Fluorescence molecular tomography: Principles and potential for pharmaceutical research

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue’s optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development

Multi-Modality Diffuse Fluorescence Imaging Applied to Preclinical Imaging in Mice

RÉSUMÉ Cette thèse vise à explorer l'information anatomique et fonctionnelle en développant de nouveaux systèmes d'imagerie de fluorescence macroscopiques à base de multi-modalité. L‘ajout de l‘imagerie anatomique à des modalités fonctionnelles telles que la fluorescence permet une meilleure visualisation et la récupération quantitative des images de fluorescence, ce qui en retour permet d'améliorer le suivi et l'évaluation des paramètres biologiques dans les tissus. Sur la base de cette motivation, la fluorescence a été combinée avec l‘imagerie ultrasonore (US) d'abord et ensuite l'imagerie par résonance magnétique (IRM). Dans les deux cas, les performances du système ont été caractérisées et la reconstruction a été évaluée par des simulations et des expérimentations sur des fantômes. Finalement, ils ont été utilisés pour des expériences d'imagerie moléculaire in vivo dans des modèles de cancer et d‘athérosclérose chez la souris. Les résultats ont été présentés dans trois articles, qui sont inclus dans cette thèse et décrits brièvement ci-dessous. Un premier article présente un système d'imagerie bimodalité combinant fluorescence à onde continue avec l‘imagerie à trois dimensions (3D) US. A l‘aide de stages X-Y motorisés, le système d'imagerie a été en mesure de recueillir l‘émission fluorescente et les échos acoustiques délimitant la surface 3D et la position des inclusions fluorescentes dans l'échantillon. Une validation sur fantômes, a montré que l'utilisation des priors anatomiques provenant des US améliorait la qualité de la reconstruction fluorescente. En outre, un étude pilote in-vivo en utilisant une souris Apo-E a évalué la faisabilité de cette approche d'imagerie double modalité pour de futures études pré-cliniques. Dans un deuxième effort, et sur la base du premier travail, nous avons amélioré le système d'imagerie par fluorescence-US au niveau des algorithmes, de la précision----------ABSTRACT This thesis aims to explore the anatomical and functional information by developing new macroscopic multi-modality fluorescence imaging schemes. Adding anatomical imaging to functional modalities such as fluorescence enables better visualization and recovery of fluorescence images, in turn, improving the monitoring and assessment of biological parameters in tissue. Based on this motivation, fluorescence was combined with ultrasound (US) imaging first and then magnetic resonance imaging (MRI). In both cases, the systems characterization and reconstruction performance were evaluated by simulations and phantom experiments. Eventually, they were applied to in vivo molecular imaging in models of cancer and atherosclerosis in mice. Results were presented in three peer-reviewed journals, which are included in this thesis and shortly described below. A first article presented a dual-modality imaging system combining continuous-wave transmission fluorescence imaging with three dimensional (3D) US imaging. Using motorized X-Y stages, the fluorescence-US imaging system was able to collect boundary fluorescent emission, and acoustic pulse-echoes delineating the 3D surface and position of fluorescent inclusions within the sample. A validation in phantoms showed that using the US anatomical priors, the fluorescent reconstruction quality was significantly improved. Furthermore, a pilot in-vivo study using an Apo-E mouse evaluated the feasibility of this dual-modality imaging approach for future animal studies. In a second endeavor, and based on the first work, we improved the fluorescence-US imaging system in terms of sampling precision and reconstruction algorithms. Specifically, now combining US imaging and profilometry, both the fluorescent target and 3D surface of sample could be obtained in order to achieve improved fluorescence reconstruction. Furthermore,

Advanced maximum entropy approaches for medical and microscopy imaging

The maximum entropy framework is a cornerstone of statistical inference, which is employed at a growing rate for constructing models capable of describing and predicting biological systems, particularly complex ones, from empirical datasets.‎ In these high-yield applications, determining exact probability distribution functions with only minimal information about data characteristics and without utilizing human subjectivity is of particular interest. In this thesis, an automated procedure of this kind for univariate and bivariate data is employed to reach this objective through combining the maximum entropy method with an appropriate optimization method. The only necessary characteristics of random variables are their continuousness and ability to be approximated as independent and identically distributed. In this work, we try to concisely present two numerical probabilistic algorithms and apply them to estimate the univariate and bivariate models of the available data. In the first case, a combination of the maximum entropy method, Newton's method, and the Bayesian maximum a posteriori approach leads to the estimation of the kinetic parameters with arterial input functions (AIFs) in cases without any measurement of the AIF. ‎The results shows that the AIF can reliably be determined from the data of dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) by maximum entropy method. Then, kinetic parameters can be obtained. By using the developed method, a good data fitting and thus a more accurate prediction of the kinetic parameters are achieved, which, in turn, leads to a more reliable application of DCE-MRI. ‎ In the bivariate case, we consider colocalization as a quantitative analysis in fluorescence microscopy imaging. The method proposed in this case is obtained by combining the Maximum Entropy Method (MEM) and a Gaussian Copula, which we call the Maximum Entropy Copula (MEC). This novel method is capable of measuring the spatial and nonlinear correlation of signals to obtain the colocalization of markers in fluorescence microscopy images. Based on the results, MEC is able to specify co- and anti-colocalization even in high-background situations.‎ ‎The main point here is that determining the joint distribution via its marginals is an important inverse problem which has one possible unique solution in case of choosing an proper copula according to Sklar's theorem. This developed combination of Gaussian copula and the univariate maximum entropy marginal distribution enables the determination of a unique bivariate distribution. Therefore, a colocalization parameter can be obtained via Kendall’s t, which is commonly employed in the copula literature. In general, the importance of applying these algorithms to biological data is attributed to the higher accuracy, faster computing rate, and lower cost of solutions in comparison to those of others. The extensive application and success of these algorithms in various contexts depend on their conceptual plainness and mathematical validity. ‎ Afterward, a probability density is estimated via enhancing trial cumulative distribution functions iteratively, in which more appropriate estimations are quantified using a scoring function that recognizes irregular fluctuations. This criterion resists under and over fitting data as an alternative to employing the Bayesian criterion. Uncertainty induced by statistical fluctuations in random samples is reflected by multiple estimates for the probability density. In addition, as a useful diagnostic for visualizing the quality of the estimated probability densities, scaled quantile residual plots are introduced. Kullback--Leibler divergence is an appropriate measure to indicate the convergence of estimations for the probability density function (PDF) to the actual PDF as sample. The findings indicate the general applicability of this method to high-yield statistical inference.Die Methode der maximalen Entropie ist ein wichtiger Bestandteil der statistischen Inferenz, die in immer stärkerem Maße für die Konstruktion von Modellen verwendet wird, die biologische Systeme, insbesondere komplexe Systeme, aus empirischen Datensätzen beschreiben und vorhersagen können. In diesen ertragreichen Anwendungen ist es von besonderem Interesse, exakte Verteilungsfunktionen mit minimaler Information über die Eigenschaften der Daten und ohne Ausnutzung menschlicher Subjektivität zu bestimmen. In dieser Arbeit wird durch eine Kombination der Maximum-Entropie-Methode mit geeigneten Optimierungsverfahren ein automatisiertes Verfahren verwendet, um dieses Ziel für univariate und bivariate Daten zu erreichen. Notwendige Eigenschaften von Zufallsvariablen sind lediglich ihre Stetigkeit und ihre Approximierbarkeit als unabhängige und identisch verteilte Variablen. In dieser Arbeit versuchen wir, zwei numerische probabilistische Algorithmen präzise zu präsentieren und sie zur Schätzung der univariaten und bivariaten Modelle der zur Verfügung stehenden Daten anzuwenden. Zunächst wird mit einer Kombination aus der Maximum-Entropie Methode, der Newton-Methode und dem Bayes'schen Maximum-A-Posteriori-Ansatz die Schätzung der kinetischen Parameter mit arteriellen Eingangsfunktionen (AIFs) in Fällen ohne Messung der AIF ermöglicht. Die Ergebnisse zeigen, dass die AIF aus den Daten der dynamischen kontrastverstärkten Magnetresonanztomographie (DCE-MRT) mit der Maximum-Entropie-Methode zuverlässig bestimmt werden kann. Anschließend können die kinetischen Parameter gewonnen werden. Durch die Anwendung der entwickelten Methode wird eine gute Datenanpassung und damit eine genauere Vorhersage der kinetischen Parameter erreicht, was wiederum zu einer zuverlässigeren Anwendung der DCE-MRT führt. Im bivariaten Fall betrachten wir die Kolokalisierung zur quantitativen Analyse in der Fluoreszenzmikroskopie-Bildgebung. Die in diesem Fall vorgeschlagene Methode ergibt sich aus der Kombination der Maximum-Entropie-Methode (MEM) und einer Gaußschen Copula, die wir Maximum-Entropie-Copula (MEC) nennen. Mit dieser neuartigen Methode kann die räumliche und nichtlineare Korrelation von Signalen gemessen werden, um die Kolokalisierung von Markern in Bildern der Fluoreszenzmikroskopie zu erhalten. Das Ergebnis zeigt, dass MEC in der Lage ist, die Ko- und Antikolokalisation auch in Situationen mit hohem Grundrauschen zu bestimmen. Der wesentliche Punkt hierbei ist, dass die Bestimmung der gemeinsamen Verteilung über ihre Marginale ein entscheidendes inverses Problem ist, das eine mögliche eindeutige Lösung im Falle der Wahl einer geeigneten Copula gemäß dem Satz von Sklar hat. Diese neu entwickelte Kombination aus Gaußscher Kopula und der univariaten Maximum Entropie Randverteilung ermöglicht die Bestimmung einer eindeutigen bivariaten Verteilung. Daher kann ein Kolokalisationsparameter über Kendall's t ermittelt werden, der üblicherweise in der Copula-Literatur verwendet wird. Die Bedeutung der Anwendung dieser Algorithmen auf biologische Daten lässt sich im Allgemeinen mit hoher Genauigkeit, schnellerer Rechengesch windigkeit und geringeren Kosten im Vergleich zu anderen Lösungen begründen. Die umfassende Anwendung und der Erfolg dieser Algorithmen in verschiedenen Kontexten hängen von ihrer konzeptionellen Eindeutigkeit und mathematischen Gültigkeit ab. Anschließend wird eine Wahrscheinlichkeitsdichte durch iterative Erweiterung von kumulativen Verteilungsfunktionen geschätzt, wobei die geeignetsten Schätzungen mit einer Scoring-Funktion quantifiziert werden, um unregelmäßige Schwankungen zu erkennen. Dieses Kriterium verhindert eine Unter- oder Überanpassung der Daten als Alternative zur Verwendung des Bayes-Kriteriums. Die durch statistische Schwankungen in Stichproben induzierte Unsicherheit wird durch mehrfache Schätzungen für die Wahrscheinlichkeitsdichte berücksichtigt. Zusätzlich werden als nützliche Diagnostik zur Visualisierung der Qualität der geschätzten Wahrscheinlichkeitsdichten skalierte Quantil-Residuen-Diagramme eingeführt. Die Kullback-Leibler-Divergenz ist ein geeignetes Maß, um die Konvergenz der Schätzungen für die Wahrscheinlichkeitsdichtefunktion (PDF) zu der tatsächlichen PDF als Stichprobe anzuzeigen. Die Ergebnisse zeigen die generelle Anwendbarkeit dieser Methode für statistische Inferenz mit hohem Ertrag.

Radiolabeled PET/MRI Nanoparticles for Tumor Imaging

The development of integrated positron emission tomography (PET)/ magnetic resonance imaging (MRI) scanners opened a new scenario for cancer diagnosis, treatment, and follow-up. Multimodal imaging combines functional and morphological information from different modalities, which, singularly, cannot provide a comprehensive pathophysiological overview. Molecular imaging exploits multimodal imaging in order to obtain information at a biological and cellular level; in this way, it is possible to track biological pathways and discover many typical tumoral features. In this context, nanoparticle-based contrast agents (CAs) can improve probe biocompatibility and biodistribution, prolonging blood half-life to achieve specific target accumulation and non-toxicity. In addition, CAs can be simultaneously delivered with drugs or, in general, therapeutic agents gathering a dual diagnostic and therapeutic effect in order to perform cancer diagnosis and treatment simultaneous. The way for personalized medicine is not so far. Herein, we report principles, characteristics, applications, and concerns of nanoparticle (NP)-based PET/MRI CAs

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

Regularized estimation and model selection in compartment models

Dynamic imaging series acquired in medical and biological research are often analyzed with the help of compartment models. Compartment models provide a parametric, nonlinear function of interpretable, kinetic parameters describing how some concentration of interest evolves over time. Aiming to estimate the kinetic parameters, this leads to a nonlinear regression problem. In many applications, the number of compartments needed in the model is not known from biological considerations but should be inferred from the data along with the kinetic parameters. As data from medical and biological experiments are often available in the form of images, the spatial data structure of the images has to be taken into account. This thesis addresses the problem of parameter estimation and model selection in compartment models. Besides a penalized maximum likelihood based approach, several Bayesian approaches-including a hierarchical model with Gaussian Markov random field priors and a model state approach with flexible model dimension-are proposed and evaluated to accomplish this task. Existing methods are extended for parameter estimation and model selection in more complex compartment models. However, in nonlinear regression and, in particular, for more complex compartment models, redundancy issues may arise. This thesis analyzes difficulties arising due to redundancy issues and proposes several approaches to alleviate those redundancy issues by regularizing the parameter space. The potential of the proposed estimation and model selection approaches is evaluated in simulation studies as well as for two in vivo imaging applications: a dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) study on breast cancer and a study on the binding behavior of molecules in living cell nuclei observed in a fluorescence recovery after photobleaching (FRAP) experiment