Fluorescence Lifetime Imaging Microscopy (FLIM) of endogenous fluorophores has recently emerged as a powerful, marker-free, and non-invasive tool for investigating cellular metabolism. This cutting-edge imaging technique provides valuable insights into cellular energy states by measuring the fluorescence lifetimes of intrinsically fluorescent redox cofactors. The lifetimes of these cofactors reflect their binding states to enzymes, thus indicating enzymatic activity within specific metabolic pathways. As a result, FLIM can help to reveal the overall redox status of the cell and, to some extent, shifts between oxidative phosphorylation and glycolysis. The application of FLIM in metabolic research has shown significant progress across a diverse range of pathological contexts, including cancer, diabetes, neurodegenerative disorders, and various forms of cardiopathology.The aim of this mini-review is to introduce the methodology of NAD(P)H and FAD/FMN FLIM, outline its underlying principles, and demonstrate its ability to reveal changes in cellular metabolism. Additionally, this mini-review highlights FLIM's potential for understanding cellular redox states, detecting metabolic shifts in various disease models, and contributing to the development of therapeutic strategies.

Department of Physiology Faculty of Science Charles University Vinicna 7 Prague 2 128 00 Czech Republic

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