Image- and Fluorescence-Based Test Shows Oxidant-Dependent Damages in Red Blood Cells and Enables Screening of Potential Protective Molecules

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape “Tirocytes”. The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.

Optimalisation de la photothérapie dynamique et de la photodétection de cancers précoces par spectroscopie résolue en temps de luminophores endogènes et exogènes

Pascal Uehlinger

The aim of this thesis is to optimize and obtain fundamental information on i) the photodetection (PD) of early stage cancers in the tracheo-bronchial tree by spectroscopic imaging of the tissue autofluorescence, ii) photodynamic therapy (PDT) applied in dermatology based on the use of protoporphyrin IX (PPIX) precursors. These two areas of photomedecine have been the subject of investigation at the air and soil pollution laboratory (LPAS) for the last 10 years. PD and PDT are areas of research that evolve in parallel, both having similar objectives: i.e. improving the patients' chances of survival by the early detection and treatment of cancer. Three main studies are presented, each in relation to a particular type of chromophore : Endogenous fluorophores generate the autofluorescence of biological tissue; some of these fluorophores can be synthesized endogenously in excess of their normal concentration following the administration of a biochemical precursor; finally, exogenous luminophores are artificially synthesized and administered to the organism where they can be used for detection. The introduction of this thesis presents several aspects of cancer as well as the theoretical background necessary for this study. At first we address time-resolved autofluorescence spectroscopy of bronchial tissue and the mechanisms at the origin of the contrasts existing between normal tissue and early stage tumours. Several hypotheses explain the reduction in intensity and spectral changes of the autofluorescence observed between early epidermal carcinoma and normal bronchial mucosa : 1) the reduction in the concentration of fluorophores in the malignant tissue, 2) the quenching of the fluorescence due to a change in the physico-chemical environment of the tumour, 3) an architectural effect such as a thickening of the malignant epithelium due to an abnormal cellular proliferation and 4) an increase in the concentration of light absorbers such as haemoglobin. In the course of our measurements no statistically significant difference in the fluorescence lifetime was observed between carcinoma in situ, moderate dysplasia and normal tissue, which leads us to conclude that hypotheses 1, 3 and/or 4 may well be at the origin of the differences in fluorescence intensity. The fluorescence lifetime decays are consistent, identifiable by a triple-exponential decay in the green and red part of the spectrum. This seems to indicate that one or two dominant fluorophores are involved which possibly, given the measured lifetimes, could be collagen and/or elastine. The second part of this thesis concerns the modulation of the production of PPIX (an endogenously induced fluorophore) in the skin, following the administration of 5-aminolevulinic acid or one of its esterified derivatives. These investigations have been carried out on the epidermis of human volunteers as well as on the epidermal equivalent (Epidex™). The final goal of these studies was on the one hand to increase the production of PPIX well below the skin surface to improve certain dermatological treatments such as the PDT of nodular basal cell carcinomas (BCC), and, on the other hand, the ability to use the results obtained with the Epidex™ model to produce PPIX specifically in hair follicles, which permits the development of a new method of hair removal by PDT. The tests using the Epidex™ model demonstrated that it is possible, by adding Desferal® (an iron chelator) or (L+) ascorbic acid iron (II) salt, to increase respectively decrease the production of PPIX as well as to modulate its elimination. These results open new perspectives such as the specific use of zinc-protoporphyrin (produced by skin cells following the dermatological use of Desferal®), and the identification of a specific precursor of PPIX for hair follicles. This precursor is an excellent "candidate" in the field of research for the development of a new method of hair removal by PDT. In the third part of this thesis we describe a preliminary study of optical sensors for the intravascular partial pressure of oxygen via the measurement of phosphorescence lifetime quenching. The phosphorescence lifetime of these species is directly related to the partial pressure of the oxygen dissolved (pO2). These oxygen sensors allow us, for instance, to measure the reduction of the pO2 during PDT, without phototoxicity. Palladium and ruthenium complexes were among the most promising compounds, and certain types of nanoparticules were used as vectors. Another interesting lead consists in the covalent modification of these "luminophores-molecules": in this context, the experiments conducted in collaboration with the "Laboratoire de photonique et interfaces" of the EPFL, demonstrate that we can drastically reduce the speed of leakage outside the vascularisation of these molecules and/or improve their retention by the nanoparticules.

EPFL2004

Design and validation of bioorthogonal probes for cellular disease models

Well-validated chemical probes enable testing of biological hypotheses, investigation of target tractability and translatability to the clinical phase. Consequently, these important tool compounds play a key role in the drug discovery process. Moreover, their use for target validation may ultimately help to decrease the attrition rate encountered by new molecular entities in clinical trials. Despite the number of publications describing well-validated chemical probes, for several reasons, it remains a difficult challenge to identify and select the appropriate high quality molecule enabling the desired biological and pharmacological studies in the relevant disease model. Various reports describe sets of principles to be fulfilled by high quality chemical probes, which mainly rely on verifying that chemical probes are potent, engage their intended targets in the relevant cellular model, have sufficient exposure at the desired site of action and express functional pharmacological activities by a selective modulation of their targets. Classical chemical probes include LMW ligands, which inform on the functional consequences of interacting with a particular bio-logical target in a model system. To get information on other parameters (such as sub-cellular distribution of target or compound) different tools may need to be used, often requiring specific chemical functionalization in order to observe the molecule using the currently available technologies. Compared to classical chemical probes, bioorthogonal probes can also be small molecules that elicit a functional response but with the additional advantage of being able to undergo reaction with a variety of chemical reporters (e.g. fluorophores) in situ, in bio-logical model systems such as cells and animals. Therefore, this increases their versatility; for example their visualization using imaging technologies (bioluminescence and fluorescence imaging) can provide important insights on compound permeability, in-tracellular distribution, and potentially co-localization with its protein target in relevant cellular disease models. Hence, the use of a bespoke bioorthogonal probe may answer key biological questions, which are usually only addressed by using multiple classical probes. Herein, we describe the design and the synthesis of bioorthogonal probes to study the inhibition of the p53-Mdm2 protein-protein interaction. Furthermore, we report the development of a set of assays based on fluorescence and bioluminescence techniques enabling the validation of these molecules in a cellular osteosarcoma model. Our approach for chemical probe design and validation in cellular disease models may be applied in principle to a wide range of novel drug discovery projects to provide early mechanistic understanding.

EPFL2019

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