Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Abstract

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.

Development of a microstructured tissue phantom with adaptable optical properties for use with microscopes and fluorescence lifetime imaging systems

“…The fluorescent beads were chosen based on the similarity of their spectral excitation and emission characteristics with those of NADH and FAD, because the phantom was codeveloped with the modification of a multiphoton tomography system for metabolic FLIM investigations. 45,46 The prepared structures are not limited to this use case, since fluorescent spheres are available in a variety of diameters and spectral excitation and emission characteristics. These properties can be chosen to match the requirements for the system to be tested.…”

Fluorescence lifetime imaging and electron microscopy: a correlative approach

“…Antimycin A-treated cells were incubated for 15 min before image acquisition and immediate fixation. FLIM measurement of NADH autofluorescence was conducted as described by Kalinina et al ( 2021 ). In order to ensure that the region of interest imaged with FLIM could be located after preparation for electron microscopy, cells were selected at specific coordinates.…”

“…Such a protocol would help to improve the understanding of physiological processes in mitochondria as well as the mechanisms behind diseases involving cell metabolism. This is especially promising for diseases where FLIM is already established as a method to investigate pathomechanisms, like neurodegenerative diseases (Gomez et al 2018 ), cancer (Kalinina et al 2021 ), eye diseases, and cardiovascular disorders (Marcu 2012 ).…”

Graphene-based phenformin carriers for cancer cell treatment: a comparative study between oxidized and pegylated pristine graphene in human cells and zebrafish

“…15,16 The redox state and subsequent fluorescence of FAD and FMN are tied to the electron flow through ETC during normal ATP production, 17–19 making the fluorescence of these co-enzymes sensitive to ETC disruption. 20–22 Interestingly, inhibition of complex I of the ETC is associated with a metabolic shift towards alternative energy pathways, such as glycolysis, leading to an increased response to chemotherapy. 2,11–13 Therefore, biguanide has the potential to revert chemotherapy resistance, 11,13 enhancing treatment outcomes.…”