Microspectrofluorometric Approach to the Study of Free/Bound Nad(p)h Ratio as Metabolic Indicator in Various Cell Types

Salmon, Jean‐Marie; Kohen, Elli; Viallet, Pierre; Hirschberg, Joseph; Wouters, A.; Kohen, Cahide; Thorell, B.

doi:10.1111/j.1751-1097.1982.tb04420.x

Cited by 105 publications

(71 citation statements)

References 35 publications

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“…After rinsing of the cells, the fluorescence intensity of the R110 intracellular fluorescence was recorded vs time in the same cell population (Figure 4). After 30 min, the fluorescence intensity returned to the level of the intrinsic cell fluorescence, presumably due, under the excitation conditions used, to flavin ring emissions (flavoproteins, flavin mononucleotides, flavin dinucleotides) (Salmon et al 1982). The rate of clearance of the R110 zwitterionic species is short, because the experiment demonstrated that R110 elimination from the CCRF-CEM cells was complete within 30 min.…”

Section: Kinetics Of R110 Elimination From Ccrf-cem Cellsmentioning

confidence: 93%

Intracellular Accumulation of Rhodamine 110 in Single Living Cells

Jeannot

Salmon

Deumié

et al. 1997

J Histochem Cytochem.

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SUMMARYTo gain a better understanding of the internalization of rhodamines, vital staining of living cells in situ by two different rhodamines, R110 and R123, was studied by microfluorometry. These dyes differ strongly in their lipophilic properties because of differences in charge distribution. Microspectrofluorometry was used to study the fluorescence emission spectra of R110-loaded cells to determine reliable loading conditions. Cell uptake and cell efflux studies of R110 were performed by numerical microfluorescence imaging. A slower uptake was observed for R110 (14 hr) vs R123 (2 hr), but the R110 efflux was much more rapid (30 min) than that of R123 ( Ͼ 24 hr). Although it appeared in the R110 and R123 co-localization study that R110 was able to accumulate in mitochondria, labeling with R110 was lower than with R123. Our results indicate that, rhodamine 110 in its acid cationic form is able to cross the plasma and mitochondrial membrane and to accumulate in cell compartments as does the cationic rhodamine 123. However, because of its acido-basic properties, R110 should be able to decrease the pH of cell compartments, depending on their ability to regulate pH. In such a model, mitochondrial pH should be more greatly decreased than cytosolic pH, leading to a lower mitochondrial accumulation of R110 than of R123. Surprisingly, these effects, which should affect the energetic state of mitochondria, do not influence cell growth, because no cytotoxic effect was observed.( J Histochem Cytochem 45:403-412, 1997 )

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Section: Kinetics Of R110 Elimination From Ccrf-cem Cellsmentioning

confidence: 93%

Intracellular Accumulation of Rhodamine 110 in Single Living Cells

Jeannot

Salmon

Deumié

et al. 1997

J Histochem Cytochem.

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“…In the present report we describe the use of FLIM to image NADH when free in solution and when bound to a dehydrogenase. NADH was chosen because the emission of NADH from cells and tissues has been widely used for studies of the redox status of tissues (13)(14)(15)(16)(17), following the pioneering observations of Chance et al (18) of the increase in NADH fluorescence under anoxic conditions. Additionally, resolution of free and bound NADH represents a challenging case for the FLIM methodology because the decay times are on the subnanosecond timescale (19).…”

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confidence: 99%

Fluorescence lifetime imaging of free and protein-bound NADH.

Lakowicz

Szmacinski

Nowaczyk

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

681

582

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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...

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“…659 0.5 pmol, have been reported by Jongkind et al (6) and Loxdale (8); in the elegant studies of Kohen and coworkers (e.g. 29), in situ measurements of NAD(P)H in various cell compartments have been reported. We also note that our work over the past 5 years has been simultaneous with that of Mroz and Lechene (9,10), who also have reported high measurement sensitivity.…”

mentioning

confidence: 66%

Histochemical Technique

Outlaw¹,

Springer²,

Tarczynski³

1985

Plant Physiol.

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Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods we describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are 'extracted' in microdroplets of assay cocktail.The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, we evaluated several parameters required for optimization; the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints.With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P)triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which has been demonstrated previously in other reports based on diverse assay approaches.In some instances, the activity of relatively few cells that are dispersed throughout an organ determine the functional competence ofthe entire organism. For example, guard cells comprise about 0. 1% of the volume of a leaf; pairs of these cells surround stomata and regulate the aperture sizes. Yet the size of these pores is a primary determinant of the rate of CO2 uptake by the leaf. To gain a biological understanding of the function of specialized cells, an investigator is challenged to develop or adapt appropriate methodology. Sometimes, the cells can be isolated in bulk from the surrounding tissue matrix and studied by conventional biochemical methodology. This approach is attractive (e.g. 36), but it is limited to tissues amenable to maceration and, unfortunately, is also potentially subject to artefacts arising from instability of cellular constituents. Alternatively, such studies may be approached using histochemical methods. In broad terms, these methods fall into two categories: (a) those that rely on high morphological resolution of the assay indication (e.g. electron microprobe, 'slide' histochemistry) and (b) (The time resolution of our software ranges from 0.7 to 125 s, depending on the reading precision one sets.) For reference, individual readings are accessed at >285 Hz and could be speeded-at the expense of signal increments-to about 10 kHz (which would still be the slowest electronic time constant but would be faster than the mechanical response of the shutters). We express precision in mol of natural substrate conver...

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Microspectrofluorometric Approach to the Study of Free/Bound Nad(p)h Ratio as Metabolic Indicator in Various Cell Types

Cited by 105 publications

References 35 publications

Intracellular Accumulation of Rhodamine 110 in Single Living Cells

Intracellular Accumulation of Rhodamine 110 in Single Living Cells

Fluorescence lifetime imaging of free and protein-bound NADH.

Histochemical Technique

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