Noniterative Biexponential Fluorescence Lifetime Imaging in the Investigation of Cellular Metabolism by Means of NAD(P)H Autofluorescence (original) (raw)
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Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
BIO-PROTOCOL
NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzymebinding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.
Scientific Reports
Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses. Applications of Fluorescence Lifetime Imaging Microscopy (FLIM) have grown exponentially in a broad range of life-sciences and industrial fields, a reflection of specific advantages over intensity-based microscopy 1-5. FLIM, when combined with FRET (Förster Resonance Energy Transfer), can establish the fraction of interacting and non-interacting donor fluorophores 6-13. Importantly, fluorescence lifetime is independent of fluorophore concentration, which makes it a valuable tool for quantitative studies in scattering and absorbing samples. Both frequency domain and time domain FLIM methods have been applied 14-16. This manuscript uses the latter, also called Time-Correlated Single Photon Counting (TCSPC) 17. Multiphoton excitation conveniently excites molecules that would otherwise require excitation in the UV region, generally injurious to live cells at longer exposure. Mitochondrial oxidative phosphorylation (OXPHOS) activity consumes NADH (increased NADH-enzyme-bound fraction) and produces FAD (diminished FAD enzyme-bound fraction). Both the co-enzymes in their reduced (NAD(P)H and FADH 2) and oxidized (NAD(P) + and FAD) forms participate in the cellular oxidation-reduction reactions critical for cell physiology. In cancer, a higher glycolytic rate is a less efficient way of producing energy (2Pyruvate + 2ATP + 2NADH) than the low glycolytic rate and mitochondrial oxidation of pyruvate (36 ATP) seen in normal cells 18. The interplay between glycolysis and OXPHOS is changed in different cancers and involvement of other pathways like elevated mitochondrial glutaminolysis is also seen in prostate cancer (PCa). The coenzymes NADH and FAD are involved in catabolic reactions of amino acid and fatty acid oxidation, glycolysis, citric acid cycle and in electron transport chain (ETC) which ultimately results in energy generation by oxidative phosphorylation (OXPHOS). NADPH is mainly involved in anabolic reactions, which use energy for biosynthesis. Previous reports have shown that Tryptophan (Trp) lifetime (as donor) is quenched through FRET
Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H
Scientific Reports, 2019
The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these tra...
Cancer Research, 2005
Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD + reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of proteinbound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD + . Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD + ratio.
2007
NAD(P)H, crucial in effective management of cellular oxidative metabolism and the principal electron donors for enzymatic reactions, is a major source of autofluorescence induced in cardiac cells following excitation by UV light. Spectrally-resolved timecorrelated single photon counting was used to simultaneously measure the fluorescence spectra and fluorescence lifetimes of NAD(P)H, following excitation by a pulsed picosecond 375 nm laser diode. Spectra, as well as fluorescence lifetimes of NADH and NADPH molecules were investigated in solution at different concentrations Effects of their respective dehydrogenation by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR) were also questioned. NAD(P)H autofluorescence recorded in vitro was compared to the one measured in freshly isolated cardiac cells. We observed a good comparability between NAD(P)H parameters recorded in solution and in cells.
Investigating mitochondrial redox state using NADH and NADPH autofluorescence
Free Radical Biology and Medicine, 2016
The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues.
Metabolic Imaging by Simultaneous FLIM of NAD(P)H and FAD
2020
We describe a metabolic imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. Two lasers, with wavelengths of 375nm and 410 nm, are multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other in the emission band of FAD. For both channels, the data analysis delivers images of the amplitudes of the decay components, a1 and a2. We show that these are robust parameters to characterize the metabolic state of cells. FLIM results obtained from excised human-bladder tissue were in perfect agreement with histology.
J Biomed Opt, 2014
Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells is a useful method for monitoring aspects of cellular metabolism, because the NADH∕NAD þ reduction-oxidation pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors: namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also measured in both treated and control cells. The combination of these two techniques gave valuable insights into cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically active cells.
Metabolic imaging by simultaneous 2-photon FLIM of NAD(P)H and FAD
Proc. SPIE, 2020
We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses two-photon excitation by a dual-wavelength femtosecond fibre laser. The two wavelengths of the laser, 780 nm and 880 nm, are multiplexed synchronously with the frames or the lines of the scan. The recording system uses two parallel TCSPC FLIM channels, detecting from 420 to 475 nm and 480 to 600 nm. By using the multiplexing functions of the TCSPC modules, separate images for NAD(P)H and FAD are recorded. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, the amplitude ratio, a1/a2, and the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system for metabolic imaging of mammalian skin.
Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping
Journal of Biophotonics, 2016
Cellular responses to oxygen tension have been studied extensively. Oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescence sensor. The simultaneous usage of FLIM of coenzymes as NAD(P)H and FAD+ and PLIM of oxygen sensors could provide information about correlation of metabolic pathways and oxygen tension. We investigated correlative NAD(P)H-FLIM and oxygen sensing-PLIM for simultaneously analyzing cell metabolism and oxygen tension. Cell metabolism and pO2 were observed under different hypoxic conditions in squamous carcinoma cell cultures and in complex ex vivo systems. Increased hypoxia induced an increase of the phosphorescence lifetime of Ru(BPY)3 and in most cases a decrease in the lifetime of NAD(P)H which is in agreement to the expected decrease of the protein-bound NAD(P)H during hypoxia. Oxygen was modulated directly in the mitochondrial membrane. Blocking of complex III and accumulation of oxygen could be observed by both the decrease of the phosphorescence lifetime of Ru(BPY)3 and a reduction of the lifetime of NAD(P)H which was a clear indication of acute changes in the redox state of the cells. For the first time simultaneous FLIM/PLIM has been shown to be able to visualize intracellular oxygen tension together with a change from oxidative to glycolytic phenotype.