Jan Schlothauer

New insights to primary photodynamic effects--Singlet oxygen kinetics in living cells.

The kinetics of chemical singlet oxygen quencher consumption inside living cells during low dose illumination was revealed via time resolved singlet oxygen luminescence detection. Deviations of the measured data from the common theoretical model for (1)O(2) kinetics forced the authors to consider a one-dimensional diffusion model for description of the kinetics of singlet oxygen generated by membrane localized photosensitizers. Our observations reconcile seemingly contradictory reports presenting different values for the efficiency of singlet oxygen interaction with cells.

Time-resolved singlet oxygen luminescence detection under photodynamic therapy relevant conditions: comparison of ex vivo application of two photosensitizer formulations

Abstract. Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F64PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F64PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers’ oxidation as well as O2 saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.

Luminescence investigation of photosensitizer distribution in skin: correlation of singlet oxygen kinetics with the microarchitecture of the epidermis

Abstract. This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.

Prospects of in vivo singlet oxygen luminescence monitoring: Kinetics at different locations on living mice

BACKGROUND Singlet oxygen observation is considered a valuable tool to assess and optimize PDT treatment. In complex systems, such as tumors in vivo, only the direct, time-resolved singlet oxygen luminescence detection can give reliable information about generation and interaction of singlet oxygen. Up to now, evaluation of kinetics was not possible due to insufficient signal-to-noise ratio. Here we present high signal-to-noise ratio singlet oxygen luminescence kinetics obtained in mouse tumor model under PDT relevant conditions. METHODS A highly optimized system based on a custom made laser diode excitation source and a high aperture multi-furcated fiber, utilizing a photomultiplier tube with a multi photon counting device was used. RESULTS Luminescence kinetics with unsurpassed signal-to-noise ratio were gained from tumor bearing nude mice in vivo upon topic application, subcutaneous injection as well as intravenous injection of different photosensitizers (chlorin e6 and dendrimer formulations of chlorin e6). Singlet oxygen kinetics in appropriate model systems are discussed to facilitate the interpretation of complex kinetics obtained from in vivo tumor tissue. CONCLUSIONS This is the first study addressing the complexity of singlet oxygen luminescence kinetics in tumor tissue. At present, further investigations are needed to fully explain the processes involved. Nevertheless, the high signal-to-noise ratio proves the applicability of direct time-resolved singlet oxygen luminescence detection as a prospective tool for monitoring photodynamic therapy.

Highly sensitive time resolved singlet oxygen luminescence detection using LEDs as the excitation source

For the first time singlet oxygen luminescence kinetics in living cells were detected at high precision using LED light for excitation. As LED technology evolves, the light intensity emitted by standard LEDs allows photosensitized singlet oxygen luminescence detection in solution and cell suspensions. We present measurements superior to those of most actual laser powered setups regarding precision of singlet oxygen kinetics in solutions and cell suspensions. Data presented here show that LED based setups allow the determination of the photosensitizer triplet and singlet oxygen decay times in vitro with an accuracy of 0.1??s. This enables monitoring of the photosensitizer efficiency and interaction with the cellular components using illumination doses small enough not to cause cell death.

Singlet oxygen kinetics inside living cells: observation of endogenous quencher consumption and consequences for the spatial resolution in time-resolved measurements

The consumption of singlet oxygen quenchers during illumination of cells incubated with photosensitizers is a serious issue for the determination of singlet oxygen kinetics in cells. Using LNCaP cells incubated with pheophorbide a it will be shown, that already an illumination of 100 nJ/cell dramatically changes the value of the observables. This finding has consequences for the spatial resolution of any time-resolved singlet oxygen measurement, since the radiative rate constant of singlet oxygen in aqueous solutions is very small.

In vivo detection of time-resolved singlet oxygen luminescence under PDT relevant conditions

For the fist time worldwide we report high amplitude NIR-Luminescence signals, measured in pig skin. The measurements are achieved with a recently developed setup that was shown to provide superior performance for 1O2- luminescence detection in vitro. This setup has been adapted to allow now the detection of singlet oxygen signals in vivo. Pig ears were used for first measurements as a widely accepted in vivo model for human skin.

Fluorescence imaging for analysis of the degradation of PV-modules

Fluorescence detection is presented as a tool for the investigation of EVA degradation that is used as encapsulant in PV-modules. The superior sensitivity of our set-up allows an early assessment of ageing processes in EVA already after 20h damp-heat exposure. A newly developed scanning system enables the laterally resolved inspection of entire PV modules. We also report the evaluation of outdoor weathered crystalline Si-modules demonstrating that besides ageing of EVA cracks of cells are well detectable, too.

Prospects of 2D-Luminescence Spectroscopy for Aging Investigations of the Embedding EVA Polymer in PV Modules: Revealing DLO Conditions

Abstract For a reliable lifetime prediction of photovoltaic (PV) modules, the investigation of degradation processes of materials in the product is a fundamental prerequisite. Especially for nondestructive characterization of the encapsulating material, suitable characterization methods are needed. We show that luminescence is a sensitive, noninvasive method for the characterization of the aging of the embedding polymer in PV modules. Luminescence exhibits a good correlation of the aging duration and different aging parameters. A very important feature in aged PV modules is complex 2-dimensional luminescence patterns, which correlate to inhomogeneous aging detected by conventional, destructive methods. The results imply that photoluminescence can be used to follow material degradation and detect inhomogeneous material aging due to diffusion-limited oxidation. Prospective applications for aging investigations of complex products arise as this method improves upon drawing a connection between accelerated aging and outdoor weathering.

Non-destructive monitoring of ethylene vinyl acetate crosslinking in PV-modules by luminescence spectroscopy

This work reports on the prospects of using luminescence spectroscopy as a non-destructive method for the characterization of ethylene vinyl acetate copolymer (EVA) crosslinking in photovoltaic (PV) modules. Luminescence has the potential to be easily applied in-line for monitoring purposes, e.g. during manufacturing. We investigate the correlation of luminescence, Raman spectroscopy and differential scanning calorimetry with the EVA crosslinking. We show that all these methods, including the luminescence method, show a good correlation with the hold time during the lamination process. Furthermore, time-dependent luminescence measurements are introduced. These make use of the fact that the luminescence decreases upon the ultraviolet irradiation during the measurement. In contrast to steady-state luminescence, this facilitates measurements that are inherently less dependent on possible interfering signal artifacts, as these may occur in industrial PV modules due to other components of the PV module.