Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 22, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/ijms22115952
Keywords
FLIM; NAD(P)H; FAD; FMN; NAD(P)H metabolic index; FLIRR index; extended FLIRR; cell metabolism; OXPHOS; glycolysis
Funding
- Ministry of Research and Development
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Metabolic FLIM is used to image bioenergetic status in cells and tissue based on fluorescence lifetime of coenzymes. A new algorithm utilizing NAD(P)H and FAD lifetimes to calculate FLIRR has been developed. The study extended FLIRR approach and introduced FLIRR1, FLIRR2, and FLIRR3 to analyze the redox state of cells with different enzymes. The significance of extended FLIRR was compared to the metabolic index, showing the highest difference for FLIRR1 in tumor and normal cells.
Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.
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