A preliminary microspectrofluorometric study of NAD(P) reduction in dibenzo(a, e) fluoranthene-treated single living cells.

The fluorescence increase, due to NAD(P) reduction, following microelectrophoretic injection of glucose 6-P (G6P) into EL2 and NCTC 8739 single living cells treated with diBenzo(ae) Fluoranthene (diB(ae)F) and non-treated, has been studied with a rapid microspectrofluorometer. This study shows the enhanced capacity of treated cells to utilize larger doses (6-10 times more) of G6P than control cells. The time course of the return to the initial fluorescence level is essentially related to the magnitude of the injection dose. There are alterations (e.g. red & blue shifts) in the fluorescence spectrum of diB(ae)F-treated cells before injection and in the increase spectrum after injection of G6P, as compared to the same spectra in the diB(ae)F-untreated cells. This is discussed in reference to the metabolization of diB(ae)F as an alternative pathway for the reoxidation of NAD(P)H.     

Rapid microspectrofluorometric studies in EL2 cells following intracellular accumulation of dibenzocarbazoles

Summary Microspectrofluorometric observations were carried out in EL2 ascites cancer cells and dibenzo(a,e)fluoranthene (diB(a,e)F)-grown EL2 cells, following treatment (5 min) with three dibenzocarbazoles (1,2,7,8; 1,2,5,6 and 3,4,5,6). After microinjection of glucose-6-P leading to reduction of NAD(P), a sequence of difference spectra (after substrate minus before) is recorded. In dibenzocarbazole-untreated cells, maximum NAD(P) reduction (emission maximum at 465–475 nm) is attained within 5 s, followed by a gradual return to initial fluorescence within 20 to 200 s (faster in the diB(a,e)F-grown). In dibenzocarbazole-treated cells there is a rather regular increase in the intensity of the difference spectrum up to 300–500 s. Initially the increase is more predominant in the region around 460–470 nm, but it gains later prominence in the shorter wavelength region (420–430 nm) characteristic of the hydrocarbon (higher and steadier increase in the 3,4,5,6, dibenzocarbazole-treated diB(a,e)F-grown). Subsequently there is a gradual decrease of fluorescence which may or may not return to initial level. The observed increase spectra require evaluation in terms of possible components (e.g. a mixture of NAD(P)H and hydrocarbon, binding changes, succession of fluorescent metabolites).     

Rapid microspectrofluorometric studies in EL2 cells following intracellular accumulation of dibenzocarbazoles.

Microspectrofluorometric observations were carried out in EL2 ascites cancer cells and dibenzo(a,e)fluoranthene (diB(a,e)F)-grown EL2 cells, following treatment (5 min) with three dibenzocarbazoles (1,2,7,8; 1,2,5,6 and 3,4,5,6). After microinjection of glucose-6-P leading to reduction of NAD(P), a sequence of difference spectra (after substrate minus before) is recorded. In dibenzocarbazole-untreated cells, maximum (NAD(P) reduction (emission maximum at 465-475 nm) is attained within 5 s, followed by a gradual return to initial fluorescence within 20 to 200 s (faster in the diB(a,e)F-grown). In dibenzocarbazole-treated cells there is a rather regular increase in the intensity of the difference spectrum up to approximately 300-500 s. Initially the increase is more predominant in the region around 460-470 nm, but it gains later prominence in the shorter wavelength region (420-430 nm) characteristic of the hydrocarbon (higher and steadier increase in the 3,4,5,6, dibenzocarbazole-treated diB(a,e)F-grown). Subsequently there is a gradual decrease of fluorescence which may or may or not return to initial level. The observed increase spectra require evaluation in terms of possible components (e.g. a mixture of NAD(P)H and hydrocarbon, binding changes, succession of fluorescent metabolites).     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells

Summary Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture of flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry.A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanom a cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modified by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10^–6 oligomycin.When atebrine at levels (10^–6 M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g. 2×10^–5 M) an irreversible increase of atebrine fluorescence is seen.The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells.

Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

The effect of intracellular oxygen on the metabolization of Benzo(a)pyrene and Benzo(k)Fluoranthene. A microspectrofluorometric analysis in single living cells

Summary The fluorescence increase which accompanies the injection of glycolytic intermediates to Benzo(a)pyrene (BP) and Benzo(k) Fluoranthene-B(k)F treated EL2 ascites cancer cells, under aerobic and anaerobic conditions, has been studied in a microspectrofluorometer. In the carcinogen-treated cells the altered fluorescence increase pattern (in reference to control cells) which is observed at aerobiosis and attributed to BP or B(k)F metabolization, is not any more observable at anaerobiosis, in which case the fluorescence increase of the carcinogen-treated cells resembles that of the controls. This difference in behavior is discussed and a comparison is initiated between the response to injection in cells treated with BP (compound with K region) or B(k)F (compound without K region).     

Neither total culture fluorescence nor intracellular fluorescence are indicative of NAD(P)H levels in Escherichia coli MG 1655

Summary The blue fluorescence emitted by microbial cells irradiated with UV light at 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels. Offprint requests to: J. E. Bailey     

Isolation of nuclei from epidermis cells of larvae of the blowfly Calliphora vicina R.-D

Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell.     

Fluorescence monitoring during cultivation of Enterobacter aerogenes at different oxygen levels

On-line monitoring of NAD(P)H fluorescence and 2D fluorescence spectroscopy was performed with Enterobacter aerogenes, a bacterium sensitive to oxygen availability. The organism was grown in a reactor under low and high dissolved oxygen concentrations and circulated through a bypass attached to the reactor. Under low dissolved oxygen concentration in the reactor, NAD(P)H fluorescence in the reactor and the bypass showed a deviation, but not when the dissolved oxygen level in the reactor was high. The pattern of growth curves was identical under low and high oxygen levels. This indicates a difference in the metabolic activity of E. aerogenes in response to oxygen. The difference spectrum of the 2D fluorescence shows that growing E. aerogenes under high dissolved oxygen levels increases the NAD(P)H content of the cells. Received: 2 March 1999 / Received revision: 25 May 1999 / Accepted: 28 May 1999     

Ultraviolet-induced autofluorescence characterization of normal and tumoral esophageal epithelium cells with quantitation of NAD(P)H.

Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P)H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett's mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve-fitting analysis. A statistically highly significant difference was observed between the average intensity of the raw spectra of the different cell types. Tumoral cells had a fluorescence intensity approximately twice as high as that of normal cells. The results of the NAD(P)H quantitation analyzed by microspectrofluorimetry on single living cells and spectrofluorimetry on cell suspensions were consistent with those obtained by biochemical cycling assays, showing that the amount of intracellular NAD(P)H is higher in tumoral cells than in normal cells. Bound NAD(P)H concentration was found to be quite stable whatever the cell type while the amount of free NAD(P)H showed a very important increase in tumoral cells.     

Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters

Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (~460 nm) using 710–720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 μm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 ± 0.17 μm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.     

NAD(P)H oscillates in pollen tubes and is correlated with tip growth

The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily (Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme (excitation, 360 nm; emission, >400 nm). The strongest signal resides 20 to 40 microm behind the apex where mitochondria (stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD(P)H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-microm increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 microm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced (peaks) and oxidized (troughs) states of the coenzyme and that an increase in the oxidized state [NAD(P)(+)] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD(P)H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.     

First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor

Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% (v/v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000.     

Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.     

NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.     

NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes

A fiber-optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic-anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose-depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine-depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate-level phosphorylation, as shown by decreases of 35-52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6-38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.

     

A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.     

Spectral characterization of NAD(P)H fluorescence in intact isolated chloroplasts and leaves: Effect of chlorophyll concentration on reabsorption of blue-green fluorescence

In the present communication we report a spectral analysis of the blue-green fluorescence related to changes in NAD(P) redox state in chloroplasts and leaves. To assess the contribution of reabsorption and the inner filter effect, we compared transmission and fluorescence at different chloroplast concentrations, and showed that reabsorption by the photosynthetic pigments (chlorophylls and carotenoids) was at the origin of the two peaks in the emission spectrum in vivo. The absence of potential green-emitting fluorophores in chloroplasts was determined by measuring variable and time-resolved fluorescence at different wavelengths. We defined the conditions which optimize the UV-excited blue-green fluorescence signal dependent on NAD(P)H, and we present an example of monitoring of NAD(P)H fluorescence in intact leaves.