More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research.

Abstract. 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. © The Authors.Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

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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In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.