Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electronmultiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi’s sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual-wavelength fluorescence images were acquired using a targeted 111In- DTPA-K(IRDye800)-c(KRGDf) to selectively detect tumor angiogenesis and an untargeted Cy5.5 to image lymphatics. After acquiring the experimental data, fluorescence image-guided surgery was performed. Dynamic, multi-wavelength fluorescence imaging was accomplished using a liquid crystal tunable filter (LCTF). Excitation light was used for reflectance images with a LCTF transmitting a shorter wavelength than the peak in the excitation light spectrum. Therefore, images can be dynamically acquired alternating frame by frame between emission and excitation light, which should enable image-guided surgery.

We have developed a simulation of the AdaptiSPECT small-animal Single Photon Emission Computed Tomography (SPECT) imaging system. The simulation system is entitled SimAdaptiSPECT and is written in C, NVIDIA CUDA, and Matlab. Using this simulation, we have accomplished an analysis of the Scanning Linear Estimation (SLE) technique for estimating tumor parameters, and calculated sensitivity information for AdaptiSPECT configurations.SimAdaptiSPECT takes, as input, simulated mouse phantoms (generated by MOBY) contained in binary files and AdaptiSPECT configuration geometry contained in ASCII text files. SimAdaptiSPECT utilizes GPU parallel processing to simulate AdaptiSPECT images. SimAdaptiSPECT also utilizes GPU parallel processing to perform 3-D image reconstruction from 2-D AdaptiSPECT camera images (real or simulated), using a novel variant of the Ordered Subsets Expectation Maximization (OSEM) algorithm. Methods for generating the inputs, such as a population of randomly varying numerical mouse phantoms with randomly varying hepatic lesions, are also discussed.

L’imagerie in vivo du petit animal est de plus en plus utilisée en pharmacologie pour identifier et caractériser l’activité de nouveaux agents anticancéreux.La première partie de ma thèse a consisté à développer des outils pour améliorer la quantification enbioluminescence. Une méthode, basée sur les caractéristiques spectrales des photons émis, a été établie pour corriger l’absorption tissulaire. La seconde, faisant appel aux méthodes de restauration d’images, avait pour but de corriger la diffusion pour augmenter la résolution. Dans un second temps, j’ai mis en place des modèles in vivo de tumeurs expérimentales bioluminescentes (un glioblastome intracérébral, un lymphome anaplasique à grandes cellules et un neuroblastome métastatique) en utilisant les méthodes d’imagerie décrites précédemment. Ces études ont permis d’étendre la caractérisation de l’activité préclinique d’un nouvel agent anticancéreux. L’objectif de la dernière partie de mon travail était de développer des sondes d’imagerie. La première sonde, un anticorps monoclonal anti-CD45 marqué avec un fluorochrome a permis la détection de cellules leucémiques humaines implantées chez la souris en utilisant l’imagerie de fluorescence. La seconde a été développée pour prédire l’entrée d’un agent anticancéreux, un conjugué spermine-podophyllotoxin, dans les cellules tumorales via les transporteurs des polyamines. La sonde synthétisée est une spermine à laquelle un groupement HYNIC a été ajouté afin de pouvoir lier un radioisotope : le Technétium 99m et ainsi réaliser un examen scintigraphique. Les résultats ont démontré la faisabilité d’une application préclinique de cette sonde. Ainsi à l’issu de cette thèse, les méthodes de traitement des signaux de bioluminescence développées sont disponibles pour améliorer l’application de l’imagerie optique en pharmacologie. Bien sûr des études supplémentaires sont encore nécessaires pour définir précisément dans quel contexte ces corrections seront les plus appropriées. / Small animal imaging is more and more used in pharmacology to identify and to characterize the activities of new antitumor agents. The first part of my thesis consisted in the development of new tools to improve the quantitation in bioluminescence. A method, based on spectral characteristics of emitted photons, has been established to correct tissue absorption. The second, using methods of image restoration had for objective to correct tissue scattering to increase the resolution. In a second part, I developed in vivo models of bioluminescent tumors (intracranial glioblastoma, a large cell anaplastic lymphoma and a metastatic neuroblastoma) using the imaging methods described previously. These studies allowed the characterization of the activity of a new antitumor agent. The aim of the last part was to develop imaging probes. The first, a monoclonal antibody antiCD45 labeled with a fluorochrome allowed the detection of human leukemic cells implanted in the mice using fluorescence imaging. The second was developed to predict the uptake of a antitumor agent, a spermine-podophyllotoxin conjugate, in tumor cells via the polyamine transport system. The synthesized probe is a spermine conjugated to a HYNIC group to bind a radioisotope: the Technetium 99m and to realize a scintigraphic examination. The results showed the feasibility of a preclinical use of this probe. So, at this end of this thesis, the developed methods of bioluminescent signal processing are available to improve the use of optical imaging in pharmacology. Of course, supplementary studies are necessary to define precisely in which context these corrections will be the most appropriate.

Multifunctional theranostic nanoparticles hold promise for enabling non-invasive image guided cancer therapy such as photothermal therapy. Human breast tumor models in which response to image guided therapy can quickly and non-invasively be determined are needed to facilitate translation and application of these technologies. We hypothesize that a system utilizing a murine light-reporter mammary tumor cell line and near-infrared nanoparticles (NIR-NP) can be used to quantify response to therapy and determine fate of nanoparticles following photothermal ablation.

Research involved in modeling human lung disease conditions has provided insight into disease development, progression, and treatment. In particular, mouse models of human pulmonary disease are increasingly utilized to characterize lung disease conditions. With advancements in small animal imaging it is now possible to investigate the phenotypic differences expressed in inbred mouse strains in vivo to investigate specific disease conditions that affect the lung. In this thesis our aim was to generate a comprehensive characterization of the normative mouse lung phenotypes in three of the most utilized strains of mice, C57BL/6, A/J, and BALB/c, through imaging techniques. The imaging techniques that we utilized in this research included micro-CT, a custom Large Image Microscope Array (LIMA) system for 3D microscopy, and classical histology. Micro-CT provided a non-destructive technique for acquiring in vivo and fixed lung images. The LIMA 3D microscopy system was utilized for direct correspondence of the gold standard histology images as well as to validate the anatomical structures and measurements that were extracted from the micro-CT images. Finally, complete lung histology slices were utilized for assessment of the peripheral airspace structures that were not resolvable using the micro-CT imaging system. Through our developed imaging acquisition and processing strategies we have been able to successful characterize important phenotypes in the mouse lung that have not previously been known as well as identify strain variations. These findings will provide the scientific community with valuable information to be better equipped and capable of pursuing new avenues of research in investigating pulmonary disease conditions that can be modeled in the mouse.

Various colloidal delivery systems, including polymeric nanoparticles, metal colloids, liposomes, and microemulsions have been reported to enhance the delivery of therapeutic agents following intranasal administration. However, the mechanisms involved in the uptake of these nanomaterials, especially those in the ultrafine size ranges (diameter < 20 nm) through nasal mucosa and their subsequent biodistribution in the body are not well characterized. The objectives of this study address the knowledge gap regarding ultrafine nanoparticle transfer in the nasal mucosa by quantifying nanoparticle uptake and biodistibution patterns in the presence and absence of known inhibitors of endocytic processes. The uptake of ~ 10 nm fluorescent quantum dots (QDs) was investigated by measuring the concentration of QDs following exposure to bovine respiratory and olfactory mucosal explants. An inductively coupled optical emission spectroscopy method was developed to measure the amount of QDs within the tissues. The results demonstrated that carboxylate-modified QDs (COOH-QDs) show ~2.5 fold greater accumulation in the epithelial and submucosal regions of the olfactory tissues compared to the respiratory tissues. Endocytic inhibitory studies showed that in respiratory tissues clathrin-dependent, macropinocytosis and caveolae-dependent endocytosis process were all involved in the uptake of COOH-QDs. Whereas in olfactory tissues, clathrin-dependent endocytosis was the major endocytic pathway involved in uptake of COOH-QDs. Additional energy-independent pathways appeared to also be active in the transfer of COOH-QDs into the olfactory mucosa. Interestingly, PEGylated quantum dots (PEG-QDs) of similar size ~15 nm were not internalized into the bovine nasal tissues. In vivo fluorescence imaging was used to study the biodistribution of quantum dots following nasal instillation in mice. These studies showed that majority of COOH-QDs remain in the nasal tissues for relatively long periods of time (up to 24 h) whereas PEG-QDs showed no such accumulation. Biodistribution studies of gold nanoparticles (~15 nm) in mice using micro-CT showed that gold nanoparticles were transferred to the posterior turbinate region and a fraction of the administered dose distributed to regions in close proximity to the olfactory bulb. Both NIR imaging and micro-CT imaging were useful tools for visualization of in vivo nanoparticle distribution. A diazepam-containing microemulsion (dispersed phase ~40 nm) was formulated to investigate the uptake mechanisms utilized for fluid-phase colloidal dispersions in the nasal mucosa. The resulting diazepam-containing microemulsion showed enhanced transfer of the drug into the bovine nasal respiratory and olfactory tissues. It is unclear if endocytosis of the fluid-phase nanodispersions played a role in drug absorption from the microemulsions in a manner similar to the uptake of solid-phase nanoparticles, however, since there was significant loss of the epithelial cell layer following exposure to the microemulsion formulation which likely altered the barrier properties of the epithelium. These studies have increased the fundamental understanding of ultrafine nanoparticle uptake in the nasal tissues and the resulting nanoparticle biodistribution patterns. While ultrafine nanoparticles may have limited application in the development of efficient drug delivery systems, an understanding of the size-dependent and tissue-dependent processes responsible for the uptake of particulates into mucosal tissues will contribute to the rational development of nanoparticulate drug delivery strategies investigating the nasal and other routes of administration.

Reconstructed tomographic image resolution of small animal PET imaging systems is improving with advances in radiation detector development. However the trend towards higher resolution systems has come with an increase in price and system complexity. Recent developments in the area of solid-state photomultiplication devices like silicon photomultiplier arrays (SPMA) are creating opportunities for new high performance tools for PET scanner design. Imaging of excised small animal organs and tissues has been used as part of post-mortem studies in order to gain detailed, high-resolution anatomical information on sacrificed animals. However, this kind of ex-vivo specimen imaging has largely been limited to ultra-high resolution μCT. The inherent limitations to PET resolution have, to date, excluded PET imaging from these ex-vivo imaging studies. In this work, we leverage the diminishing physical size of current generation SPMA designs to create a very small, simple, and high-resolution prototype detector system targeting ex-vivo tomographic imaging of small animal organs and tissues. We investigate sensitivity, spatial resolution, and the reconstructed image quality of a prototype small animal PET scanner designed specifically for imaging of excised murine tissue and organs. We aim to demonstrate that a cost-effective silicon photomultiplier (SiPM) array based design with thin crystals (2 mm) to minimize depth of interaction errors might be able to achieve sub-millimeter resolution. We hypothesize that the substantial decrease in sensitivity associated with the thin crystals can be compensated for with increased solid angle detection, longer acquisitions, higher activity and wider acceptance energy windows (due to minimal scatter from excised organs). The constructed system has a functional field of view (FoV) of 40 mm diameter, which is adequate for most small animal specimen studies. We perform both analytical (3D-FBP) and iterative (ML-EM) methods in order to reconstruct tomographic images. Results demonstrate good agreement between the simulation and the prototype. Our detector system with pixelated crystals is able to separate small objects as close as 1.25 mm apart, whereas spatial resolution converges to the theoretical limit of 1.6 mm (half the size of the smallest detecting element), which is to comparable to the spatial resolution of the existing commercial small animal PET systems. Better system spatial resolution is achievable with new generation SiPM detector boards with 1 mm x 1 mm cell dimensions. We demonstrate through Monte Carlo simulations that it is possible to achieve sub-millimeter spatial image resolution (0.7 mm for our scanner) in complex objects using monolithic crystals and exploiting the light-sharing mechanism among the neighboring detector cells. Results also suggest that scanner (or object) rotation minimizes artifacts arising from poor angular sampling, which is even more significant in smaller PET designs as the gaps between the sensitive regions of the detector have a more exaggerated effect on the overall reconstructed image quality when the design is more compact. Sensitivity of the system, on the other hand, can be doubled by adding two additional detector heads resulting in a, fully closed, 4π geometry.

Herein we present the development of a fluorescence-enhanced, frequency-domain radiative transport reconstruction system designed for small animal optical tomography. The system includes a time-dependent data acquisition instrument, a radiative transport based forward model for prediction of time-dependent propagation of photons in small, non-diffuse volumes, and an algorithm which utilizes the forward model to reconstruct fluorescent yields from air/tissue boundary measurements. The major components of the instrumentation include a charge coupled device camera, an image intensifier, signal generators, and an optical switch. Time-dependent data were obtained in the frequency-domain using homodyne techniques on phantoms with 0.2% to 3% intralipid solutions. Through collaboration with Transpire, Inc., a fluorescence-enhanced, frequency-domain, radiative transport equation (RTE) solver was developed. This solver incorporates the discrete ordinates, source iteration with diffusion synthetic acceleration, and linear discontinuous finite element differencing schemes, to predict accurately the fluence of excitation and emission photons in diffuse and transport limited systems. Additional techniques such as the first scattered distributed source method and integral transport theory are used to model the numerical apertures of fiber optic sources and detectors. The accuracy of the RTE solver was validated against diffusion and Monte Carlo predictions and experimental data. The comparisons were favorable in both the diffusion and transport limits, with average errors of the RTE predictions, as compared to experimental data, typically being less than 8% in amplitude and 7% in phase. These average errors are similar to those of the Monte Carlo and diffusion predictions. Synthetic data from a virtual mouse were used to demonstrate the feasibility of using the RTE solver for reconstructing fluorescent heterogeneities in small, non-diffuse volumes. The current version of the RTE solver limits the reconstruction to one iteration and the reconstruction of marginally diffuse, frequency-domain experimental data using RTE was not successful. Multiple iterations using a diffusion solver successfully reconstructed the fluorescent heterogeneities, indicating that, when available, multiple iterations of the RTE based solver should also reconstruct the heterogeneities.

Herein we present the development of a fluorescence-enhanced, frequency-domain radiative transport reconstruction system designed for small animal optical tomography. The system includes a time-dependent data acquisition instrument, a radiative transport based forward model for prediction of time-dependent propagation of photons in small, non-diffuse volumes, and an algorithm which utilizes the forward model to reconstruct fluorescent yields from air/tissue boundary measurements. The major components of the instrumentation include a charge coupled device camera, an image intensifier, signal generators, and an optical switch. Time-dependent data were obtained in the frequency-domain using homodyne techniques on phantoms with 0.2% to 3% intralipid solutions. Through collaboration with Transpire, Inc., a fluorescence-enhanced, frequency-domain, radiative transport equation (RTE) solver was developed. This solver incorporates the discrete ordinates, source iteration with diffusion synthetic acceleration, and linear discontinuous finite element differencing schemes, to predict accurately the fluence of excitation and emission photons in diffuse and transport limited systems. Additional techniques such as the first scattered distributed source method and integral transport theory are used to model the numerical apertures of fiber optic sources and detectors. The accuracy of the RTE solver was validated against diffusion and Monte Carlo predictions and experimental data. The comparisons were favorable in both the diffusion and transport limits, with average errors of the RTE predictions, as compared to experimental data, typically being less than 8% in amplitude and 7% in phase. These average errors are similar to those of the Monte Carlo and diffusion predictions. Synthetic data from a virtual mouse were used to demonstrate the feasibility of using the RTE solver for reconstructing fluorescent heterogeneities in small, non-diffuse volumes. The current version of the RTE solver limits the reconstruction to one iteration and the reconstruction of marginally diffuse, frequency-domain experimental data using RTE was not successful. Multiple iterations using a diffusion solver successfully reconstructed the fluorescent heterogeneities, indicating that, when available, multiple iterations of the RTE based solver should also reconstruct the heterogeneities.

No description available.