Optical fiber-based setup for in vivo measurement of the delayed fluorescence lifetime of oxygen sensors

A new optical-fiber-based spectrofluorometer for in vivo or in vitro detection of delayed fluorescence is presented and characterized. This compact setup is designed so that it can be readily adapted for future clinical use. Optical excitation is done with a nitrogen laser-pumped, tunable dye laser, emitting in the UV-vis part of the spectrum. Excitation and luminescence signals are carried to and from the biological tissues under investigation, located out of the setup enclosure, by a single optical fiber. These measurements, as well as measurements performed without a fiber on in vitro samples in a thermostable quartz cell, in a controlled-atmosphere enclosure, are possible due to the efficient collection of the laser-induced luminescence light which is collected and focused on the detector with a high aperture parabolic mirror. The detection is based on a gated photomultiplier which allows for time-resolved measurements of the delayed fluorescence intensity. Thus, relevant luminescence lifetimes, typically in the sub-microsecond-to-millisecond range, can be measured with near total rejection of the sample's prompt fluorescence. The instrument spectral and temporal resolution, as well as its sensitivity, is characterized and measurement examples are presented. The primary application foreseen for this setup is the monitoring and adjustment of the light dose delivered during photodynamic therapy. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3558846]

Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo

Emmanuel Louis Arthur Gerelli, Veronika Huntosova, Georges Wagnières, Matthieu Zellweger

The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm(2), quite possibly as low as a fraction 1J/cm(2) at 405nm. This leads to an increased accuracy of pO2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts.

Elsevier Science Sa2016

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

A general framework for non-exponential delayed fluorescence and phosphorescence decay analysis, illustrated on Protoporphyrin IX

Emmanuel Louis Arthur Gerelli, Aurelien Stephen Arnaud Gregor, Jaroslava Joniová, Georges Wagnières

Delayed fluorescence (DF) is a long-lived luminescence process used in a variety of applications ranging from oxygen sensing in biological tissues to organic Light Emitting Diodes. In common cases, DF results from the deexcitation of the first excited triplet state via the first excited singlet state of the chromophore, which produces a mono-exponential light signal whose amplitude and lifetime give an insight into the probed environment. However, non-linear de-excitation reactions such as triplet-triplet annihilation, which can cause decays to lose their mono-exponential nature, are often neglected. In this work, we derive a global framework to properly interpret decays resulting from a combination of linear and non-linear de-excitation processes. We show why the standard method of using multi-exponential models when decays are not mono-exponential is not always relevant, nor accurate. First, we explain why the triplet de-excitation and light production processes should be analyzed individually: we introduce novel concepts to precisely describe these two processes, namely the deactivation pathway - the reaction which mainly contributes to the triplet state de-excitation - and the measurement pathway - the reaction which is responsible for light production. We derive explicit fitting functions which allow the experimenter to estimate the reaction rates and excited state concentrations in the system. To validate our formalism, we analyze the in vitro Transient Triplet Absorption and DF of Protoporphyrin IX, a well-known biological aromatic molecule used in photodynamic therapy, cancer photodetection and oxygen sensing, which produces DF through various mechanisms depending on concentration and excitation intensity. We also identify the precise assumptions necessary to conclude that triplet-triplet annihilation DF should follow a monoexponential decay with a lifetime of half the triplet state lifetime. Finally, we describe why the commonly used definitions of triplet / DF lifetime are ill-defined in the case where second-order reactions contribute to the deactivation process, and why the fitting of precise mixed-orders DF kinetics should be preferred in this case. This work could allow the correct interpretation of various long-lived luminescence processes and facilitate their understanding.