In combination with laser scanning microscopes, optical imaging technologies based on time correlated single photon counting (TCSPC) are successfully used in fluorescence lifetime imaging microscopy (FLIM) providing monitoring of intracellular intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)). Due to oxygen-dependent quenching of the phosphorescence of some compounds including transition metal complexes, the phosphorescence lifetime imaging microscopy (PLIM) can be used for evaluation of oxygen partial pressure (pO2). Using a multi-channel FLIM/PLIM system, we were able to monitor pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells and tissues.
Otical imaging techniques based on time correlated single photon counting (TCSPC) has found wide applications in medicine and biology. Non-invasive and information-rich fluorescence lifetime imaging microscopy (FLIM) is successfully used for monitoring fluorescent intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD+ (flavin adenine dinucleotide) in living cells and tissues. The ratio between proteinbound and free coenzymes gives an information about the balance between oxidative phosphorylation and glycolysis in the cells. The changes of the ratio reflects major cellular disorders. A correlation exists between metabolic activity, redox ratio and fluorescence lifetime during stem cell differentiation, neurodegenerative diseases, and carcinogenesis.<p> </p> 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 (pO<sub>2</sub>). Using a two-channel FLIM/PLIM system we monitored intrinsic pO<sub>2</sub> by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells.<p> </p> 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.
Alterations of cellular bioenergetics are a common feature in most neurodegenerative disorders. However, there is a selective vulnerability of different brain regions, cell types, and even mitochondrial populations to these metabolic disturbances. Thus, the aim of our study was to establish and validate an in vivo metabolic imaging technique to screen for mitochondrial function on the subcellular level. Based on nicotinamide adenine dinucleotide (phosphate) fluorescence lifetime imaging microscopy [NAD(P)H FLIM], we performed a quantitative correlation to high-resolution respirometry. Thereby, we revealed mitochondrial matrix pH as a decisive factor in imaging NAD(P)H redox state. By combining both parameters, we illustrate a quantitative, high-resolution assessment of mitochondrial function in metabolically modified cells as well as in an amyloid precursor protein-overexpressing model of Alzheimer’s disease. Our metabolic imaging technique provides the basis for dissecting mitochondrial deficits not only in a range of neurodegenerative diseases, shedding light onto bioenergetic failures of cells remaining in their metabolic microenvironment.