A multi-parameter and simultaneous study of energy metabolism, oxygenation and intracellular pH using metabolic FLIM and PLIM of novel iridium phosphorescent complexes (SIMOPI)
Final Report Abstract
The development of metabolic disorders causes significant changes in the energy metabolism and mitochondrial respiration of cells. This can have an adverse influence on other factors such as cellular pH level and/or oxygen tension. Glycolytic metabolic pathways, reverse pH gradient and hypoxic status are often combined. Therefore, a parallel study was done showing the influence of different oxygenation of cells on the alteration of energy metabolism, followed by possible induction of abnormal cellular acidic or alkaline stress that finally inhibits cellular respiration. Our project was an agreed collaboration between the University of Ulm and the St. Petersburg State University on studies demonstrating the relationship between mitochondrial energy metabolism, intracellular and mitochondrial pH and oxygen levels in normal and cancer cells using time resolved fluorescence and phosphorescence lifetime imaging methods. The influence of aerated and hypoxic micro-environmental conditions was studied using phosphorescent sensors which were either oxygen or hydrogen ion sensitive. As sensors novel synthesized complexes based on Ir(III), where iridium is in oxidation state III, were used. The complexes were synthesized by the group in St. Petersburg and transferred to Ulm. Additionally, synthesized derivatives of Indirubin by Daniela dos Santos were found to possess a delayed fluorescence lifetime which was oxygen dependent. Simultaneously, cell metabolism was monitored by the fluorescence lifetime of metabolic coenzymes as NADH and FAD. The research group in Ulm, which had extensive knowledge and experience with FLIM/PLIM techniques to study energy metabolism and associated changes in pH as well as cellular oxygenation developed new algorithms to investigate metabolic patterns. FLIM of FAD and FMN was introduced as a new measure and the phasor algorithm for cell segmentation and metabolic patterns. The research group in St. Petersburg with experience in synthesis and applications of novel phosphorescent transition metal complexes, provided molecules based on Ir(III). Metabolic patterns were studied in normal and tumor cells under different oxygen levels. In addition, possible alterations in pH were simultaneously investigated using FLIM of NADH and FAD and PLIM of pH dependent iridium sensors. Twophoton excitation was performed using femtosecond pulsed lasers and fluorescence/phosphorescence imaging detection with TCSPC techniques. Simultaneous FLIM/PLIM detection was possible due to the unique technical improvement of our system allowing time modulation of the pulsed laser beam. The overall result of our project was a knowledge on the correlation between energy metabolism, oxygen tension, and intracellular pH levels. Moreover, the phasor pattern segmentation allowed for a correlation between important intracellular metabolic energy states and pH/oxygen distribution, revealing distinct microenvironmental profiles between tumoral and non-tumoral cells.
Publications
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Functional Hybrid Materials for Phototheranostic Applications: From Organic to Inorganic Nanostructure. PhD Thesis: Presented to the University of Ulm
Nilanjon Naskar
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Bromine indirubin FLIM/PLIM sensors to measure oxygen in normoxic and hypoxic PDT conditions. Photodiagnosis and Photodynamic Therapy, 45, 103964.
dos Santos, D.N.S.; Naskar, N.; Delgado-Pinar, E.; Reess, K.; Seixas de Melo, J.S. & Rueck, A.
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Metabolic FLIM and oxygen PLIM/dFLIM in translational research. Multiphoton Microscopy in the Biomedical Sciences XXIV, 39. SPIE.
Rueck, Angelika C.; Wieland, Johannes; Reess, Kirsten; dos Santos, Daniela & Naskar, Nilanjon
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Metabolic imaging with FLIM/PLIM indirubin derivatives: an ultrafast window into cellular metabolism and oxygen consumption. Multiphoton Microscopy in the Biomedical Sciences XXIV, 40. SPIE.
N. S. dos Santos, Daniela; Naskar, Nilanjon; Reess, Kirsten; Delgado-Pinar, Estefanía; Seixas de Melo, João Sérgio & Rueck, Angelika
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PhD Thesis: Presented to the University of Coimbra on the 20th of November 2024 Title: „Unveiling the excited state deactivation mechanisms of indirubin and its role in metabolic and oxygen imaging in living cells”
Daniela Nobre Sarmento dos Santos
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Advanced metabolic FLIM/PLIM to investigate metabolic pattern developments in various disorders and treatments. Multiphoton Microscopy in the Biomedical Sciences XXV, 13. SPIE.
Rueck, Angelika C.; Wieland, Johannes; Reess, Kirsten; von Einem, Bjorn & dos Santos, Daniela
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Beyond the microscope: targeting metabolic regions through FLIM algorithms and phasor pattern segmentation. Multiphoton Microscopy in the Biomedical Sciences XXV, 38. SPIE.
N. S. dos Santos, Daniela; Werner, Katharina; Reess, Kirsten; Wieland, Johannes & Rueck, Angelika C.
