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In our investigation, we employed a combination of 2-photon (2P) excited FLIM and PLIM techniques, along with timecorrelated single-photon counting (TCSPC) detection. Through this, we made a significant discovery of bromine indirubin derivatives that exhibit a PLIM/dFLIM signal in two living cell lines. Notably, indirubin is a natural dye, and though renowned for its anti-tumor properties, its mechanism of action remains to be fully investigated.
Under normoxic conditions, the PLIM signal of indirubin exhibited a value of 62 ns living cells, while under hypoxic conditions, it increased significantly to 107ns. This observation demonstrates the potential of these indirubins as highly reliable oxygen consumption sensors. Moreover, our investigation revealed that bromine indirubin had a profound impact on cellular metabolism, prompting a shift from oxidative phosphorylation to glycolysis.
Through our research, we aim to demonstrate that these techniques offer valuable insights into cellular metabolism, covering the way for deeper understanding and potential breakthroughs in various fields of biology and medicine.
The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.
A multichannel FLIM detection system was designed for monitoring the redox state of NAD(P)H and FAD+ and other intrinsic fluorophores as protoporphyrin IX. In addition, the unique upgrade is useful to perform FLIM and PLIM (phosphorescence lifetime imaging microscopy) simultaneously. PLIM is a promising method to investigate oxygen sensing in biomedical samples. In detail, the oxygen-dependent quenching of phosphorescence of some compounds as transition metal complexes enables measuring of oxygen partial pressure (pO2). Using a two-channel FLIM/PLIM system we monitored intrinsic pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells.
Physico-chemical properties of oxygen sensitive probes define certain parameters including their localisation. We present results of some ruthenium based complexes including those specifically bound to mitochondria.
Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect the underlying metabolic mechanisms are therefore of prominent interest. They offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways. Moreover oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NADH and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.
Here we present a setup, which is based on two-photon microscopy and multi-dimensional time-correlated single-photoncounting (TCSPC). The presented method allows consecutive NADH and FAD imaging with high specificity and minimal side effects. In addition the setup is useful for simultaneous monitoring of NADH FLIM and intracellular oxygen partial pressure (pO2). With the two-channel FLIM/PLIM system we could show that pO2 is investigated simultaneously with different metabolic parameters in living cells.
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