The project is dedicated to the investigation of the pH sensor based on the iridium (III) complex. The sensor is studied in vivo in the PLIM (Phosphorescence Lifetime Imaging) channel with simultaneous measurement of the lifetimes in the FLIM (Fluorescence Lifetime Imaging) channel of the nicotinamide adenine dinucleotide (NADH). The sensor is initially tested in solutions. Solutions with various concentrations are prepared and studied using a luminescent microscope. Optimal research conditions are determined. After studying the solutions, research is carried out on cell cultures Studies with nigericin make it possible to equalize the pH of the intra- and intercellular space. The investigation is conducted with it in buffer solutions simultaneously measuring the lifetimes of NADH in the FLIM channel.
Various diseases affect the metabolism of cells. Metabolic changes are often associated with changes in oxygen levels and pH values, which are important indicators of the metabolic state of cells. In this case, cancer cells are more acidic than healthy. This project visualizes simultaneously the fluorescence lifetime (FLIM) of metabolic coenzymes of nicotinamide adenine dinucleotide (NADH) along with the visualization of the phosphorescence lifetime (PLIM) of sensors based on iridium complex, which is sensitive to pH, is investigated.
The lifetimes of the complex were studied in solution with varying concentrations. The influence of the medium on the lifetime of the complex in solution was investigated. The lifetimes of the complex and nicotinamide adenine dinucleotide were measured in vivo at different pH values.
The lifetimes of the complex (c = 100 μM) were measured in a solution of PBS (Phosphate Buffered Saline), DMEM (Dulbecco's Modified Eagle Medium). Lifetimes have changed slightly. The influence of concentration effects in PBS solutions with concentrations of 100 and 500 µM was studied. The difference in values can be caused by the aggregation of the complex at high concentrations.
The lifetimes of the sensor (50 μM) and nicotinamide adenine dinucleotide were studied on the SCC-4 oral cancer cell line according to the research protocol with nigericin and valinomycin in various buffers (pH = 5.5, 6.5, 7.5). The results showed similar ambiguous trends in the lifetimes of the complex and NADH.
Simultaneous visualization of the fluorescence lifetime (FLIM) of metabolic coenzymes of nicotinamide adenine dinucleotide (NADH) along with the visualization of the phosphorescence lifetime (PLIM) of sensor based on iridium (III) complex was carried out.
Kirill Maksimovich Kuznetsov coordinated experiments between the Russian and German sides supervised the implementation of research protocols. Nilanjon Naskar carried out work on a luminescent microscope, prepared samples. Dr. Angelika Rück supervised the implementation of the research protocols, provided expertise. Kirsten Reeß performed the preparation of the SCC-4 cell line.
The study of intracellular processes requires knowledge of not only intracellular metabolic processes but also the basics of luminescence for the correct interpretation of changes in emission parameters. The contribution of the German side as specialists in biological processes as well as in technical and hardware equipment is no less important than the contribution of the Russian side, which provides sensors based on iridium (III) complexes, as well as advising on changes in photophysical properties based on previous experience.
The German side is intensively studying objects using PLIM/FLIM microscopy in order to understand metabolic pathways and associated intracellular pH changes. The Russian side is actively engaged in the synthesis and investigation of pH sensors in solution. It is the collaboration of specialists in the field of luminescence microscopy and the chemistry of luminescent compounds that make it possible to study the issues of intracellular processes using novel previously unpublished sensors.
| Short title | FLIM / PLIM study of iridium (III) complex and NADH |
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| Acronym | GRISC 2021_2 |
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| Status | Finished |
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| Effective start/end date | 20/11/21 → 20/12/21 |
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