Fluorescence lifetime imaging of free and protein-bound NADH.

Abstract

We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challengi case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetimeimages are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images, each obtained with various phase shifts of the ginmodulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe an imang procedure that allows speyific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.The phenomenon of fluorescence is widely utilized for research in the biosciences. These applications have been focused on two divergent types of measurements, timeresolved fluorescence and fluorescence microscopy. In the former, one takes advantage of the high information content ofthe time-dependent fluorescence decays to uncover details about the structure and dynamics of macromolecules (1-3). Such measurements are performed almost exclusively by using picosecond laser sources coupled with high-speed photodetectors. In contrast, fluorescence microscopy, in combination with dyes, stains, fluorophores, or fluorophorelabeled antibodies, is most often used to determine the localization of species of interest, usually proteins or other macromolecules (4-6). The acquisition of two-dimensional (2D) fluorescence images is typically accomplished with low-speed accumulating detectors. Consequently, the high information content of time-resolved fluorescence is not usually available for studies of microscopic biological samples. This is particularly disadvantageous when one considers the sensitivity of fluorescence decay times to chemical and environmental factors of interest, such as local pH, cation concentration, oxygen, and polarity, to name a few.In the present report we combine measurements of fluorescence lifetimes with 2D imaging to create images in which the lifetimes at each pixel are used to create contrast in the images. While there have been reports...

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

“…The lifetime decay curve of each pixel was fit to a double-exponential decay model (19), where the free parameters are the short and long lifetime components ( 1 and 2 , respectively), and the relative contributions of the lifetime components (␣ 1 and ␣ 2, where ␣ 1 ϩ ␣ 2 ϭ 100%). The presence of two distinctly different lifetimes for free and protein-bound NADH (10,18), and for free and protein-bound FAD (11,29) indicates that NADH and FAD fluorescence decay curves are best fit to a double-exponential decay model. When the scattering contribution was fixed at zero, the 2 value of the fit was minimized.…”

“…NADH has a short and long lifetime component, respectively, depending on whether it is in a free or protein-bound state (10), and FAD has a short and long lifetime component, respectively, depending on whether it is in a protein-bound or free state (11). The shorter fluorescence lifetimes of both protein-bound FAD and free NADH are due to dynamic quenching by the adenine moiety (12,13).…”

NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

“…NAD(P)H is fluorescent and has been visualized by exciting at 340 to 365 nm and detecting emission at 400 to 500 nm (Lakowicz et al, 1992;Paul and Schneckenburger, 1996;Danø et al, 1999;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). To visualize the fluorescent signal, we used an inverted wide-field fluorescence microscope (Nikon TE-300), equipped with a CCD camera (CoolSnap HQ; Roper Instruments) mounted on the Kö hler port, and a Xenon excitatory light source (DG-4; Sutter Instruments).…”

“…Their high energy status and reducing power drive many key biosynthetic reactions and ATP production. Because NAD(P)H but not NAD(P) 1 possesses an endogenous fluorescence, it is possible to detect the reduced form in living cells; this property has been widely exploited (Lakowicz et al, 1992;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). Particularly pertinent are the oscillations in NADH that have been observed in yeast cells, which serve as a marker for changes in metabolism (Ghosh and Chance, 1964;Goldbeter, 1996;Danø et al, 1999).…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] . In the last years, FLIM gained on interest in the biomedicine, as well 27,49,78,[91][92][93][94][95][96] .…”