Novel Molecular Probes for Non-invasive Optical Imaging of Fatty Acid and Triglyceride Uptake in Living Animals

Molecular imaging allows noninvasive visualization of biological processes in their native environment within living systems. It can give a better understanding of fundamental biology, help in identifying disease mechanisms and visualizing pathological tissues, and enable monitoring disease progression and studying the drug efficacy in intact living organisms. Among various imaging modalities, bioluminescence and fluorescence imaging are powerful techniques that have become essential methods for non-invasive real-time studies of biological processes in vivo. The aim of my thesis was the development of novel fluorescent and bioluminescent probes for in vivo imaging of fatty acid and triglyceride uptake. The goal of my first project was the development of a near infrared fluorescent fatty acid probe for glioma imaging. Some tumors such as glioma may rely on the uptake of extracellular fatty acids. Fatty acids can therefore be considered as targeting molecules for these tumors and could be used to develop tumor-imaging probes. The probe design is based on a near-infrared fluorophore indocyanine green (ICG) that is conjugated to a long-chain fatty acid palmitic acid. The conjugation of ICG to a fatty acid (ICG-FA) was envisioned to improve cell permeability of the fluorophore and its accumulation in glioma. The ICG-FA probe was first evaluated for its ability to mimic the uptake of natural fatty acids in cells and was then applied for glioma imaging studies in vivo, where it showed significant tumor accumulation. As a proof-of-concept, the probe was tested for intraoperative image-guided surgery in a canine patent with mastocytoma, where it allowed intraoperative fluorescence tumor imaging and provided optical guidance for a surgeon during tumor resection. The aim of my second project was the development of bioluminescent triglyceride probes for real-time non-invasive imaging of triglyceride uptake in vivo. Applying an optimized stategy for generating conjugates with luciferin, I developed several bioluminescent triglyceride probes. These imaging tools were validated in cells and mice and were shown to mimic the absorption of natural triglycerides. The developed probes enabled sensitive non-invasive real-time imaging and quantification of triglyceride absorption in living mice. Futhermore, these probes were successfully used to demonstrate the effects of the anti-obesity drug orlistat and the influence of the gut microbiota on the intestinal absorption of triglycerides in vivo. Considering the high importance of triglycerides for human health and nutrition and their implication in several pathologies such as obesity, metabolic syndrome, type 2 diabetes and cancer, the bioluminescent triglyceride probes could offer valuable imaging tools for preclinical research.

A comprehensive reconstruction of the fatty acid biosynthesis in Saccharomyces cerevisiae

Vassily Hatzimanikatis, Iro Oikonomidi, Georgios Savoglidis

The role of lipids in eukaryotic cells is of high importance. They are one of the main components of cell membranes, act as storage of high potential energy as well as being a scaffold of signaling proteins or participate in signaling themselves. Many diseases are associated with alterations in the lipid distribution in the cell and the composition of membrane domains. Metabolic syndrome, obesity, atherosclerosis, as well as Alzheimer’s, Huttington diseases and cancer, all originate from alterations in some stage of lipid biosynthesis. Therefore, advancement of knowledge in the field of lipid metabolism will provide novel insights for further biomedical research and potential strategies for drug development. Fatty acids are the backbone of the lipid metabolism. Being lipids themselves they also participate in the biosynthesis of the majority of the lipid classes. Thus, the study of their synthesis and biotransformation in a cell constitutes the basic step towards a systematic investigation of the phenomena attributed to lipids. In the current study we reconstructed a comprehensive representation of the biosynthesis of fatty acids in a model eukaryote such as Saccharomyces cerevisiae. The model accounts for approximately 300 reactions and 230 metabolites, taking part in 3 compartments of the cell, describing the steps of synthesis, elongation and β-oxidation of fatty acids. We make use of other systems approaches in order to identify the input fluxes to the model and we explore the diverse capabilities of the network with respect to flux profiles and distribution of fatty acids.

2013

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

An endoscopic fluorescence imaging system for simultaneous visual examination and photodetection of cancers

André Studzinski, Hubert van den Bergh, Georges Wagnières

We describe the design and performance tested during six years of clinical trials of a fluorescence endoscope for the detection and delineation of cancers in several hollow organs. The apparatus is based on the imaging of the laser-induced fluorescence that differs between a tumor and its surrounding normal tissue. The tests are carried out in the upper aerodigestive tract, the tracheobronchial tree, the esophagus, and the colon. In the three former cases an exogenous dye is used (Photofrin II), whereas in the latter case fluorescein molecules conjugated with monoclonal antibodies directed against carcinoembryonic antigen are injected. The decrease of native tissue autofluorescence observed in early cancers is also used for detecting lesions in the tracheobronchial tree. The fluorescence contrast between the tumor and surrounding normal tissue is enhanced by real time image processing. This is done by simultaneously recording the fluorescenceimage in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Moreover, the device that is described below allows for an immediate observation of the endoscopic area under white light illumination during fluorescence detection in order to localize the origin of the “positive” fluorescence signals. Typical results obtained in the tracheobronchial tree and in the colon are presented and the sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.

1997

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

EPFL2006