Monitoring pH and extracellular acidification in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins (OEP and OEPK) embedded in a proton-permeable polymeric matrix together with a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.
The recently designed Tpx3Cam camera based PLIM (Phosphorescence Lifetime IMaging) macro-imager was tested using an array of phosphorescent chemical and biological samples. A series of sensor materials prepared by incorporating the phosphorescent O2-sensitive dye, PtBP, into five polymers with different O2 permeability were imaged along with several commercial and non-commercial sensors based on PtBP and PtOEPK dyes. The PLIM images showed good lifetime contrast between the different materials, and phosphorescence lifetime values obtained were consistent with those measured by alternative methods. A panel of live tissues samples stained with PtBP based nanoparticle probe were also prepared and imaged under resting conditions and upon inhibition of respiration. The macro-imager showed promising results as a tool for PLIM of O2 in chemical and biological samples.
KEYWORDS: Oxygen, Sensors, Phosphorescence, Medical research, Tissues, Live cell imaging, 3D modeling, Tissue optics, Hypoxia, In vitro testing, Imaging systems, Control systems, Solid state electronics
A variety of in vitro and ex-vivo cell and tissue models are being used in biomedical research, but for many of them control of the cellular microenvironment, particularly oxygenation state and intracellular O2 levels, is inadequate. Since O2 is a key parameter and biomarker of cellular function, implementation of reliable in situ control and knowledge of actual O2 levels in different compartments of biological samples is of critical importance. The versatile and flexible technology of O2 sensing and imaging based on phosphorescence quenching provides such capabilities. In recent years, various O2 sensing systems, which operate with solid-state sensors, soluble probes or imaging (nano)sensors in conjunction with portable handheld instruments, commercial plate readers or live cell imaging platforms, have been developed, which are suitable for routine use in many research labs to perform a range of important analytical and biomedical tasks. Here we overview the available O2 sensing solutions, their analytical features, and describe how they can be integrated in the current paradigm of biomedical research. Representative examples of the use of such systems in complex physiological studies with advanced tissue and disease models are given, in which they provide strict environmental control of dissolved and gaseous O2 (macroscopically and microscopically, by point measurements and high-resolution imaging in 2D and 3D), and important information about cellular function and changes in tissue metabolism under different conditions and treatments.
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