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1 September 2008 Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells
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Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was ~1.3 ns. The NADH average fluorescence lifetime increased to ~3.5 ns within 15 min after 1 μM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.



Cancer is among the world’s leading causes of death. To improve the success rate of all cancer treatments including chemo, radiation, and photodynamic therapies, rapid and reliable readouts of treatment efficacy is in high demand. Direct monitoring of cell death, which is classically categorized into apoptosis and necrosis, is one way to measure treatment efficacy.1 Preclinical and clinical studies have shown that the detection of apoptosis or cell death in general correlates with tumor response.2, 3, 4 Specifically, monitoring of apoptosis can be useful in the design of apoptosis-favored treatment protocols, which could effectively kill target cells without inducing necrotic response, that have been preclinically demonstrated in the treatment of brain tumors using low dose photodynamic therapy.5, 6

There is a range of noninvasive in vivo imaging/detection techniques to detect apoptosis such as magnetic resonance imaging (MRI) and spectroscopy, nuclear imaging [e.g., single-photon emission computed tomography and positron emission tomography (PET)], ultrasound, and optical imaging.7 Molecular markers are generally used. The 40kDa vesicle-associated protein, annexin V, is probably the most widely used apoptosis marker in molecular imaging. Radio-, (F18) -, superparamagnetic iron oxide particles-, and fluorescence-labeled annexin V have been used in nuclear imaging,4, 8 PET,9 MRI,10 and optical imaging,11 respectively. Caspases play a crucial role in the early phases of apoptosis and thus are the other apoptosis targets of molecular probes. Caspase 3-specific-cleavable reporter probe was used in luciferase-based bioluminescence imaging.12 Besides molecular targeting, ultrasound measured the scattering power from tissues that showed changes due to nuclear condensation at the late stage of apoptotic process.13 Diffusion MRI measured lipid droplets or apparent diffusion coefficient of water to indirectly monitor cell death.14 Among these methods, ultrasound and diffusion MRI do not involve exogenous molecules and thus are clinically transferable. Diffusion MRI has been demonstrated to monitor therapeutic response in clinical trials.14, 15 Molecular targeting using radioactively labeled annexin V has been used to detect apoptosis in clinics although currently no clinical trial further evaluates its potential to assess the outcome of cancer therapy. Bioluminescence imaging is not readily transferable to clinical practice.

Mitochondria are known to be one of the key regulators in cell death particularly in apoptosis. Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in glycolysis and oxidative energy metabolism that acts as a principal electron and proton donor in mitochondria. It has also been widely accepted as a convenient noninvasive optical probe of metabolic state through the measurement of NADH fluorescence intensity.16, 17, 18 Recently, the NADH fluorescence lifetime (τ) measurement has been used to monitor cell metabolic activities19, 20 based on (i) the lifetime changes of two major lifetime components (i.e., free NADH exhibiting a shorter lifetime, τ10.4to0.5ns and bound NADH exhibiting a longer lifetime, τ22to3ns ) and (ii) the ratio of relative amplitudes of two major lifetime components. One advantage of NADH fluorescence lifetime measurement over intensity measurement is its sensitivity to the changes between free and bound form. The change in lifetime of NADH fluorescence from free to bound form differs as much as 10times ,21, 22, 23 but the spectral shift of the NADH fluorescence from free to bound form is relatively small ( 10to20nm blueshift) as compared with the width of the NADH fluorescence spectrum (150nm) .20

The aim of the present study was to investigate the potential of using the NADH lifetime measurement as a noninvasive probe of cell death. Similar to ultrasound and diffusion MRI methods, NADH signal is intrinsic so that it can be directly applied in a clinical setting once its utility of imaging cell death is demonstrated. Unlike ultrasound and diffusion MRI methods that indirectly probe cell death information based on cell morphological change (i.e., imaging nuclear condensation in ultrasound and cell swelling/shrinkage in diffusion MRI), NADH fluorescence lifetime method has a potential to detect the change of biochemical functions during cell death. We applied two-photon fluorescence lifetime imaging microscopy (FLIM) to map NADH fluorescence lifetime of unstained live HeLa cells and 143B osteosarcoma cells before and after treatment with staurosporine (STS) and hydrogen peroxide (H2O2) . The STS-induced cell death has been well established to be executed by the mitochondria-mediated apoptotic pathway.24, 25, 26, 27, 28, 29. High doses of H2O2 have been documented as a necrosis inducer.30 Our results demonstrated that the average NADH fluorescence lifetime increased shortly after 1μM STS treatment, but no significant lifetime change was observed after 1mM H2O2 treatment of HeLa and 143B cells. These findings suggest that the average NADH lifetime changes may be a valuable indicator for apoptosis and for differentiation of apoptosis from necrosis of tissue cells in a noninvasive manner.


Materials and Methods


Cell Cultures and Experiments

Human osteosarcoma 143B and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen Corp., Carlsbad, California) containing 100unitsml penicillin G, 100μgml streptomycin sulfate, 0.5μgml amphotericin B, and 5% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C in a humidified atmosphere with 5% CO2 . At 24h before fluorescence lifetime imaging and drug treatments, cells at a density of 2×104cellscm2 were seeded onto 24mm diameter round glass coverslips (Paul Marienfeld GmbH & Co., Lauda-Konigshofen, Germany) that had been coated with FBS. These coverslips were then kept in dishes and cultured in DMEM inside an incubator for 24h . After 24h , cells were completely attached onto the coverslip, ready to grow (in the early log phase of cell proliferation curve), and ready for fluorescence imaging. Immediately before fluorescence imaging, cells were washed twice using phosphate-buffered saline solution and then transferred to a cell chamber designed for viewing live cell specimens. A 1 mL aliquot of 5 mM HEPES ( N -2-hydroxyethylpiperazine- N -2-ethanesulfonic acid) buffer ( 5mM KCl, 140mM NaCl, 2mM CaCl2 , 1mM MgCl2 , 10mM glucose, pH 7.4) was added into the cell chamber to nourish cells and to avoid light absorption of the red color of the culture medium. Fluorescence lifetime images of grouped cells were acquired at 1 to 3 sites per coverslip before treatment. The field of view (FOV) of each image was 100×100μm . The FLIM system was equipped with a microscope cage incubator (Oko-lab, Naples, Italy) to maintain the optimal temperature during experiments. To optimize the NADH fluorescence intensity and to acquire high photon counts for statistical processing of the data, the imaging was conducted at room temperature (20°Cto23°C) .31 Additional measurements were performed at the physiological temperature (35°Cto37°C) in both controls and STS-treated cells to ascertain that the results of NADH lifetime measurements were not affected by the room temperature conditions.

Our previous experience with STS-induced apoptosis showed that the activation of caspase 3 occurred at 8h or later after 25nM STS treatment and the apoptotic bodies appeared at 24h after treatment.32 Maeno 29 showed that the release of cytochrome c from mitochondria and the activation of caspase 3 occurred at 2to4h after 4μM STS treatment of HeLa cells. Thus we randomly chose a medium dose (1μM) of STS (Sigma-Aldrich, St. Louis, Missouri). Hydrogen peroxide is known to induce both apoptosis and necrosis depending on the concentration (e.g., 10to100μM for apoptosis, and 1to10mM for necrosis) used.30 We chose to use 1mM H2O2 (Sigma-Aldrich) for necrosis induction. Time-lapsed fluorescence lifetime images were obtained at the same site (same FOV) before, immediately after (0to15 min) , and up to 10h after treatment with STS or H2O2 . Controls are fluorescence lifetime images of cells without and before treatment.


Caspase 3 Activity Assay

Cells were disintegrated in 100μl lysis buffer [ 12.5mM Tris-HCl, 1mM dithiothreitol, 0.125mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, and an aliquot of complete protease inhibitor mixture (Roche Applied Sciences, Mannheim, Germany), pH 7.0] on ice for 30min and centrifuged at 9000g for 10min at 4°C . A 50μg aliquot of protein was incubated with 20μM Ac-DEVD-AFC (Calbiochem, San Diego, California), a fluorescent substrate of caspase 3, in 500 μl of assay buffer [50 mM Tris-HCl, 1 mM EDTA, and 10 mM EGTA (ethyleneglycol-bis-(β-aminoethylether)- N , N , N , N -tetraacetic acid), pH 7.0] at 37°C for 30min in the dark. The fluorescence intensity was determined by spectrofluorometry (Hitachi F-3000, Tokyo, Japan) at an excitation wavelength of 380nm and an emission wavelength of 508nm as described previously.32, 33



Time-domain FLIM was performed with a 60×1.45 numerical apenture PlanApochromat oil objective lens (Olympus Corp., Tokyo, Japan) on a modified two-photon laser scanning microscope (FV300 with the IX71 inverted microscope, Olympus Corp.) as described previously.34 In this study, samples were excited at 750nm (two-photon) by a mode-locked Ti:Sapphire Mira F-900 laser, pumped by a solid-state continuous wave 532nm Verdi laser (both from Coherent Inc., Santa Clara, California). The scanning speed of the FV300 was controlled externally by a function generator (AFG310, Tektronix Inc., Beaverton, Oregon).

Fluorescence photons were detected in a non-descanned mode by a photon-counting photomultiplier (H7422P-40; Hamamatsu Photonics K.K., Hamamatsu, Japan). Time-resolved detection was conducted by the single-photon-counting SPC-830 printed circuit board (Becker & Hickl GmbH, Berlin, Germany). A bandpass filter of 450±40nm (Edmund Optics Inc., Barrington, New Jersey) was inserted in the emission path for the detection of NADH that emits with the maximum at 450nm .35 Additional short pass and IR cutoff filters were used to reject reflected or scattered excitation light at 750nm . The average laser power measured at the focal plane of the objective was 3to5mW , which was lower than the reported laser power of two-photon damage36 and was found to be optimal for the prevention of photobleaching. All the images were taken at 256×256pixels resolution with the acquisition time in the range of 900to1800s for enough photon count statistics at the given laser power for further data analysis. To compare the emitted fluorescence intensity between controls and treated cells, we adapted the photon count from the peak time channel (and with fixed binning parameters) of the recorded decay curve at the brightest point of a FLIM image.


FLIM Data Analysis

Data were analyzed with the commercially available SPCImage version 2.8 software package (Becker & Hickl GmbH) via a mathematical convolution of a model function and the instrument response function (IRF), and fitting to the experiment data. To calculate the lifetime from the composite decays of NADH, we convolved an IRF, Iinstr , with a double-exponential model function, defined in Eq. 1, with offset correction for the ambient light and/or dark noise I0 to obtain calculated lifetime decay function Ic(t) in Eq. 2

Eq. 1


Eq. 2

Here, a1etτ1 and a2etτ2 represent the contributed fluorescence decays from free and bound NADH, respectively; τ1 and τ2 represent their corresponding lifetimes; and a1 and a2 are the corresponding relative amplitudes. Iinstr was calculated automatically by SPCImage from the rising edge of the fluorescence decay.

The model parameters (i.e., ai and τi ) were derived by SPCImage software by fitting the calculated decay Ic(tk) , defined in Eq. 2, to the actual data Ia(tk) through minimizing the goodness-of-fit χR2 function defined in Eq. 3 using the Levenberg-Marquardt search algorithm

Eq. 3

Here n is the number of the data (time) points (equal to 256 in this study), and p is the number of the model parameters.37




NADH Fluorescence Intensity and Lifetime Images of Intact Cells

Figure 1 shows representative images, fitting curves, and lifetime distribution from control 143B cells. It includes a fluorescence intensity image [Fig. 1a], fluorescence lifetime image [Fig. 1b] using two-photon FLIM, the corresponding fluorescence decay curves (measured: blue; fit: red), the fitting residuals [Fig. 1c], and normalized lifetime histograms [Fig. 1d] of the 256×256 lifetime image as shown in Fig. 1b. This entire image was acquired over a period of 1300s . In control cells, the average photon count collected over the given period of time was approximately 600 in the peak time channel.

Fig. 1

Measurement of the intensity and average lifetime of NADH fluorescence in 143B cells. The intensity and average lifetime images of NADH fluorescence in 143B cells are shown in gray (a) and color scale (b), respectively, using two-photon FLIM with the excitation wavelength at 750nm . The FOV of each image is 100×100μm . The scale bar in (a) is 20μm . A representative fluorescence lifetime decay curve (blue curve in the top panel), the fit (red curve in the top panel), and the residuals (bottom panel) are shown (c). The normalized histograms over the 256 x 256 pixel lifetime image shown in (b) were plotted for τ1 , τ2 , and τave (d).


Figure 1a shows that the fluorescence signals in the cytoplasm displayed punctuate perinuclear patterns, which were attributed to mitochondria-associated NADH.19, 38 No fluorescence was seen in the nuclei or on the nuclear membrane. In Fig. 1b, each pixel represents the average lifetime (τave) of the short (τ1) and the long (τ2) lifetime components weighted by their relative contributions a1 and a2 , respectively, such that τave=(a1τ1+a2τ2)(a1+a2) . The scale of the color mapping was chosen for later comparison with FLIM images of treated cells so that the blue color represents the longer lifetime (maximum 4000ps ) and the red color presents the shorter lifetime (minimum 200ps ). Overall, the NADH fluorescence in the control 143B cells exhibited an average lifetime (τave) of 1300ps , which corresponds to the yellowish color in the selected color scale. The lifetime across the entire FOV or even within a single cell was not homogeneous: some pixels tend to be greenish and some tend to show orange color. Figure 1c demonstrates the fitting curve obtained from Fig. 1b, with the goodness-of-fit residuals shown in the bottom panel of Fig. 1c. Finally, the distribution of the averaged lifetime (τave) from all pixels shown in Fig. 1b is presented in Fig. 1d, which depicts the normalized histogram (Hnorm) of τave . The histogram shows a peak (τpeak) at 1300ps with a full width at half maximum 500ps . This broad distribution illustrates the inhomogeneous color distribution observed in Fig. 1b. To compare with previously published results regarding the values of the NADH short (τ1) and long (τ2) lifetime components, we obtained τ1 and τ2 from the entire image pixels by SPCImage software and plotted their normalized histogram in the same figure [Fig. 1d]. The histograms of τ1 and τ2 showed peaks at 600 and 3000ps , respectively. These results are close to the previously published lifetime values of free (400to500ps) and bound NADH (2000to3000ps) .22, 23 The observation that τ2 had a broader distribution than τ1 may be attributed to the wide distribution of lifetimes of NADH molecules bound to different proteins.39, 40

We acquired NADH lifetime images from a total of 35 sites of control HeLa cells (from 15 coverslips) and 11 sites of control 143B osteosarcoma cells (from 8 coverslips). The values of τpeak from all of the lifetime images were analyzed, recorded, and averaged. Table 1 summarizes the total number of sites (Nsite) and the corresponding mean±standard error (SE) of the τpeak values from control HeLa cells and control 143B cells. The results showed that HeLa and 143B cells had the average τpeak values equal to 1360±24 (N=35) and 1260±49ps (N=11) , respectively. The two mean values are not significantly different as judged by two-tailed Student’s t test (pvalue=0.09) . The mean value of all control τpeak values is 1336±22ps , which will be used later in Figs. 4 and 6 for comparison.

Table 1

Summary of the number of sites (Nsite) of NADH lifetime images acquired from the number of coverslips (Ncoverslip) in control, STS-treated, and H2O2 -treated cells. The number of sites is the same as the number of coverslips in treated cells because of time sequence measurements at the same site. The corresponding means (±SE) of the peaks (τpeak) of the average lifetime (τave) distributions were calculated and listed with the unit of picoseconds (ps). τpeak was defined as the peak position (or the maximal value) of the τave histogram from each 256×256 pixel lifetime image. For STS-treated cells, only the lifetime at the first time point (0to15min) was listed. For H2O2 -treated cells, the lifetimes at both 0 to 15 and 0to25min after treatments ( Nsite at each time point is 2) were averaged and listed to account for a larger number of the samples for averaging.

CellsControls 0to15Min After STS Treatment 0to25Min After H2O2 Treatment
HeLa Nsite (Ncoverslip) 35 (15)6 (6)4 (4)
Mean±SE 1359±24 3570±56 1514±51
143B Nsite (Ncoverslip) 11 (8)5 (5)N/A
Mean±SE 1260±49 3448±76 N/A


Cell Morphological Change and Caspase 3 Activation Confirmed STS-Induced Apoptosis and H2O2 -Induced Necrosis

Although STS-induced apoptosis and high dose H2O2 -induced necrosis have been well documented, we observed the cell morphology and measured caspase 3 activity to confirm the cell death pathway at the drug dose we chose. Figure 2a shows the light microscopic images of HeLa cells 15min and 5h after either H2O2 or STS treatment. All the images were taken at the same magnification. Cell swelling or eruption, a typical feature when cells are under necrotic pathway, was observed in both 15min and 5h after H2O2 treatment. In contrast, the typical feature of apoptotic cells, cell shrinkage, was observed in STS-treated cells. Figure 2b shows the normalized caspase 3 activities of HeLa cells before (controls), 15min after, and 2h after either H2O2 or STS treatment. Caspase 3 activity was significantly higher at 2h after STS treatment compared with controls, indicating that STS-treated cells were undergoing apoptosis. However, no significant increase of caspase 3 activity was observed in cells at 15min or 2h after H2O2 treatment.

Fig. 2

The morphology (a) and normalized caspase 3 activity (b) of HeLa cells after treatment with H2O2 and STS, respectively. The FOV of each image is 180×180μm . The scale bar in (a) is 30μm .



NADH Fluorescence Lifetime Increased Immediately after STS-Induced Apoptosis

Figure 3 shows the representative FLIM images (in color mapping) before, 0 to 15, 30 to 45, and 60to75min after 1μM STS treatment at the same FOV of HeLa cells. All the figures are displayed using the same scale of color bar as in Fig. 1b that the minimal and maximal lifetime is 200ps (red) and 4000ps (blue), respectively. Similar to Fig. 1b, control HeLa cells [Fig. 3a] had a1300ps lifetime (yellowish image in the selected color scale). The acquisition time of this control image was 900s or 15min . Immediately after STS treatment, we continuously acquired FLIM images with the acquisition time of 15min per image for up to 90min (the images acquired at 15 to 30, 45 to 50, and 75to90min after treatment are not shown) at the same site to trace the response of the specimen. Apparently, the color-coded lifetime image exhibited blueshift (increased lifetime) at 0to15min [Fig. 3b] after treatment, and then exhibited redshift at 60to75min [Fig. 3d]. The nuclear areas tended to decrease in size with the increment of treatment time. A ringlike structure surrounding the nucleus [arrow head in Fig. 3c] was observed. In addition, the overall intensity of NADH fluorescence significantly increased immediately after STS treatment so that the laser power was decreased to escape the photomultiplier tube saturation. The recorded average photon count for the STS-treated cells was more than 1000, approximately twice higher than that in controls (600 counts), in the peak time channel of the recorded decays.

Fig. 3

Effect of staurosporine treatment on the FLIM images of HeLa cells. The FLIM images of HeLa cells from the same sites before (a), 0to15min (b), 30to45min (c), and 60to75min (d) after treatment with 1 μM STS. The FOV of each image is 100×100μm . The scale bar in (a) is 20μm .


The distributions of the average lifetime (τave) [Figs. 3a, 3b, 3c, 3d] were plotted on the top panel of Fig. 4 . The curve labels (a) to (d) correspond to the results of Figs. 3a, 3b, 3c, 3d, respectively. An immediate lifetime increase after STS treatment was observed that the histogram shifted from the lower lifetime [curve (a)] to the higher lifetime [curve (b)] values. Then, the lifetime decreased and the histogram shifted toward the controls and became broader. The peak (i.e., maximal) value of τave histograms, τpeak , increased from 1300ps [controls, curve (a)] to 3500ps at 0to15min after STS treatment [curve (b)], and then decreased to 2200ps at 60to75min .

Fig. 4

Effect of staurosporine treatment on the distributions of the average lifetime (τave) of the FLIM images of HeLa and 143B cells. The top panel shows the plot of the distributions of the average lifetime (τave) corresponding to the FLIM images in Figs. 3a, 3b, 3c, 3d. The bottom panel shows the plot of the mean and standard error (SE) of the τpeak as the function of time in all STS-treated cells. The number of sites acquired at each time point was labeled below each data point. The mean τpeak value (±SE) of controls (i.e., 1336±22ps ) was plotted (star) in the same figure.


Similar results were repeatedly observed in a total of 6 sites (6 coverslips) of HeLa cells and 5 sites (5 coverslips) of 143B cells and are summarized in the bottom plot of Fig. 4. Here the number of sites is the same as the number of coverslips because time sequence measurements were performed at the same site of cells. The bottom panel of Fig. 4 plotted the mean and standard error (SE) of the τpeak at all time points over all STS-treated HeLa and 143B cells. The number of sites acquired was decreased as a function of time and was labeled below each data point in the figure. The time point was assigned as the mean value of the acquisition period of time. For example, 7.5min represents the data point acquired at 0to15min after treatment, except that the latest point was labeled as 600to675min . The mean τpeak value (±SE) of all controls (i.e., 1336±22ps ) was plotted (star) in the same figure for comparison. The mean τpeak values (±SE) for HeLa and 143B cells at 0to15min after STS treatment are listed in Table 1. The mean τpeak values at 0to15min after STS treatment for HeLa or 143B cells are significantly different from those values of the controls ( pvalue=1.4×1016 ). The two mean values of HeLa and 143B cells (i.e., 3570±56 versus 3448±76 ) are not significantly different (pvalue=0.23) .


No NADH Fluorescence Lifetime Change in High Dose H2O2 -Induced Necrosis

Figure 5 shows the representative FLIM images (in the same color mapping as previous FLIM images of controls and STS-treated cells) before, 0 to 15, 30 to 50, and 50to70min after treatment with 1mM H2O2 , taken from the same FOV of HeLa cells. In contrast to the FLIM images of STS-treated cells shown in Fig. 3, all images in Fig. 5 tend to be yellowish in the same selected color scale, which indicates that there was no significant lifetime change after H2O2 treatment. The fluorescence intensity or photon counts tended to decrease after H2O2 treatment [as seen in the changes of signal-to-background noise from Figs. 5a, 5b, 5c, 5d], which in part is due to the NADH oxidation16 by H2O2 . The recorded average photon count for the H2O2 -treated cells was 100 to 200 (i.e., 3 to 6 times lower than that of controls) in the peak time channel.

Fig. 5

Effect of H2O2 treatment on the FLIM images of HeLa cells. The FLIM images of HeLa cells were recorded from the same sites before (a), 0to15min (b), 30to45min (c), and 45to60min (d) after treatment of the cells with 1mMH2O2 . The FOV of each image is 100×100μm . The scale bar in (a) is 20μm .


The top panel of Fig. 6 plotted the normalized histogram of τave of NADH lifetime images shown in Figs. 5a, 5b, 5c, 5d. The curves (b) to (d) overlapped with one another. There is slight difference between curve (a) and curves (b) to (d). Similar to the bottom panel of Fig. 4, the bottom panel of Fig. 6 plotted the mean error and standard error of the τpeak at all time points over all H2O2 -treated HeLa cells. The time point was assigned the same way as in Fig. 4 except the latest time point labeled as 135to185min . The mean τpeak value (±SE) of controls (i.e., 1336±22 ) was plotted (star) in the same figure for comparison. The number of sites acquired at each time point was labeled below each data point in the figure. We observed no significant difference in τpeak at all time points. To get more data points for averaging and comparing with the results of controls and apoptotic cells in Table 1, the mean τpeak values (±SE) for both time points at 0to15min (N=2) and 0to25min (N=2) after treatment were calculated and listed in Table 1. The mean τpeak values at 0to25min after H2O2 treatment in HeLa cells are marginally different from the values of control HeLa cells (pvalue=0.05) .

Fig. 6

Effect of H2O2 treatment on the distributions of the average lifetime (τave) of the FLIM images of HeLa cells. The top panel plotted the distributions of the average lifetime (τave) corresponding to the FLIM images in Figs. 5a, 5b, 5c, 5d. The bottom panel plotted the mean and standard error (SE) of the τpeak as a function of time in all H2O2 -treated cells. The number of sites acquired at each time point was labeled below each data point. The mean τpeak value (±SE) of the controls (i.e., 1336±22ps ) was plotted (star) in the same figure.




In live cells, NADH, reduced NADH phosphsate (NADPH), and flavins [or flavin adenine dinucleotide (FAD)] are intrinsic fluorophores that have sufficient concentration to yield detectable fluorescence signals under two-photon excitation at 740nm .39 The flavin signals that emit fluorescence maxima at 550nm can be spectrally separated from NAD(P)H signals that emit fluorescence maxima at 450nm . Although NADH and NADPH have nondiscriminating spectral features,41 NADH dominates the intrinsic fluorescence of live cells because the contribution of NADPH to the intrinsic fluorescence is small42 and the quantum yield of mitochondrial NADH is 1.25 to 2.5 times higher than that of NADPH.43 In this study, we used a 450±40nm bandpass emission filter to selectively collect the NADH fluorescence signals, although we used two-photon excitation at 750nm , instead of 740nm used in other published studies, due to our instrument limitation. Recently, Wu 20 reported that increase of flavin fluorescence contributed to both lifetime components ( τ1 and τ2 ) and the ratio of the corresponding amplitudes (a1a2) in the fluorescence decay when the single photon excitation wavelength increased from 365to405nm (equivalent to wavelengths from 730to810nm in two-photon excitation) and when a 10nm bandpass emission filter (λem=447to457nm) was used. In their study, the τ1 , τ2 , and a1a2 of control SiHa cells were found to change, not consistently increase or decrease, ∼6% to 12%. However, Skala 44 showed that there was no measurable fluorescence at 900nm excitation, which is optimal for the two-photon excitation of flavins,35 with their two-photon FLIM system, neither did FAD contribute to their observed lifetime at 780nm excitation in the hamster cheek pouch. Thus, we concluded that the flavin fluorescence contribution to our results using two-photon excitation wavelength at 750nm was negligible.

To confirm that the difference in lifetime changes between controls and STS and H2O2 treatments is not due to long image acquisition time (15to20min) and/or room temperature condition, we performed additional FLIM measurements using shorter acquisition time (5to10min) and/or at the physiological temperature maintained in the microscope cage incubator (35°Cto37°C) . Similar results of the NADH lifetime values, with lower photon counts and thus noisier τave histogram plots, have been observed using 100sto10min acquisition times in controls, STS-treated, and H2O2 -treated cells (data not shown). Table 2 lists the NADH lifetime acquired under physiological condition and using short image acquisition time (100s) , respectively, in control and STS-treated cells. From 4 measurements of HeLa cells taken using shorter acquisition time (100s) , lifetime increased from 1396±17ps before to 3402±83ps at 0to15min after treatment. From measurements of both HeLa ( Nsite=3to4) and 143B ( Nsite=1to3) cells taken at the physiological temperature, we observed the same trends of changes in lifetime and intensity in STS-treated cells including increased τave from 1306±91 (HeLa) and 1165 (143B) ps before to 3544±103 (HeLa) and 3545±31 (143B) ps at 0to15min after treatment, decreased τave thereafter, increased NADH fluorescence intensity after treatment (indicated by using lower input laser power and detecting higher photon counts after STS treatment), shrinkage of nuclei, and the appearance of perinuclear rings [arrowhead in Fig. 3d].

Table 2

The NADH lifetime acquired under physiological condition and using short image acquisition time (100s) , respectively, in control and STS-treated cells. The corresponding means (±SE) of the peaks (τpeak) of the average lifetime (τave) distributions were calculated and listed with the unit of picoseconds (ps). τpeak was defined as the peak position (or the maximal value) of the τave histogram from each 256×256 pixel lifetime image. For STS-treated cells, only the lifetime at the time point 0to15min was listed.

Cells Controls 0to15Min After STS Treatment
PhysiologyShort ImageAcquisitionPhysiologyShort ImageAcquisition
HeLa Mean±SE (Nsite) 1306±91 (4) 1396±17 (4) 3544±103 (3) 3402±83 (4)
143B Mean±SE (Nsite) 1192 (1) 3545±31 (3)

Similar results of increased NADH fluorescence intensity were reported by Levitt 45 and by Liang 46 Levitt 45 observed that intense fluorescence was progressively confined to a gradually smaller perinuclear cytoplasmic region in the cells that had been treated with cisplatin. Their results suggest that this strongly fluorescent, highly metabolically active perinuclear ring represents a subpopulation of mitochondria that are mobilized in response to the apoptotic stimulus and may provide the energy required to execute the final step of apoptosis.

The increase in the average lifetime of NADH fluorescence after STS-induced apoptosis (Fig. 3) may be attributed to two possible mechanisms based on the lifetime distribution analysis. One mechanism is that the portion of bound NADH to total amounts of NADH is higher than that of free NADH [i.e., a2(a1+a2)> a1(a1+a2) such that τave=(a1τ1+a2τ2) (a1+a2) increased] assuming their lifetimes (i.e., τ1 and τ2 ) remain the same. The other mechanism is that the bound NADH lifetime τ2 increases, assuming that the free NADH lifetime τ1 does not change, due to microenvironment changes that NADH binds to different enzymes during apoptosis. We examined the histograms of τ1 and τ2 in several FLIM images of STS-treated cells at all time points and found that both mechanisms are possible (data not shown). For example, at 0to15min after STS treatment, the histogram of τ1 , τ2 , and τave are almost identical [curve (b) in Fig. 4] that single exponential fit was applicable in this case. This indicates that all free NADH (i.e., the short lifetime component) became a bound form (i.e., the long lifetime component). At later time points after STS treatment, some τ2 histograms became broader (in both longer and shorter lifetime directions), which indicates that some NADH may bind to different enzymes to broaden the lifetime distribution. Vishwasrao 39 reported three types of enzyme-bound NADHs using four component analyses in hippocampal slices. They found that the ratio between free NADH and individual enzyme-bound species of NADH changed significantly under hypoxia, which indicates a redistribution of the protein-bound NADH to enzyme binding sites.

The STS-induced apoptotic pathway has been well established to be due to internal signals and involve mitochondrial membrane potential (ΔΨ) change, cytochrome c release, caspase 3 activation, and production of reactive oxygen species.24, 25, 26 In this study, we observed increased NADH fluorescence lifetime at relatively early time points (within 15min after 1μM STS treatment) as compared with the activation of caspase 3 at 2h after 1μM STS treatment [Fig. 2b] and cytochrome c release and the caspase 3 activation at 2to4h after higher dose (4μM) STS treatment as described in the study by Maeno 29 Thus we have hypothesized that the immediately increased NADH lifetime may be related to early apoptotic activities. Annexin V was used to indicate early apoptosis47, 48 to stain the exposure of phosphatidylserine (PS) on the external leaflet of the plasma membrane. We performed annexin V fluorescence imaging (data not shown) and observed that annexin V binding, quantified by averaging binding percentages over five different 100×100μm FOVs, appeared at 15min after 1μM STS treatment (binding percentage<2% ). The binding percentage increased as time increased to be 4% and 50% at 1h and 5h after STS treatment, respectively. Lugli 48 showed that the appearance of PS exposure (annexin V–positive) was correlated to intermediate ΔΨ in quercetin-induced apoptosis of U937 cells, which could be important to maintain adenosine triphosphate (ATP) production for the activation of caspases and induction of cell death. Halestrap, 49, 50 showed that the mitochondrial permeability transition pore (MPTP) opening can be transient and thus does not cause ATP depletion, and then the cell dies by apoptosis. If the MPTPs stay open, ATP is depleted and the cells die by necrosis.50 Furthermore, it was demonstrated that the cytosolic ATP level was increased in HeLa cells immediately after treatment with 4μM STS.51 It remains unclear whether our observations that NADH lifetime increased shortly after apoptosis and NADH redistribution are related to NADH binding to different proteins in mitochondria for more efficient generation of ATP in the target cells. Further studies are needed to reveal the relationship between NADH lifetime dynamics, mitochondrial membrane potential, MPTP opening, and ATP production/maintenance in the early phase of apoptosis.



Nicotinamide adenine dinucleotide is a principal electron and proton donor in mitochondria, which are known to be one of the key regulators in cell death particularly in apoptosis. We have demonstrated that NADH fluorescence lifetime significantly changed immediately after 1μM STS-induced apoptosis, but not 1mM H2O2 -induced necrosis in HeLa and 143B osteosarcoma cells. This increased lifetime was in the early phase of apoptosis and was in part due to NADH redistribution from free to bound form in mitochondria possibly for more efficient generation of ATP in the target cells. Our findings suggest that the NADH lifetime changes may be a valuable noninvasive marker for the detection of apoptosis.


We acknowledge the imaging core facility of National Yang-Ming University, Professor Der-Ming Yang who provided culture chamber, and Professors Britton Chance, Chi-Hung Lin, and Arthur Chiou for valuable discussions. This work was supported by a new faculty start-up fund from National Yang-Ming University, the “Aim for Top University Plan” from the Ministry of Education of Taiwan, and Grant Nos. NSC 94-2321-B-010-004-YC and NSC 95-2112-M-010-002 from the National Science Council of Taiwan.



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©(2008) Society of Photo-Optical Instrumentation Engineers (SPIE)
Hsing-Wen Wang, Vladimir Ghukasyan, Chien-Tsun Chen, Yau-Huei Wei, Han-Wen Guo, Jia-Sin Yu, and Fu-Jen Kao "Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells," Journal of Biomedical Optics 13(5), 054011 (1 September 2008).
Published: 1 September 2008

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