Characterization of NADH lifetime at different cell densities in a culture

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1 Characterization of NADH lifetime at different cell densities in a culture Vladimir Ghukasyan, Fu-Jen Kao * Institute of Biophotonics Engineering, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Taipei 112, Taiwan ABSTRACT One of the major intrinsic fluorophores, reduced nicotinamide dinucleotide (NADH) is as sensitive non-invasive indicator of the cellular energy metabolism, whereas measurement of its fluorescence lifetime has been demonstrated to derive more information from the cells, than its spectrum, providing with the information on free and enzyme-bound states dynamics of the NADH as well as its environment. This attractiveness of NADH as a non-invasive indicator served as a basis for the rapid increase in it studies, which resulted in a number of diagnostic methods for a range of pathological conditions, utilizing NADH. Given this growing importance of NADH thorough characterization of its lifetime dynamics is of high importance. We have conducted a series of NADH lifetime measurements at different cell density in the early logarithmic growth phase. The results has shown that the decrease in both short and long lifetime compounds is the earlier event cell culture growth, than the changes in NADH lifetime components preexponential factors ratio. Keywords: autofluorescence, fluorescence lifetime, TCSPC, cell culture 1. INTRODUCTION Reduced nicotinamide dinucleotide (NADH) is a sensitive non-invasive indicator of the cellular energy metabolism. NADH serves as a co-enzyme and a principal electron donor within the cell for both oxidative phosphorylation (aerobic respiration) and glycolysis (anaerobic respiration). For oxidative phosphorylation, which accounts for the majority of the energy carrier adenosine triphosphate (ATP), the NADH is one of the key compounds. The oxidized form of the NADH, NAD+, is kept at higher concentrations in the cells, than the reduced form, thus favoring the hydride transfer from a substrate to NAD+. The transfer of electrons between the substrate and NAD+ is known to be catalyzed by more than 200 enzymes, NADH produced then leaves the enzyme surface and carries electrons to their point of entry into the respiratory chain in mitochondria, to which it participates as a part of the enzymes complex (complex I)[1]. It is well known that mitochondrial dysfunction is involved in many diseases, such as ischemia, hypoxemia, Parkinson s disease, Alzheimer s disease, and in apoptotic process. Therefore, the possibility of monitoring the mitochondrial NADH redox state in cells is of greater importance. NADH is one of the major intrinsic fluorophores, absorbing at 340 nm and emitting at 460 nm. The absorbance of the oxidized species differs from that of NADH, moreover NAD+ is not fluorescent, and therefore changes in the ratio of [NADH] to [NAD+] can be observed by the optical methods. The role of the intrinsic fluorophore NADH as the principal electron donor in glycolytic and oxidative energy metabolism makes it a convenient non-invasive fluorescent probe of metabolic state [2]. However, the fluorescence intensity quantum yield depends heavily on the fluorophore environment which makes it difficult to apply the technique to define the dynamics of the NADH amount in tissue. Moreover, discriminating the main states of NADH bound to enzymes, or free in cytoplasm, is a challenging task, since the spectrum of the both species differs only by 20 nm. The parameter itself is an important characteristic since it has been shown that dehydrogenases kinetics depend on the quantity of the locally available free NADH [3]. The fluorescence lifetime, which is an average time the fluorophore molecules spend in the excited state levels before decaying to the ground state, provides a method for a reliable discrimination of free and bound NADH molecules: early experiments with NADH in solution have demonstrated that its mean lifetime in aqueous solution becomes about 10-fold longer when the molecule binds to a protein like lactate dehydrogenase [4]. However, both free and bound forms of * fjkao@ym.edu.tw; phone ; fax Multiphoton Microscopy in the Biomedical Sciences VIII, edited by Ammasi Periasamy, Peter T. C. So, Proc. of SPIE Vol. 6860, 68602B, (2008) /08/$18 doi: / SPIE Digital Library -- Subscriber Archive Copy Proc. of SPIE Vol B-1

2 NADH are usually multiexponential with shorter components that are comparable with the free species. Four different pools of NADH lifetime have been found by various methods in solution and intact cells: free NADH pool with a lifetime of ns, a second short lifetime component of ns, and two long lifetime pools of and 6-8 ns correspondingly [5,6]. At this, the first lifetime component is attributed to free, and the rest three to bound NADH. Free NADH in aqueous solution at room temperature exhibits a bi-exponential fluorescence decay with fluorescence lifetime components of ~0.3 and ~0.7 ns [7,8] and a mean fluorescence lifetime of ~0.4 ns. The shorter lifetime of free NADH is caused by the dynamic quenching by the adenine moiety [9]. Protein bound NADH exhibits a threeexponential fluorescence decay and the shorter lifetime component can be comparable to that of the long lifetime component of free NADH. The fluorescence lifetime of protein-bound NADH depends on the enzyme to which it is bound and this suggests that it can be probed by the lifetime of this fluorophore. Therefore, the common practice of fitting a bi-exponential decay profile to a mixture of free and protein bound NADH exhibiting 4 decay components (or more, with multiple bound proteins) is a considerable simplification of the underlying dynamics. However, due to the low quantum yield and heavy dependence of the results reliability on the count statistics, the double-exponential model is currently commonly accepted. Several attempts to explain the decay parameters were made. The two components of NADH and NADPH in an aqueous solution were assigned to different conformers: folded and unfolded, whereas different lifetimes of the longer lifetime components originate from the species bound to different enzymes [10]. At the same time, with the parallel lifetime and anisotropy measurements it has been demonstrated that bound forms can exhibit a long lifetime as well, thus resulting in a mean short lifetime component shifted to the longer range as these bound species exhibit a short lifetime component with the average values of 600 ps. The first attempt to apply fluorescence lifetime imaging for the observation of NADH has been presented in 1992 in [11]. To record fluorescence lifetime changes gated image intensifiers, modulation techniques and gated photon counting can be used. In conjunction with a scanning microscope, most of these methods have serious drawbacks. In contrast, state-of-the-art time-correlated single photon counting systems (TCSPC) reach count rates in the MHz range and therefore are able to record decay functions within a few ms. TCSPC method has a high detection efficiency, a time resolution limited only by the transit time spread of the detector and directly delivers the decay functions in the time domain [12,13]. The fluorescence lifetime imaging microscopy (FLIM) of NADH is intensively used currently in a wide range of studies and diagnosis techniques. In particular, the technique has been applied in the early diagnostics of the human breast cancer [14], non-invasive glucose sensing [15], etc. 2. MATERIALS AND METHODS 2.1 Cell culture and sample preparation Human cervical cancer HeLa cell culture was grown in high glucose Dulbecco s Modified Eagle s Medium (DMEM) with 5% FBS. 25cc flasks with the cells were kept in the incubator at 37 C and CO2 5% flow to maintain the physiological ph level. 12 hours prior to the first measurement the cells were removed from the flasks by trypsinization and plated in the concentration of 1x10 4 /ml on the 24 mm round cover slips coated with fetal bovine serum to increase the cells attachment to the non-charged glass. Right before the imaging session the cover slips with the cells attached were twice washed with PBS and replaced to a custom made chamber, where there were glued by vacuum grease at the edges to the metallic ring with a hole diameter of 22 mm. The ring was covered with a plastic cup which held further added 1 ml of DMEM above the cells. At this the major part of the cover slip was accessible for the lens of the inverted microscope. All the measurements were conducted in the microscope stage incubator (H201, Okolab Inc.), maintaining physiological temperature and perfusion with the 5% CO 2. To exclude the effect of serum starvation, the measurements were conducted for several hours upon addition of fresh media to the chamber. After the first measurement (Day 1 measurement hereafter) the rest were taken each 24 hours. To reduce the photobleaching effect the laser power at which the photon counts wouldn t decrease significantly during long measurements (15-25 min in this study) was chosen and kept at the same level for all further measurements. 2.2 System Setup The measurements were conducted on our time-resolved two-photon fluorescence spectroscopy system, built around the modified laser-scanning microscope (inverted IX71 equipped with the FV300 scanning unit, Olympus Corp.). Proc. of SPIE Vol B-2

3 The sample was excited at 740 nm by a mode-locked femtosecond Ti:sapphire Mira F-900 laser, operated in two-photon mode at 76MHz frequency. The beam was fed into the scanning unit with the high-precision alignment and scanned across the sample at the speed, controlled externally by a function generator (AFG310, Tektronix Inc.). The 100x1.4 numerical aperture (NA) PlanApochromat lens (Olympus Inc.) focused the beam at the cell samples, collected the autofluorescence signals and directed further in a non-descanned mode to the photon-counting photomultiplier (H7422- P40, Hamamatsu Photonics K.K.). The 447 ±30 nm filter (FF02-447/60-25, Semrock) along with the IR cut-off filter (Edmund Optics Inc.) installed in the detection channel provided with the recording of the NADH/NADPH signal only and minimized the impact of the flavins, which were further filtered out by applying the threshold parameter. Signals synchronization and building of the time-resolved data matrix of the image was conducted by the PC-board based Time- Correlated Single Photon Counting System (TCSPC) (SPC830, Becker&Hickl GmbH). All the images were taken at 256x256 pixels resolution with the acquisition times ranging from 900 to 1200 s. The instrument response function (IRF) of the setup was measured from the second harmonic generated by the periodically poled lithium niobate (PPLN) crystal. Due to the weak SHG signal at the given wavelength the IRF signal was accumulated for 10 s with further removal of the noise background. The measured full width at half maximum of the IRF was ~320 ps and represented the IRF of the whole system. 2.3 FLIM data analysis Data analysis via model function fitting along with the IRF deconvolution and color coding was conducted with the commercially available SPCImage software package (v. 2.9, Becker&Hickl, GmbH). Given the decay nature of the NAD(P)H a two-exponential model function was applied for the fitting to the actual data by iterative non-least squares reconvolution: n I( t) = Iinstr ( t) I 0 + ai ( exp( t τ i )) = dt (1) i 1 where I instr is the instrument response, calculated from the PPLN crystal second harmonic generation, I 0 is the baseline offset, and a i (exp(-t/t i )) represents main components to the fluorescence decay. The a i intensity coefficients have relative amplitudes and demonstrate the correlation between components fractions populations. The reduced goodness-of-fit parameter has been calculated by the equation n 2 2 χ R = [ I ( tk ) I C ( tk )] I ( tk ) ( n p) (2) k = 1 where I(t k ) - is the actual experimental data, I c (t k ) - calculated decay as described above, n - number of the data (time) 2 points (we used 256 time channels), and p - number of the model parameters. The χ was minimized by the Levenberg- R Marquardt search algorithm. All the images were analyzed with a two-exponential decay model. To remove the background signal either the regions of interest were marked so that only the pixels inside the region were used to generate the lifetime distribution histogram, or a threshold parameter was used. The physiological temperature decreased the fluorescence intensity exhibited by the NADH, so that both long collection time and pixels binning were applied to obtain a statistically sufficient photon counts per decay curve. The binning factor n determines number of the adjacent pixels the signal from which is summed together to obtain a single decay curve, whereas the number is equal (2n + 1) 2. A factor of 2 was applied in our analysis, so that a signal from 25 adjacent pixels was used to derive a single decay curve for further fitting. The method decreases the resolution, which, however, wasn t important in our study. All the pixels within the specified region or with the number of the photon counts higher than that defined by the threshold parameter were then analyzed and the lifetime distribution histograms built. For all the fittings the pixels, exhibiting χ value more than 1.4 were excluded from further analysis. The representative for the fittings is given on Fig. 1 below. Image statistics For each sample we have taken measurements from 5 different spots separated from each other by at least 400 µm. For the comparison purposes the peak values of the histograms were derived and standard deviation from 5 spots calculated as for each sample measurement. 2 r Proc. of SPIE Vol B-3

4 I!I! 3. RESULTS [is Tl:Jl7 mc it a x 1fly U- 2 The data was fit to a two-exponential decay curve with short and long lifetime components similar to the values reported earlier. A representative for all the images curve, measured from a pixel of a TCSPC-generated image exhibits a good fit with the χ r 2 value close to the ideal value of 1 and the residues fluctuating around the 0 axis value. The Gaussian distribution of the χ r 2 value as for the whole image stretches from 0.8 to 1.4 in average Fig. 1. Fluorescence lifetime decay curve (blue dots), calculated model function (red line) and instrument response function (green line) of a single pixel from an image obtained with a TCSPC system from the HeLa cells sample. The good quality of fitting is evidenced by the χ r 2 value (whereas 1.0 is an ideal value) and distribution of residuals (black line below the decay curve), with even distribution around the 0 axis. The results of the measurement are summarized in Table 1 below. The data included gives the dynamics of the NADH lifetime measured from the cells at initial (12 hours upon plating, signified as Day 1), and early logarithmic stage of cell culture growth (Day 2 Day 4). The table shows the fluorescence lifetimes of short (τ 1 ) and long (τ 2 ) lifetime components as well as their relative contributions during the whole period of observation (a 1 and a 2 correspondingly). We have observed a decrease of the short lifetime component over the whole term of measurements. The Day 1 measurement resulted in a τ 1 value of 484±21 ps with almost equal distribution within the cells as revealed by the color coding. No statistically significant change was observed on the day 2 with a continuous decrease of the component to 475±7 on the day 3 and 459±13ps on the last day, illustrated on the graph on Fig. 2. At this, the shorter lifetimes of the τ 1 exhibited gradual accumulation in the close perinuclear area. In some cases we also observed appearance of bright pixels with shorter than the average lifetimes, distributed either in a punctate manner or in a grouped way. Previously these high-intensity spots were attributed to lipofuscin [16]. The signal wasn t dominating in neither of the cases and thus no correction was made to remove it from the analysis. The long lifetime component (τ 2 ) exhibited lifetime of 2616±40 ps for the measurements on the day 1 after cells plating. On the day 2 a slight increase of the component value to 2658±39 ps was observed with further continuous decrease to 2601±39 ps on the third and 2559±60 ps on the fourth days. No distinguishable trend in the lifetime distribution pattern similar to that, exhibited by the τ 1, was detected for the long lifetime component. The results are illustrated on Fig. 3. For the illustration purposes we have chosen broader range for the color coding mapping, than these for the τ 1 which is explained by a wider full-width at half maximum (FWHM) of the τ 2 lifetime distribution histogram obtained from the whole image with the values, as compared to that of τ 1 (800 ps versus 800 ps correspondingly). Table 1. Distribution of the NADH fluorescence lifetimes and ratios over the period of observation. τ 1 Days τ 2 a 1 a 2 (ps) (ps) (%) (%) 1 484± ± ± ± ± ± ± ± ±7 2601± ± ± ± ± ± ±1.3 Proc. of SPIE Vol B-4

5 Day 1 Day 2 ) Day 4 Day 3 Lifen. 470 ps 520 ps Fig. 2. Changes of the NADH short lifetime component during the period of observation (left) and the distribution of the τ1 inside the cells (right). Statistically insignificant decrease is observed at the second day of observation with continuous decrease over the next two days. the color coding reveals also some pattern in the distribution of the τ1 in cells with the almost even distribution for the first day of observation and then accumulation of the shorter range lifetime in the close perinuclear area (green regions). I 2600 Day Day e 2000 Day Day l000 2 Days Fig. 3. Changes of the NADH long lifetime component during the period of observation (left) and the distribution of the τ2 inside the cells (right). Slight increase observed on the day 2 and continuous decrease over the next two days of observation. The distribution of the component within the cells is mostly even. Proc. of SPIE Vol B-5

6 No significant changes in the dynamics of the pre-exponential factors a 1 and a 2 have been observed for the whole period of observation as depicted in Table 1 and Fig. 4 below. The majority of the fluorescence originates from the short lifetime component with the ratio of 72-76% and long lifetime component at 25-28% correspondingly. During the period of observation the a 1 factor was just fluctuating from 72 (day 1) to 76 (day2), then back to 72 (day3) and 74 (day 4) with the long lifetime exhibiting, correspondingly, 28, 24, 28 and 26% for the same time of observation Lifetime (ps) Days Fig. 4. Distribution of the fluorescence lifetimes ratios over the period of observation. The ratio of τ 1 (a 1 ) is presented as white bar, value of the τ 2 ratio (a 2 ) - with the grey bar. 4. DISCUSSION As a result of a bi-exponential fitting a histogram of the lifetime distribution over the images have been obtained with the average peaks of (the data not shown). The distribution of the short-lifetime component was in narrower range than that of the long-lifetime, which resulted in the lifetime distributions histograms with average FWHM of 200 and 800 ps correspondingly (data not shown) The difference is explained with the different spectroscopic properties of NADH bound to a range of different enzymes. The dynamics of the lifetime observed are similar to that reported previously for the effect of cell density on the NADH fluorescence lifetime dynamics. Measured from the MCF10A human breast cells plated at densities, corresponding to initial (25,000 cells/mm 2 ), mid (100,000 cells/mm 2, logarithmic) and (1,000,000 cells/mm 2, confluent) point on the cellular growth curve [17]. A dramatic decrease between the early and confluent points has been observed for the long-lifetime component (from 3.106±0.105 ns to 1.723±0.019 ns) as well as smaller decrease in free lifetime component (from 0.423±0.023 ns to 0.350±0.007 ns). Additionally, the ratio of free and protein-bound NADH (a 1 :a 2 ) increased by more than a factor of 2 from the early to confluent phase of the growth curve from 1.175±0.145 to 2.702± Similar results have been obtained also for the cells, treated with the electron transport inhibitor KCN and cells, undergoing serum starvation. The KCN, along with rotenone cause the reduction of NADH in complex I and in mitochondria under aerobic conditions. In both cases decrease in short and long lifetime components have been observed as well as increase of the a1:a2 ratio. Interestingly, for the experiment with the serum starvation no significant change of the short lifetime component as compared with the control cells has been observed for the cell culture in confluency (1,000,000 cells/mm 2 ) in this work. On the contrary, when treated with KCN, the long lifetime component have exhibited in confluence, wasn t significantly different from that measured from the control cells. Whereas both of the inhibitors are contradictory, the hypoxia has been shown by several groups as a factor to decrease the lifetime and reduce the bound form contribution to the overall NADH fluorescence intensity and thus increasing the a 1 :a 2 ratio. Proc. of SPIE Vol B-6

7 Among the other effects of the increasing cell density on the metabolism, decrease in oxygen consumption, net lactate production, NAD content and redox-potential decrease has been demonstrated earlier, whereas all the events were shown to happen long before cessation of proliferation and thus may influence commitment of cells to pass through a further cell cycle by very indirect mechanisms only [18]. The decrease is believed to be caused directly by cell-cell contacts rather than cell proliferation, which ceases at least two division cycles after the most cells contacted the neighbouring cell. The delay can be explained by the hypothesis of Kajstura and Korhoda, postulating depletion of basic metabolites for macomolecular synthesis by the reduction of respiration (citric acid cycle) [19]. Combined fluorescence lifetime and anisotropy decay measurements have revealed that at hypoxia, the fluorescence lifetimes of NADH may change as a result of the changes in the relative populations of the three enzyme bound species, indicating a redistribution of NADH among different enzymes, at the same time preserving the free and bound forms ratio [6]. Our results suggest that the reorganization of the bound NADH pool is the earliest event in the chain of metabolism-related processes in the cells with the increasing density in culture. and thus the decrease in the lifetime should be taken into consideration when relying on NADH as a sensitive diagnostic tool. 5. ACKNOWLEDGEMENTS This work was financially supported by the National Science Council, Taiwan, under the grants NSC M and NSC B REFERENCES D.L. Nelson, and M. M. Cox, Lehninger Princples of Biochemistry, W.H. Freeman and Co., New York, B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Intracellular oxidation-reduction states in vivo: The microfluorometry of pyridine nucleotide gives a continuous measurement of the oxidation state, Science 137(3529), , D. H. Williamson, Patricia Lund, and H. A. Krebs, The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver, Biochem. J. 103, , A. Gafni, and L. Brand, Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase, Biochemistry 15, , M. Wakita, G. Nishimura, and M. Tamura, Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ, J. Biochem. 118, , H. D. Vishwasrao, A.A. Heikal, K. A. Kasischke, and W. W. Webb, Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy, J. Biol. Chem. 280(26), , S. H. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, , M. E. Couprie, F. Mérola, P. Tauc, D. Garzella, A. Delboulbé, T. Hara, and M. Billardon, First use of the UV Super-ACO free-electron laser: Fluorescence decays and rotational dynamics of the NADH coenzyme, Rev. Sci. Instrum. 65, , J.R. Lakowicz, Principles of fluorescence spectroscopy, Plenum Press, New York, A. J. W. G. Visser, and A. van Hoek, The fluorescence decay of reduced nicotinamides in aqueous solution after excitation with a uv-mode locked Ar ion laser, Photochem. Photobiol. 33, 35-40, J. R.Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L Johnson, Fluorescence lifetime imaging of free and protein-bound NADH, Proc. Natl. Acad. Sci. U.S.A. 89, , W. Becker, Advanced Time-Correlated Single Photon Counting Techniques, Springer, Berlin, W. Becker, A. Bergmann, M.A. Hink, K. König, K. Benndorf, and C. Biskup, Fluorescence lifetime imaging by time-correlated single-photon Counting, Microsc. Res. Tech., 63(1), 58-66, P. J. Atdrous, J. Siegel, P. M. W. French, S. Shousha, E-N. Lalani, and G. W.H. Stamp, Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer, J. Path. 199, , Proc. of SPIE Vol B-7

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