Fluorimetric oxygen sensor with an efficient optical read-out for in vitro cell models

Abstract

This paper presents a phase fluorimetric sensor for the monitoring of the oxygen concentration in in vitro cell models. The sensing surface of the sensor consists of oxygen sensitive fluorescent dyes (platinum(II) octaethylporphyrinketone) embedded in a thin polystyrene film. In order to optimize the optical read-out scheme of the sensor, we carried out electromagnetic simulations of a fluorescently doped polystyrene film deposited on a glass-water interface. The simulation results showed highly anisotropic angular emission distribution with the maximum irradiance being at super critical angles, which attracts tailored optical designs to maximize the fluorescence collection efficiency. For this purpose, we applied an efficient optical read-out scheme based on an in-contact parabolic lens. The use of parabolic lens also facilitates confocal total internal reflection excitation from the substrate side. This makes the excitation effective and insensitive to biofouling or other optical changes in the sensing surface and, more importantly, greatly reduces the amount of excitation power radiated into the cell culture chamber. Experimental results show that when applied together with phase fluorimetric lifetime sensing, this optical scheme allows one to use thin films (<500nm), dilute dye-polymer ratios (0.025%), low power LED excitation (<2mW), and yet achieve over 40dB signal-to-noise ratio at 10Hz data rate at physiologically relevant oxygen concentrations. These features are important in cell studies, as the potential cytotoxicity of the dyes and the sensing method (i.e. the production of singlet oxygen) are mitigated by the low dye content and excitation power. In addition, thin and dilute polystyrene films are highly transparent and facilitate optical microscopy. We conclude the study by presenting experimental results where the device is applied together with on-line microscopy to an oxygen tension stress study of beating cardiomyocyte cultures.