Fluorescence lifetime imaging (FLIM) of metabolic coenzymes, as NAD(P)H and FAD and oxygen induced phosphorescence lifetime imaging (PLIM) of phosphorescent drugs or delayed FLIM (dFLIM) is now widely accepted to be one of the most important imaging methods for cell metabolism and oxygen. Different algorithms are developed and optimized for simultaneous FLIM/PLIM/dFLIM to get reproducible results. The interaction of light, drug and oxygen during PDT (photodynamic therapy) induces a cellular response, which could be followed by various techniques. The aim of this work was to simultaneously image the cellular response and oxygen consumption in living cells with subcellular resolution.
Metabolic Fluorescence Lifetime Imaging Microscopy (FLIM) algorithms have emerged as powerful tools for unraveling the intricate dynamics of cellular and tissue metabolism. FLIM algorithms offer unique insights into cellular metabolic processes that transcend traditional imaging methods. By combining Metabolic FLIM with Phosphorescence Lifetime Imaging Microscopy (PLIM), it becomes possible to assess cellular metabolic states, such as oxygen consumption, redox states, pH levels, and energy production pathways.
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.
FLIM of metabolic coenzymes, as NAD(P)H and FAD, is now widely accepted to be one of the most important imaging methods for cell metabolism. However, new algorithms are needed to circumvent various problems and to image cell metabolism and redox state from fluorescence lifetimes. The significance of a metabolic index based on NAD(P)H FLIM will be explained and compared with the fluorescence lifetime induced redox ratio (FLIRR). The importance of FMN will be discussed and the FLIRR approach will be extended. Using a three channel TCSPC system simultaneous metabolic NADH/FAD/FMN FLIM and oxygen PLIM/dFLIM (delayed fluorescence) could be realized.
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