Metabolic Imaging by NAD(P)H and FAD FLIM
NAD(P)H (nicotinamide adenine (pyridine) dinucleotide) and FAD (flavin adenine dinucleotide) are coencymes involved in the cell metabolism. Both NAD(P)H and FAD are fluorescent. FAD and, especially, NAD(P)H are unique in the sense that their fluorescence intensities and fluorescence decay functions bear direct information on the metabolic state of the cells: The fluorescence lifetimes of NAD(P)H and FAD depend on the binding to proteins. Unbound NAD(P)H has a fluorescence lifetime of about 0.3 to 0.4 ns. Bound NAD(P)H has a lifetime of about 1.2 ns . For FAD the effect of binding is opposite: Bound FAD has a lifetime of a few 100 ps, unbound FAD of a few ns.
Importantly, the ratios of the amounts of bound and unbound NAD(P)H and of bound and unbound FAD depend on the type of the metabolism. A shift from glycolysis to oxidative phosphorylation or back results in a change in the unbound/bound ratios: Glycolysis yields high a1/a2 for NAD(P)H and low a1/a2 for FAD, oxydative phosphorylation yields low a1/a2 for NAD(P)H and high a1/a2 for FAD. The bound/unbound ratios directly reflect the ‘Warburg Effect’: In normal cells oxidative phosphorylation dominates, in cancer cells reductive glycolysis. Normal cells and cancer cells can therefore be distinguished by their a1/a2 ratios, or by a1 alone (because a1+a2=1). It should be noted that changes in a1 or a1/a2 are implicitly present also in the amplitude-weighted lifetime, tm and the intensity-weighted lifetime, ti. However, the lifetimes of the decay components themselves are influenced by other molecular-environment parameters, such as mitochondrial pH. The lifetime can therefore not be used as an absolute discrimination parameter.
Excitation and Emission Wavelengths
Approximate excitation and emission spectra of NAD(P)H and FAD are shown in the figure below. The figure shows that the fluorescence signals from NAD(P)H and FAD can only be separated if different excitation wavelengths and different detection wavelengths are used.
Suitable one-photon excitation wavelengths are 350 to 375 nm for NADH and 405 to 420 nm for FAD. Two photon-excitation wavelengths are 750 nm and 920 nm, respectively. Detection wavelength intervals are about 425 to 475 nm for NAD(P)H and 475 to 600 nm for FAD.
Simultaneous Measurement of NAD(P)H and FAD
To minimise the influence of photobleaching, focus drift, an possible physiological changes it is desirable to record the FLIM data of NAD(P)H and FAD quasi simultaneously. This can be obtained by laser multiplexing and multiplexed TCSPC: The lasers are multiplexed synchronously with the scanner, either line by line or frame by frame. The signals in the two emission wavelength intervals are recorded by two parallel FLIM channels. All the required functions are implemented in the bh DCS-120 confocal scanning FLIM system. Metabolic FLIM with the DCS-120 is thus only a matter of using the right lasers and choosing the right setup parameters. With bh FLIM systems attached to other laser scanning microscopes metabolic FLIM can, in principles be performed as well if the right lasers are used and are prepared for multiplexing.
The images shown below were recorded with a DCS-120 system with BDL-SMN lasers of 375 nm and 405 nm wavelength. The lasers were multiplexed frame by frame, and the images recorded in the two channels of the DCS-120 system.
The data shown above demonstrate show that the distribution of the amplitude of the fast decay component, a1, in the NAD(P)H and in the FAD images are different for normal cells and tumor cells. For more information please see bh TCSPC Handbook and application note ‘Metabolic Imaging with the DCS-120 Confocal FLIM System: Simultaneous FLIM of NAD(P)H and FAD’.
References Related to Metabolic Imaging
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