrin IX as a fluorescent agent, experiments were performed on tissue phantoms comprised of typical in-vivo tumor to normal tissue contrast ratios, ranging from 3.5:1 up to 10:1. It was found that tomographic imaging was able to recover interior inclusions with high contrast relative to the background; however, simple planar fluorescence imaging provided a superior contrast to noise ratio. Overall, FRI performed optimally when the object was located on or close to the surface and, perhaps most importantly, FDOT was able to recover specific depth information about the location of embedded regions. The results indicate that an optimal system for localizing embedded fluorescent regions should combine fluorescence reflectance imaging for high sensitivity and sub-surface tomography for depth detection, thereby allowing more accurate localization in all three directions within the tissue

Imaging of Glioma Tumor with Endogenous Fluorescence Tomography

Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX. Fluorescence tomography provides a tool for preclinical molecular contrast agent assessment in oncology.1, 2, 3, 4 Systems have advanced in complexity to where noncontact imaging,5 automated boundary recovery,6 and sophisticated internal tissue shapes can be included in the recovered images. The translation of this work to humans will require molecular contrast agents that are amenable to regulatory approval and maintain tumor specificity in humans, where often nonspecific uptake of molecular imaging agents can decrease their utility. In this study, a new fluorescence tomography system coupled to microCT7 was used to illustrate diagnostic detection of orthotopic glioma tumors that were not apparent in the microCT images, using endogenous fluorescent contrast from protoporphyrin IX (PpIX). Glioma tumors provide significant endogenous fluorescence from PpIX,8, 9, 10, 11 and this is enhanced when the subject imaged has been administered aminolevulinic acid (ALA). The endogenous production process of PpIX is known to stem from the administered, ALA bypassing the regulatory inhibition of ALA synthase, allowing the heme synthesis pathway to proceed uninhibited. Since there is a limited supply of iron in the body, this process produces overabundance of PpIX rather than heme, and many tumors have been shown to have high yields of PpIX. Clinical trials with PpIX fluorescence guided resection of tumors have shown significant promise,12 and yet deep tissue imaging with PpIX fluorescence has not been exploited in clinical use. Early studies have shown that detection of these tumors with PpIX is feasible,13, 14 but no tomographic imaging has been used. This limitation in development has largely been caused by problems in wavelength filtering and low signal intensity, as well as background fluorescence from the skin limiting sensitivity to deeper structures. In the system developed and used here, this feasibility is demonstrated by imaging a human xenograft glioma model. To solve the sensitivity problem and study the ability to diagnostically image PpIX in vivo, time-correlated single-photon counting was used in the fluorescence tomography system, which provides maximum sensitivity. Figure 1a shows the system designed to match up with a microCT, allowing both x-ray structural and optical functional imaging sequentially. Lens-coupled detection of signals is acquired from the mouse using five time-resolved photomultiplier tubes (H7422P-50, Hamamatsu, Japan) with single-photon counting electronics (SPC-134 modules, Becker and Hickl GmbH, Germany). The system has fan-beam transmission geometry similar to a standard CT scanner, with single source delivery of a1-mW role= presentation \u3e1-mW pulsed diode laser light at 635nm role= presentation \u3e635nm , collimated to a 1-mm role= presentation \u3e1-mm effective area on the animal. The five detection lenses were arranged in an arc, each with 22.5-deg role= presentation \u3e22.5-deg angular separation, centered directly on the opposite side of the animal with long working distance pickup,7 allowing noncontact measurement of the diffuse light through the animal. The diffuse intensity signals collected at each of the five channels were then translated via 400-μm role= presentation \u3e400-μm fibers and split using beamsplitters to be directed toward the fluorescence (95%) and transmission (5%) channel detectors. A 650-nm role= presentation \u3e650-nm long-pass filter was used in the fluorescence channels to isolate the signal, and in the transmitted intensity signals, a neutral density filter (2 OD) was used to attenuate the signals. This latter filtering was necessary to ensure that the fluorescence and transmission. Intensity signals fell within the same dynamic range, allowing a single 1s role= presentation \u3e1s acquisition for each detector. Scans were then performed by rotating the fan-beam around the specimen to 32 locations. A GE eXplore Locus SP scanner (GE Healthcare, London, Ontario, Canada) that incorporated a detector with 94-micronpixel role= presentation \u3e94-micronpixel resolution, a 80-kV role= presentation \u3e80-kV peak voltage, and a tube current of 450μAs role= presentation \u3e450μAs , was used in acquiring the microCT data, as displayed in Fig. 2 . In this example, since soft tissue was being imaged, the CT data was largely used to image the exterior of the animal, although in future studies, it could be used to isolate the cranium region as well

Split operator method for fluorescence diffuse optical tomography using anisotropic diffusion regularisation with prior anatomical information

Fluorescence diffuse optical tomography (fDOT) is an imaging modality that provides images of the fluorochrome distribution within the object of study. The image reconstruction problem is ill-posed and highly underdetermined and, therefore, regularisation techniques need to be used. In this paper we use a nonlinear anisotropic diffusion regularisation term that incorporates anatomical prior information. We introduce a split operator method that reduces the nonlinear inverse problem to two simpler problems, allowing fast and efficient solution of the fDOT problem. We tested our method using simulated, phantom and ex-vivo mouse data, and found that it provides reconstructions with better spatial localisation and size of fluorochrome inclusions than using the standard Tikhonov penalty term

High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation

Vision 20/20: Molecular-guided surgical oncology based upon tumor metabolism or immunologic phenotype: Technological pathways for point of care imaging and intervention

Surgical guidance with fluorescence has been demonstrated in individual clinical trials for decades, but the scientific and commercial conditions exist today for a dramatic increase in clinical value. In the past decade, increased use of indocyanine green based visualization of vascular flow, biliary function, and tissue perfusion has spawned a robust growth in commercial systems that have near-infrared emission imaging and video display capabilities. This recent history combined with major preclinical innovations in fluorescent-labeled molecular probes, has the potential for a shift in surgical practice toward resection guidance based upon molecular information in addition to conventional visual and palpable cues. Most surgical subspecialties already have treatment management decisions partially based upon the immunohistochemical phenotype of the cancer, as assessed from molecular pathology of the biopsy tissue. This phenotyping can inform the surgical resection process by spatial mapping of these features. Further integration of the diagnostic and therapeutic value of tumor metabolism sensing molecules or immune binding agents directly into the surgical process can help this field mature. Maximal value to the patient would come from identifying the spatial patterns of molecular expression in vivo that are well known to exist. However, as each molecular agent is advanced into trials, the performance of the imaging system can have a critical impact on the success. For example, use of pre-existing commercial imaging systems are not well suited to image receptor targeted fluorophores because of the lower concentrations expected, requiring orders of magnitude more sensitivity. Additionally the imaging system needs the appropriate dynamic range and image processing features to view molecular probes or therapeutics that may have nonspecific uptake or pharmacokinetic issues which lead to limitations in contrast. Imaging systems need to be chosen based upon objective performance criteria, and issues around calibration, validation, and interpretation need to be established before a clinical trial starts. Finally, as early phase trials become more established, the costs associated with failures can be crippling to the field, and so judicious use of phase 0 trials with microdose levels of agents is one viable paradigm to help the field advance, but this places high sensitivity requirements on the imaging systems used. Molecular-guided surgery has truly transformative potential, and several key challenges are outlined here with the goal of seeing efficient advancement with ideal choices. The focus of this vision 20/20 paper is on the technological aspects that are needed to be paired with these agents

Automatic exposure control and estimation of effective system noise in diffuse fluorescence tomography

A diffuse fluorescence tomography system, based upon time-correlated single photon counting, is presented with an automated algorithm to allow dynamic range variation through exposure control. This automated exposure control allows the upper and lower detection levels of fluorophore to be extended by an order of magnitude beyond the previously published performance and benefits in a slight decrease in system effective noise. The effective noise level is used as a metric to characterize the system performance, integrating both model-mismatch and calibration bias errors into a single parameter. This effective error is near 7% of the reconstructed fluorescent yield value, when imaging in just few minutes. Quantifying protoporphyrin IX concentrations down to 50 ng/ml is possible, for tumor-sized regions. This fluorophore has very low fluorescence yield, but high biological relevance for tumor imaging, given that it is produced in the mitochondria, and upregulated in many tumor types