journal of photochemistry and photobiology b:...

Journal of Photochemistry and Photobiology B: Biology 95 (2009) 46–57

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Two-photon autofluorescence dynamics imaging reveals sensitivityof intracellular NADH concentration and conformation to cell physiologyat the single-cell level

Qianru Yu a, Ahmed A. Heikal a,b,*

a Department of Bioengineering, The Pennsylvania State University, 231 Hallowell Building, University Park, PA 16802, USAb The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 August 2008Received in revised form 17 December 2008Accepted 17 December 2008Available online 25 December 2008

Keywords:NADHHs578THs578BstTwo-photon FLIMAssociated anisotropyDehydrogenase

1011-1344/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2008.12.010

* Corresponding author. Address: Department of Binia State University, 231 Hallowell Building, UnivTel.: +1 814 865 8093; fax: +1 814 863 0490.

E-mail address: [email protected] (A.A. Heikal).

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphor-ylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrin-sic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancerpathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarizationimaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative anal-ysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathologyand inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developednon-invasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 lM) to be�1.8-fold higher than in breast normal cells (99 ± 37 lM). Such analyses indicate changes in energymetabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activityusing KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates popu-lation fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADHin normal breast cells. These fractions are statistically different from those in breast cancer cells (free:0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochon-drial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells.These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than inten-sity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer,diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in livingcells, without the need for cell destruction as in conventional biochemical assays.

� 2009 Elsevier B.V. All rights reserved.

1. Introduction

Reduced nicotinamide adenine dinucleotide, NADH, is an essen-tial cofactor for oxidation–reduction (redox) reactions and energymetabolism in living cells [1–3]. In the cytoplasm of eukaryoticcells, glycolysis involves ten reactions that include the reductionof NAD+ during the oxidation of glyceraldehyde 3-phosphate,which is catalyzed by glyceraldehyde 3-phosphate dehydrogenase.The net transformation reaction of one glucose molecule into twomolecules of pyruvate includes the generation of two NADH andtwo adenosine triphosphate (ATP) molecules [3]. Under aerobicconditions, the high-energy electrons from the cytosolic NADHare shuttled to the mitochondria using glycerol-3-phosphate and

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malate-aspartate shuttles [3,4]. In the electron transport chain(ETC) of the inner membrane of mitochondria, NADH (fluorescent)is oxidized to NAD+ (not fluorescent), which eventually leads to themajority of ATP production via the oxidative phosphorylationpathway [1–3]. Under anaerobic conditions, however, NAD+ isregenerated by the reduction of pyruvate to lactate, which is cata-lyzed by lactate dehydrogenase (LDH) [5]. Anaerobic glycolysis isoften predominant in tumors and causes elevated levels of lacticacid as well as increased LDH activity [6]. It has also been shownthat cancer cells exhibit impaired mitochondrial metabolism,which skews the activities of key enzymes such as LDH and mito-chondrial malate dehydrogenase (mMDH) [5,7,8]. The concentra-tion and distribution of intrinsic NADH in living cells aresensitive to cell physiology [9,10] and pathology [11]. As a result,there is a great potential for cellular NADH as a natural biomarkerfor a range of cellular processes such as apoptosis [12], redox reac-tions [12,13], and mitochondrial anomalies associated with cancer[1,5,14,15] and neurodegenerative diseases [16]. Conventional bio-

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Q. Yu, A.A. Heikal / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 46–57 47

chemical methods have provided the bulk of information concern-ing NADH concentration in cell lysates as a snapshot of the meta-bolic and redox state of cells, but without morphological context[11,12,17,18].

Contrary to biochemical techniques, however, fluorescence-based approaches provide a non-invasive alternative for autofluo-rescence imaging in live cells or tissues. The pioneering work ofBritton Chance has opened the door for using cellular autofluores-cence as a biomarker for respiratory activities and mitochondrialfunctionality using 360 nm excitation [19–21]. Further studieshave shown a blue shift in NADH emission as a result of bindingwith proteins in several cell lines, including keratinocytes [22],bronchial tissues [23], and brain slices [9]. Unfortunately, such ashift is difficult to use as an indicator for quantitative analysis offree and bound NADH due to the broad spectral profile of NADH[9]. It is worth mentioning that reduced nicotinamide adeninedinucleotide phosphate (NADPH) is used almost exclusively forreductive biosynthesis of fatty acids and steroids, whereas NADHis used primarily for ATP generation [3,12]. Unfortunately, it is dif-ficult to differentiate between NADH and NADPH spectroscopicallydue to their similar photophysical properties. However, some evi-dence exists that intracellular NADH levels are higher than that ofNADPH [17,24]. There are some inconsistencies, however, concern-ing the sensitivity of these pyridine nucleotides autofluorescenceto cell pathology as a function of experimental techniques and celllines [25,26]. While the use of one-photon excitation is a commonpractice for fluorescence microscopy [27], it suffers from extendedphotobleaching, scattering, and limited penetration depth in turbidbiological samples [28]. In addition, ultraviolet (UV) excitation ofNADH and tryptophan residues in proteins may cause DNA muta-tions [29] and cellular photodamage [30]. Multi-photon micros-copy overcomes some of these challenges [31–37], with the two-photon (2P) excitation cross-section of NADH reported recently[38–40]. Currently, 2P-fluorescence microscopy of intrinsic NADHhas been used to monitor energy metabolism in macrophages, pan-creatic islet cells, skeletal muscle cells [41–43], brain slices [9,40],and cardiomyocytes [38].

Fluorescence lifetime imaging microscopy (FLIM) is sensitiveto the conformational changes and surroundings of a fluorophore[44]. For example, frequency-domain FLIM has been shown to besensitive to free and enzyme-bound NADH conformations insolution [45]. Recently, 2P-FLIM studies of human breast cell(MCF-10A) [10] and hair cells in isolated cochlear preparations[46] were reported for monitoring the metabolic activities usingcellular autofluorescence. However, it is generally difficult to as-sign multiple exponential fluorescence decays to specific molec-ular origins [47]. To overcome such challenges, Vishwasraoet al. used a combination of time-resolved fluorescence andanisotropy measurements of intrinsic NADH in brain slices tomonitor cellular response to hypoxia [9]. Using sample scanningmode, free and three enzyme-bound species of intrinsic NADHwere identified in brain tissues to explain the observed multiex-ponential fluorescence decays and associated anisotropies [9].Tissues, however, are more complex environment that may in-clude other fluorescent species such as collagen and elastin[36,48]. In addition, imaging throughout thick tissues may sufferfrom scattering and other optical artifacts due to refractive indexmismatch [49], which may influence polarization anisotropyimaging.

In this report, a non-invasive two-photon fluorescence dynam-ics assay is used to convert intracellular NADH fluorescence to ac-tual concentration as well as molecular conformation (i.e., free andenzyme-bound fluorescence fractions) in living cells. In this assay,2P-FLIM is used to quantify the fluorescence quantum yield varia-tion within individual cells. In addition, 2P-fluorescence anisotropyimaging is used to obtain the population fractions of free and en-

zyme-bound NADH using cellular autofluorescence. Human nor-mal (Hs578Bst) and cancerous (Hs578T) breast cells are used as amodel system with the epithelial Hs578T cell line is derived froma rare infiltrating ductal carcinoma of a 74-year-old female patient.Unlike most breast cancer cell lines, which express estrogen recep-tors, Hs578T represents the scarce transformed breast cell linesthat are estrogen receptor negative. The corresponding myoepithe-lial normal cells were obtained from the same patient at a locationperipheral to the tumor [34]. In addition to cell pathology, thefunctional response of cellular NADH to potassium cyanide (KCN)is also examined in the normal Hs578Bst cells. As a control, thebinding kinetics of NADH with both mMDH and LDH, in solution,is also investigated using the same assay. These comparative stud-ies in solution, under controlled NADH–enzyme mixing, enabled usto (i) understand the observed differences between the intracellu-lar NADH fluorescence lifetime, as compared with the free cofactorin solution, (ii) mimic observed associated anisotropy of intracellu-lar NADH, and (iii) directly quantify the fluorescence fraction ofintracellular free and enzyme-bound NADH using the inherent dif-ferences in their molecular sizes.

2. Materials and methods

2.1. Cell culture and treatment

Breast cancer cell line (Hs578T), its non-transformed counter-part (Hs578Bst), and the recommended culture media were ob-tained from American Type Culture Collection (ATCC). Cancercells were grown in Dulbecco’s modified eagle’s medium (DMEM)with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (PS). Normal cells were grown in modified DMEM,supplemented with 10% (v/v) FBS, 1% (v/v) PS and 30 ng/mL epider-mal growth factor (Sigma). Cells were cultured in T-75 flasks (BDBiosciences) in a 37 �C incubator with 5% CO2 and allowed to reach70–90% confluence before passage. Cells were plated into glass bot-tom Petri dishes (MatTek Corporation) and incubated overnightbefore imaging. Tyrodes buffer (135 mM NaCl, 5 mM KCl, 1 mMMgCl2, 1.8 mM CaCl2, 20 mM HEPES and 5 mM glucose) was usedto replace the media and to wash the cells three times prior toimaging of intracellular NADH autofluorescence. Cell morphologywas monitored before and after each measurement using differen-tial interference contrast (DIC) microscopy to ensure cell integrity.Both normal and cancer cells underwent a maximum of ten pas-sages. To monitor the drug-induced fluorescence change of NADHin normal cells, the electron transport chain inhibitor, potassiumcyanide (KCN, Fluka), was dissolved in phosphate buffered solu-tion, PBS (pH 7.4), at a final concentration of 5 mM in the Petri dishwhere the cells were incubated and imaged. 2P-FLIM images wererecorded subsequently after �8 min following KCN application.

Rhodamine 123 (Rh123; Invitrogen) was used as a mitochon-drial marker [50] to examine cell morphology and mitochondrialdistribution of breast cancer and normal cells. For our solutionstudies, free NADH was purchased from Sigma without furtherpurification. NADH was dissolved in Tris buffer (100 mM, pH 8.5),and stored as a stock solution of 500 lM. Both mMDH and L-LDH, purchased as crystalline suspensions in ammonium sulfatesolution from Sigma, were dialyzed three times against Tris buffer(100 mM, pH 8.5) at 4 �C, and then concentrated by centrifugation.Concentrations of LDH and mMDH were determined by measuringabsorbance in a UV–vis spectrophotometer (DU800, BeckmanCoulter). The extinction coefficients (at 280 nm) are7.2 � 104 M�1 cm�1 (mMDH) and 16.3 � 104 M�1 cm�1 (LDH),respectively [51]. NADH concentrations of 226 lM (for mMDH)and 183 lM (for LDH) were titrated with variable enzyme concen-tration to ensure detectable NADH fluorescence signal.

48 Q. Yu, A.A. Heikal / Journal of Photochemistry and Photobiology B: Biology 95 (2009) 46–57

2.2. Confocal and two-photon fluorescence micro-spectroscopy

The experimental setup and data analysis were described indetail elsewhere [52]. Briefly, time-resolved fluorescence lifetime(at magic angle of 54.7�) and anisotropy experiments were per-formed using a femtosecond (�120 fs at 76 or 4.22 MHz,740 nm) laser system (Coherent) consisting of a solid-state laserpumped with a 10-W diode laser. Laser pulses were steered to-ward a modified laser scanning confocal system (Olympus) beforebeing focused on the sample using a dichroic mirror (670DLPC)and a microscope objective (60�, 1.2NA, water immersion). Theepi-fluorescence was detected simultaneously using two micro-channel plate photomultiplier tubes (R3809U-50; Hamamatsu)following a beam splitter, Glan–Thompson polarizers, and filters(690SP and BGG22: 350–550 nm filter, Chroma). The detected sig-nals were amplified and then fed into a single photon countingmodule (SPC830; Becker & Hickl). Data acquisition and non-linearleast-square fitting analysis were carried out using SPCImage(Becker & Hickl).

2.3. Quantitative calculation of intracellular NADH concentration

The protocol for monitoring extrinsic fluorophore concentrationin vivo with 2P-FLIM and intensity images was reported earlier[53]. In brief, the time-averaged 2P-fluorescence from a given pixel(x,y), hF2P(x,y)i, is defined as [37,39]:

F2Pðx; yÞh i ¼8nk3Uðx; yÞnðkÞCðx; yÞr2Pgp IðtÞh i2

2RpspðpNAÞ3; ð1Þ

where U and C are the fluorescence quantum yield and concentra-tion of the fluorophore in a given pixel, respectively. The observedsignal is also dependent on detection efficiency (nðkÞ) and the 2P-excitation cross-section (r2P) of a fluorophore, as well as the exci-tation pulses (full width half maximum, sP, and repetition rate RP,and the time-averaged laser intensity hI(t)i). The dimensionless gP-factor depends on the temporal profile of the laser pulse (e.g.,gP = 0.66 for a Gaussian pulse shape) [37]. The spatial profile oftwo-photon pulsed excitation can be written as 8nðk=pNAÞ3 underthick-sample approximation [39]. The detection efficiency andother system parameters can be difficult to quantify. Thus, we usedfree NADH at a known concentration as a reference for quantita-tively imaging the spatial distribution of NADH concentration inliving cells. Because the fluorescence quantum yield is linearly pro-portional to the fluorescence lifetime (sfl) in a given pixel(U(x,y) = krsfl(x,y), where kr is the radiative rate constant), thedependence of the 2P-fluorescence signal on concentration canbe simplified as:

F2Pðx; yÞh i ¼ sflðx; yÞwðx; yÞCðx; yÞ; ð2Þ

where the system parameter, wðx; yÞ ¼ 4nk3krnðkÞr2PgpI2=

p3NA3RPsP , can be cancelled out using the free NADH in solutionwith a known concentration as a reference, such that [53]:

CcellNADHðx; yÞ ¼

Fcell2P ðx; yÞ

Fsol2Pðx; yÞ

ssolNADH

scellNADHðx; yÞ

CsolNADH: ð3Þ

For simplicity, we assume that the sensitivity of the radiativerate constant (kr) to the cellular environment is negligible, eventhough a slight blue spectral shift of NADH has been observed uponenzyme binding [9]. The accuracy of this approach can be furtherimproved by considering the refractive index variation throughoutlive cells [54,55], as well as accurate knowledge of the two-photonexcitation cross-section of both free and enzyme-bound NADH[38,40].

2.4. Data analysis

Fluorescence lifetime analysis is performed using the SPCImagesoftware package (Becker & Hickl), where the deconvoluted fluo-rescence intensity decay per pixel, F2P(x,y; t), is generally fit as[52]:

F2Pðx; y; tÞ ¼X

i

aiðx; yÞ � exp½�t=siðx; yÞ�; ð4Þ

where ai and si are the amplitude and lifetime of the ith fluores-cence component, respectively, and

Piai is normalized to unity.

For a multiexponential fluorescence decay, the average fluorescencelifetime is give by �sfl ¼

Piaisi, while the relative contribution of the

ith decay component to the total fluorescence signal can be calcu-lated as follows [47,53]:

Fiðx; y; tÞ ¼ aiðx; yÞsiðx; yÞX

i

,aiðx; yÞsiðx; yÞ: ð5Þ

All 2P-FLIM images reported here were detected at magic-angle(54.7�) polarization to eliminate rotational effects [47]. Typical2P-FLIM images were recorded using 740 nm, �120 fs pulses,76 MHz (with an average power 63 mW), 256 � 256 pixels, 64 timechannels per pixel, and 259 ps/channel. For presentation purpose,the resolution of the color-coded lifetime bar is chosen to highlightthe autofluorescence lifetime (i.e., quantum yield) heterogeneitythroughout the cell. The corresponding pixel–lifetime histogramfor each FLIM image is also provided to reflect the overall distribu-tion of average lifetime per pixel throughout the cell. DIC imageswere recorded before and after the FLIM measurements to assessthe cellular viability and photodamage, which is negligible[33,36,37] but should not be ruled completely [46]. In pseudo-singlepoint lifetime measurements, the laser pulses (4.22 MHz, averagepower 6300 lW) were scanned over the entire cell(s) and the auto-fluorescence signal was recorded in 1024 channels, with �12.2 ps/channel, and thus higher signal-to-noise ratio as well temporalresolution.

An image processing algorithm was also developed to calcu-late steady-state 2P-fluorescence anisotropy images [52,53] formapping the dipole-moment orientation of fluorescent moleculesunder local restrictions of the cellular environment. 2P-fluores-cence anisotropy, r(x,y; t), is obtained using two fluorescenceintensity images recorded simultaneously at parallel, I||(x,y; t),and perpendicular, I\(x,y; t), polarizations with respect to theelectric vector of the excitation laser, and is defined as[47,53,56]:

rðx; y; tÞ ¼ Ikðx; y; tÞ � GI?ðx; y; tÞIkðx; y; tÞ þ 2GI?ðx; y; tÞ : ð6Þ

The G-factor, which indicates the sensitivities of the detection chan-nels to polarization, is estimated using the tail-matching approach[56] with either free NADH or coumarin in solution. In these anisot-ropy images, the steady-state anisotropy per pixel, which yields theorientation angle (d) between the absorbing and emitting dipoles[47,56], is given by:

rðx; yÞ ¼ 2c3 cos2 d� 12ð2cþ 3Þ ; ð7Þ

where c is the number of excitation photons (c = 1 for 1P and c = 2for 2P) [56]. Time-resolved fluorescence anisotropy, r(t), of anensemble of non-interactive fluorophores with different excited-state lifetimes and hydrodynamic volumes, can be described as anassociated anisotropy [9,47,56,57]:

rðtÞ ¼Pm

i aie�t=si � bie�t=uiPmi aie�t=si

; ð8Þ


where the pre-exponential factor (bi) represents the initial anisot-ropy of the ith species with its size-dependent rotational time con-stant (uj). As mentioned above (Eq. (5)), the contribution of the ithspecies to the total fluorescence signal is dependent on both theamplitude fraction (ai) and fluorescence lifetime component (si).The time-resolved fluorescence in both pseudo-single point (no pix-el-to-pixel resolution) and FLIM are measured independently usingmagic-angel polarization (54.7�) detection. The associated anisot-ropy curve of a fluorophore, in two different environments, canalternatively be written as a function of its ground-state molar frac-tion [58]. In this case, one would expect that small, fast rotatingspecies (e.g., free NADH) are dominant at early times (6300 ps) ofthe associated anisotropy decay. In contrast, the large species(e.g., enzyme-bound NADH) would rotate much slower and there-fore dominate at later times during rotational diffusion. The rota-tional time of a spherically-shaped molecule is related to thehydrodynamic volume by the Stokes–Einstein equation [47,53,56]:

uðx; yÞ ¼ gðx; yÞVðx; yÞkBT

; ð9Þ

where g, V, kB and T are the viscosity of the local environment, thehydrodynamic volume, the Boltzmann constant and the absolutetemperature, respectively.

3. Results

3.1. Confocal microscopy reveals distinct morphology of breast cancercells

Confocal and DIC imaging of Rh123-stained mitochondria inboth normal and cancer breast cells (Fig. 1) were used for qualita-tive assessment of the mitochondrial distribution and cell mor-phology in both cell lines. Sub-confluent normal Hs578Bst cellsexhibit an apparently smaller nuclear-to-cytoplasmic size ratio

Fig. 1. Normal and cancerous breast cells exhibit different morphology andmitochondrial distribution. Confocal and DIC images of Rh123-stained normal(Hs578Bst) cells exhibit spindle-like in shape, with the mitochondria distributedthroughout the cell (A,B). Cancer cells (Hs578T), however, are polygonal in shapewith a peri-nuclear mitochondrial distribution (C,D). The confocal images wererecorded using 488 nm excitation and a 525/30 emission filter. The cells wereincubated with 0.5 lM mitochondrial marker Rh123 for �15 min at 37 �C andrinsed with PBS three times before imaging in Tyrodes buffer. Scale bar = 20 lM.

with a spindle-like cell shape (Fig. 1A and B), as compared withthe polygonal shaped Hs578T cancer cells (Fig. 1C and D). Cancercells also exhibit a peri-nuclear mitochondrial distribution as com-pared with normal cells that have a fibrous-shaped, inter-con-nected, and extended mitochondrial distribution.

3.2. Cellular autofluorescence exhibits a heterogeneous fluorescencelifetime (i.e., quantum yield) as revealed by 2P-FLIM

Fluorescence lifetime is a sensitive probe to changes in themolecular conformation and surrounding environment. 2P-FLIMalso allows for the conversion of a fluorescence intensity imageto a molecular concentration image by quantifying the variationof fluorescence lifetime (i.e., quantum yield) within living cells ina calibrated microscope. Care must be taken in FLIM analysis,which depends on the signal-to-noise per pixel, pixel binning,and fitting parameters such as offset and scattering contribution.To overcome these challenges, we used the same fitting procedurefor comparative FLIM analysis when possible. In addition, pseudo-single point measurements serve as a point of reference.

Typical laser scanning 2P intensity and FLIM images of normalbreast cells are shown in Fig. 2A and B, along with their pixel–life-time histogram (Fig. 2E). The corresponding 2P-FLIM image histo-

Fig. 2. Two-photon autofluorescence lifetime imaging reveals heterogeneousenvironment of the intracellular NADH, which is distinct in breast cancer andnormal cells. The 2P intensity (A,C) and FLIM (B,D) images of breast normal (A,B)and cancerous (C,D) cells reveal different autofluorescence lifetime distribution asshown in the corresponding pixel–lifetime histograms (E). The lifetime distributionhistogram of normal (E, open circle) and cancer (E, solid circle) cells can bedescribed as a double–Gaussian (see text). These images were recorded using740 nm excitation (�3 mW at the sample), 256 � 256 pixels, 120 s collection timeper image, and 690sp and BGG22 (475/150) emission filters. In these FLIM imageanalysis, similar pixel binning (3), threshold (15), scatter (<3%, floating), biexpo-nential fit, and pixel-intensity weighting were used. [See Fig. 3 and SupplementaryFig. S1 for pixel-to-pixel analyses of autofluorescence intensity and lifetime inHs578Bst and Hs578T, respectively.] Scale bar = 10 lm.


gram (Fig. 2E, open circles) reveals a heterogeneous average life-time distribution of intracellular NADH and can be fit using doubleGaussian with lifetime bands at 1.0 ns (Gaussian distributionwidth, x1 = 0.26 ns, amplitude = 4778, 53%) and 0.67 ns(x2 = 0.27 ns, amplitude = 4285, 47%). Using this approach, weestimate the average lifetime per image of normal cells to be0.9 ± 0.2 ns (n = 5). Using pixel-to-pixel (3 images, 20 pixels per im-age) analysis in 2P-FLIM images, the 2P-autofluorescence of intra-cellular NADH autofluorescence decays biexponentially (apparentmitochondrial NADH: s1 = 0.6 ± 0.1 ns, a1 = 0.71 ± 0.04, s2 = 3.4 ±0.5 ns, a2 = 0.29 ± 0.04, n = 33 pixels, and �sfl ¼ 1:3� 0:2ns) inHs578Bst cells (Fig. 3A and B). Using high resolution 2P-FLIM imag-ing, the cytosolic autofluorescence (s1 = 0.6 ± 0.1 ns, a1 = 0.75 ±0.05, s2 = 3.3 ± 0.6 ns, a2 = 0.25 ± 0.05, and �sfl ¼ 1:2� 0:1ns) alsodecays biexponentially. In both the cytoplasm and apparent mito-

Fig. 3. Pixel-to-pixel autofluorescence lifetime and intensity analyses of FLIMreveal two emitting species of intracellular NADH in normal Hs578Bst cells, whichis predominantly in the mitochondria. (A) The two emitting species of intracellularautofluorescence have distinct excited-state lifetimes (s1 = 0.6 ± 0.1 ns,s2 = 3.4 ± 0.5 ns, n = 33 pixels in 3 FLIM images). (B) While the amplitude fractionof the fast decay components is large (a1 � 0.71 ± 0.04), its contribution (gray) tothe overall fluorescence intensity is small (F1 = 31 ± 4%) compared with 69 ± 9%from the species with a longer lifetime. (C) Using high zoom imaging, pixel-to-pixeltime-averaged intensity analyses indicate that the intracellular NADH autofluores-cence in Hs578Bst is predominantly (86 ± 5%) from apparent mitochondria, ascompared with 14 ± 4% from the cytosol. [See Supplementary Fig. S1 for similaranalyses on breast cancer cells.]

chondria, the relative contribution of the species with a longer life-time, as calculated using Eq. (5), contribute �69% of the detectedautofluorescence signal, as compared with �31% contribution fromthe other species with a short lifetime (Fig. 3B). Using pixel-to-pix-el intensity analyses (4 images, 20 pixels per image), the apparentmitochondrial NADH autofluorescence in normal cells is dominant(86 ± 10%), with a minor contribution (14 ± 6%) from the cytosol(Fig. 3C).

Comparative 2P-FLIM measurements on cancer cells were alsoconducted (Fig. 2C and D) to examine the sensitivity of intrinsicNADH dynamics to cell pathology, using the same experimentaland analytical approaches. Double Gaussian fit of the correspond-ing average lifetime histogram (Fig. 2E, solid circles) of the 2P-FLIMimage yields mean lifetimes of 0.93 ns (amplitude = 3724,x1 = 0.23 ns) and 0.55 ns (amplitude = 3902, x2 = 0.32 ns). As a re-sult the estimate average lifetime per image is 0.7 ± 0.2 ns (n = 5).In addition, pixel-to-pixel fluorescence decay analysis of 2P-FLIMimages (Supplementary Fig. S1) reveals that 2P-fluorescence ofintrinsic NADH per pixel decays biexponentially (apparent mito-chondrial NADH: s1 = 0.52 ± 0.05 ns, a1 = 0.72 ± 0.05, s2 = 2.4 ±0.3 ns, a2 = 0.28 ± 0.05, n = 23 pixels, and �sfl ¼ 1:0� 0:1ns). The ob-served trend of longer NADH lifetime distribution in breast normal(n = 8) versus cancer (n = 12) cells is consistent among these repre-sentative data and is statistically significant (Student’s t-test,p < 0.05). In addition, the species with a shorter lifetime contrib-utes 38 ± 2% of the total autofluorescence signal as compared with62 ± 7% of the long-lifetime species.

To overcome the low temporal resolution and signal-to-noiseratio of 2P-FLIM, pseudo-single point lifetime measurements werealso performed. In this modality, the laser pulses (740 nm,4.2 MHz, average power of 6300 lW) were scanned over thewhole cell (i.e., without the pixel-to-pixel resolution) and the auto-fluorescence signal was continuously collected and stored in 1024channels (instead of 64 channels in FLIM images). The autofluores-cence of normal cells (n = 6) decays as triexponential, with an aver-age lifetime of 0.83 ± 0.05 ns (Table 1, Supplementary Fig. S2). Theobserved triexponential decay pattern of intrinsic NADH agreeswith recent studies on 3T3-L1 adipocytes and 3T3 fibroblast cells[44] using ultraviolet excitation. The autofluorescence of cancercells also decays as a triexponential (n = 6), with an estimated aver-age lifetime of 0.75 ± 0.05 ns (Table 1, Supplementary Fig. S2). Stu-dent’s t-tests show that the average autofluorescence lifetime innormal and cancer cells, using pseudo-single point measurementsis slightly different (p � 0.05). Under the same experimental condi-tions, the single point 2P-fluorescence of free NADH (pH 7.4) de-cays as a biexponential, with s1 = 0.36 ns (a1 = 0.82), s2 = 0.75 ns(a2 = 0.18), and �sfl ¼ 0:43 ns, which is consistent with literaturevalues [9] as well as 2P-FLIM measurements (s1 = 0.34 ns,a1 = 0.79, s2 = 0.89 ns, a2 = 0.21, and an average lifetime of�0.46 ns). The multiexponential fluorescence decays of free NADHare likely due to different molecular conformations (e.g., extendedversus folded) [9,59], which makes the assignment of cellularNADH as free and enzyme-bound more difficult using only fluores-cence lifetime measurements [9]. In addition, the heterogeneity oflocal pH, microviscosity, and refractive index [54,55] in cell envi-ronment may contribute to the observed changes in intracellularNADH autofluorescence lifetime.

3.3. Electron transport chain inhibitor (KCN) induces a functionalintracellular NADH response

We further conducted 2P-FLIM measurements on normal breastcells (n = 4) under resting conditions and pharmacological manip-ulation of the ETC using the complex IV inhibitor KCN (Fig. 4).Fig. 4 shows representative 2P intensity (Fig. 4A) and FLIM(Fig. 4B) images before and after KCN treatment (8 min, Fig. 4C

Table 1Summary of the fitting parameters for time-resolved autofluorescence of intrinsic NADH in both normal (Hs578Bst) and cancer (Hs578T) breast cells. These pseudo-single pointautofluorescence measurements on living cells were carried out using magic angle (54.7�) detection. Comparative results for free NADH in PBS solution (pH 7.4, at roomtemperature) are also shown as a function of the relative concentration ratio of mMDH ([NADH] � 226 lM) and LDH ([NADH] � 183 lM).

s1(ps) a1 s2(ps) a1 s3(ns) a1 �sflðnsÞ

(A) IntracellularHs578Bst (n = 6) 97(38)a 0.38(6) 648(75) 0.48(6) 3.45(7) 0.14(1) 0.83(5)b

Hs578T (n = 6) 75(11) 0.35(6) 564(14) 0.53(6) 3.4(2) 0.123(5) 0.75(5)

Free NADHIn PBS (pH 7.4) 356(9) 0.82(3) 754(9) 0.18(3) – – 0.43(3)

[NADH]:[mMDH]16:1 356 0.42 650 0.51 1.57 0.06 0.558:1 293 0.32 677 0.75 1.49 0.11 0.644:1 299 0.19 692 0.66 1.45 0.15 0.742.6:1 333 0.17 752 0.69 1.50 0.14 0.792:1 303 0.16 793 0.74 1.69 0.10 0.801.3:1 365 0.17 791 0.73 1.65 0.11 0.811:1 304 0.14 767 0.74 1.57 0.12 0.81

[NADH]:[LDH]32:1 233 0.44 508 0.52 1.59 0.04 0.4316:1 213 0.43 533 0.1 1.62 0.06 0.468:1 227 0.42 603 0.47 1.66 0.11 0.575:1 243 0.39 671 0.45 1.67 0.16 0.674:1 263 0.35 728 0.45 1.7 0.20 0.752:1 324 0.23 999 0.58 1.97 0.19 1.021:1 338 0.22 1081 0.67 2.53 0.11 1.08

a The numbers in parentheses represent the standard deviation of the last digit(s).b The fitting parameters shown here correspond to v2

6 1.2 and homogeneous residual around its zero-value.


and D). These FLIM images were analyzed using the same fittingconstraints (e.g., pixel binning 5, threshold 15 counts, weightedpixel intensity, and improved matrix calculations). In addition tothe heterogeneity of intracellular NADH lifetime (Fig. 4B and D),we have also carried out pixel-to-pixel analyses of the time-aver-aged autofluorescence intensity (3 images, 15 pixels per image)in both apparent mitochondria and the cytoplasm (Fig. 4E). The rel-ative changes of cytosolic and mitochondrial autofluorescenceintensity are (43 ± 8)% and (39 ± 12)%, respectively, upon the respi-ratory chain inhibition using KCN (Fig. 4F). Such functional re-sponse of breast normal cells (Hs578Bst) to ETC inhibition alsoindicates that the 2P-autofluorescence can be assigned to intrinsicNADH, under our excitation/detection conditions [9,10,13,38,60].

3.4. Intracellular NADH concentration imaging in intact, live cellsusing 2P-autofluorescence dynamics assay

Intracellular NADH concentration is an important biochemicalcriterion for many physiological and pathological events in cellularmetabolism that are indispensable to life. 2P-FLIM of cellular auto-fluorescence indicates the variation in fluorescence lifetime (i.e.,quantum yield) in the heterogeneous cell environment. As a result,it is essential to account for the differences in fluorescence quan-tum yield prior to converting fluorescence intensity images to con-centration images in living cells. Using two-photon intensity andlifetime imaging in a calibrated microscope, concentration imagesof intrinsic NADH in normal (Fig. 5A) and cancer breast cells wereobtained. As shown in this representative image of a photoselectedcross-section of a normal breast cell, the relative concentration ofintracellular NADH varies significantly from pixel-to-pixelthroughout the cell. Free NADH (PBS, pH 7.4) of known concentra-tion was used, under the same experimental conditions, as a refer-ence for calibrating our microscope. The averaged NADHconcentration in breast cancer cells is 168 ± 49 lM (n = 7), com-pared to 99 ± 37 lM (n = 7) in normal counterpart (Fig. 5B). Theconcentration between normal and cancer cells are statistically dif-ferent (Fig. 5B; Student’s t-test, p < 0.05). Recently, Kasischke et al.[40] reported an enhancement (by a factor of ten) of the two-pho-

ton excitation cross-section of mMDH-bound NADH. Accordingly,these concentration estimates, at the single-cell level, should beweighted by the molar fraction of enzyme-bound NADH in livingcells (see below).

3.5. Autofluorescence anisotropy provides direct evidence for multiplemolecular conformations of intracellular NADH at the single-cell level

Since NADH is a cofactor for enzymes that catalyze redox reac-tions in cells, it is important to quantify the population fractions offree and enzyme-bound conformations under different physiolog-ical conditions. There have been recent attempts to use FLIM aloneas a contrasting factor between NADH conformations [10,13].However, direct correlation between different fluorescence decaycomponents and structural conformations is not straightforward[9], especially with multiexponential decays of both free and en-zyme-bound NADH in solution (see below). Here, we use comple-mentary steady-state autofluorescence anisotropy imaging toassess the restrictive nature of the cellular microenvironment tointrinsic NADH. Importantly, we also employ pseudo-single point,time-resolved 2P-autofluorescence anisotropy on living cells fordirect assessment of free and enzyme-bound NADH populationsat the single-cell level for the first time, to the best of ourknowledge.

From the steady-state perspective, typical 2P-autofluorescencepolarization images (i.e., parallel and perpendicular with respectto the laser polarization) are shown in Fig. 6. These parallel(Fig. 6A) and perpendicular (Fig. 6B) polarization images of cellularNADH were recorded simultaneously to minimize possible fluctu-ations in laser intensity and cell movement. The steady-state 2P-anisotropy images (Fig. 6C), calculated using a MATLAB-basedalgorithm, reveal heterogeneous (Fig. 6C, color code) orientationorder and environmental restrictions of intracellular NADH in liv-ing cells. The estimated average (from cell to cell) initial anisotro-pies per cell are 0.32 ± 0.05 (n = 6) and 0.30 ± 0.03 (n = 6) fornormal and cancer cells, respectively. These average values of thesteady-state 2P-autofluorescence anisotropy are significantly low-er than the theoretical maximum, 0.57 [56], which may be attrib-

Fig. 4. Intracellular NADH level is sensitive to the inhibition of complex IV in theETC using KCN. These two-photon autofluorescence intensity (A,C) and FLIM (B,D)images were recorded before (A,B) and after (C,D: 8 min) the addition of KCN tonormal breast cells (Hs578Bst) in a Petri dish. These images were recorded using740 nm excitation and analyzed using similar fitting constraints (Binning 5,Threshold 15 counts, and Improved Matrix calculation). (E) Pixel-to-pixel analysesof the time-averaged autofluorescence intensity (23 pixels per image) in bothapparent mitochondria (gray column) and the cytoplasm (white column). (F) Therelative changes of cytosolic and mitochondrial autofluorescence intensity are43 ± 8% and 39 ± 12%, respectively, upon the respiratory chain inhibition using KCN.

Fig. 5. Intrinsic NADH concentration in living cells as revealed by 2P-autofluores-cence intensity and lifetime images. A typical concentration image (A) of intracel-lular NADH in a breast normal cell under physiological conditions. Thisconcentration imaging of cellular NADH was carried out after taking into consid-eration the fluorescence lifetime (i.e., quantum yield) variation in cellularcompartments and using a calibrated microscope (see text). Using this assay, ourstatistical analysis (B) indicates that the average NADH concentration in breastnormal cells is 99 ± 37 lM (n = 7), compared with 168 ± 49 lM (n = 7) in thetransformed counterpart.


uted to the rotational flexibility of intrinsic NADH [61], intramolec-ular energy transfer in cellular microenvironments, and opticaldepolarization due to the high NA objective [62,63]. On average,the corresponding angle between the absorption and emission di-poles of NADH are 33 ± 3� (normal, n = 6) and 34 ± 2� (cancer,n = 6).

From the rotational dynamic perspective, however, time-re-solved autofluorescence anisotropy of native NADH reveals anassociated anisotropy, which directly indicates the presence oftwo emitting species with different hydrodynamic volumes andfluorescence properties, at the single-cell level (Fig. 7A). The asso-ciated anisotropy curves in breast cancer (Fig. 7A) and normal cells(n = 10) are described using Eq. (8) and the fitting parameters aresummarized in Table 2. At early times of rotational diffusion, the

contribution of free NADH is prevalent compared with the diffu-sion of enzyme-bound molecules that dominates at a later timedue to the molecular size differences (Fig. 7A). In these analyses,the slow (>30 ns) rotational time is much longer than the corre-sponding average autofluorescence lifetime (0.75–1.5 ns) of cellu-lar NADH and, therefore, less accurate. The rotational timeconstants of free and enzyme-bound NADH were almost fixed witha variable amplitude fraction. In addition, the magic-angle fluores-cence decay parameters of cellular NADH autofluorescence wereused as measured to minimize the number of floating fittingparameters. The results are compared with control experimentson NADH as a function of the mMDH (Fig. 7B) and LDH (Fig. 7C)concentrations (see below).

3.6. Enzyme–NADH binding in solution mimics the rotational diffusionof cellular autofluorescence

To elucidate the underlying mechanism of autofluorescencelifetime enhancement in living cells, single point (i.e., no laserscanning) time-resolved fluorescence measurement were carriedout on NADH (PBS, pH 7.4, at room temperature) as a function ofLDH and mMDH concentrations. In addition, complementarytime-resolved fluorescence anisotropy was conducted under con-trolled molar fractions of NADH and enzymes. These control mea-surements provide a point of reference concerning the associated

Fig. 7. Time-resolved associated anisotropy of intrinsic NADH autofluorescence, atthe single-cell level, indicates multiple emitting species with different hydrody-namic volumes and fluorescence properties. Typical time-resolved fluorescenceanisotropy in a cancer cell is shown (A). These pseudo-single point measurementswere performed by scanning the laser (740 nm) over the whole cell, while bothparallel and perpendicular polarizations were recorded simultaneously using 1024channels (12.4 ps/channel). The G-factor (1.14) was estimated by the tail-matchingapproach using a coumarin sample. Comparative measurements were alsoconducted on NADH titrated with mMDH (B; [NADH]:[mMDH] = 1: 0, 16:1, 8:1,4:1, 2.7:1, and 2:1; from bottom up) and LDH (C; [NADH]:[LDH] = 1: 0, 32:1, 16:1,8:1, 5.3:1, 4:1, and 2:1; from bottom up). The concentration ratios (B;[NADH]:[mMDH] = 16:1 and C; [NADH]:[LDH] = 8:1–5.3:1) gave the closest resem-blance to the cellular autofluorescence curves (gray curve), which is also shown inboth Fig. 7B and C for visual comparison.

Fig. 6. Steady-state 2P-fluorescence anisotropy imaging of cellular autofluores-cence indicates a heterogeneous and restrictive cell environment surroundingintracellular NADH. Parallel (A) and perpendicular (B) polarized-autofluorescenceimages of a single cancer cell, using two-channels with a Glan–Thompson polarizerat each channel, were recorded simultaneously. These polarization-analyzedimages were recorded in 256 � 256 pixels with 120 s acquisition time. An imageprocessing routine was developed, using MATLAB software, for calculating theanisotropy images (C). The estimated average steady-state anisotropy of this imageis �0.32. Similar steady-state anisotropy images of cellular autofluorescence innormal breast cells were also obtained (data not shown).


anisotropy of cellular NADH and the analyses of free and enzyme-bound populations. In these experiments, we used fixed NADHconcentrations (226 lM and 182 lM for mMDH and LDH titra-tions, respectively) that would yield detectable two-photon fluo-rescence with good signal-to-noise ratio, while the concentrationratio of the enzymes (mMDH or LDH) was varied accordingly.NADH was titrated with mMDH and LDH at [NADH]:[enzyme] con-centration ratios ranging from 16:1 to 1:1 for both time-resolved2P-fluorescence and anisotropy measurements. Under the excita-tion and detection conditions used here, only the 2P-fluorescenceof NADH (both free and enzyme-bound) was measured (i.e., nofluorescence from free mMDH or LDH was detected).

The 2P-fluorescence of free and enzyme-bound NADH decays asmultiexponential (Table 1 and Supplementary Fig. S2) with anaverage lifetime that increases as the binding sites of mMDH andLDH become occupied (e.g., at [NADH]:[enzyme] �30:1–2:1 ratio).The equilibrium constant (�3.8 lM) of NADH with mMDH hasbeen reported [64]. When the two binding sites of mMDH [9,65]are fully occupied, the changes in 2P-fluorescence and lifetime ofNADH–mMDH complex are 2-fold larger (Supplementary Fig. S2)compared with the free cofactor in solution [9]. At a[NADH]:[mMDH] ratio of 16:1, which closely mimics the associ-ated anisotropy of cellular autofluorescence (Fig. 7A), the 2P-fluo-rescence decays as a triexponential with s1 = 276 ps (a1 = 0.42),s2 = 650 ps (a2 = 0.51), s3 = 1.57 ns (a3 = 0.06), and �sfl � 550 ps.Similar studies were carried on NADH–LDH binding kinetics (Table1 and Supplementary Fig. S2C), revealing a sigmoidal enhancement

of average fluorescence lifetime as a function of LDH concentration.The 2P-fluorescence and lifetime enhancement of NADH increasesby a factor of three when fully bound with LDH. The lifetimeenhancement due to enzyme binding of NADH is more pronouncedthan the viscosity effects, as measured in PBS with �22% glycerol(data not shown).

Time-resolved fluorescence anisotropy of NADH–mMDH, withall binding sites occupied, decays as a single exponential with arotational time of �30 ns and an estimated hydrodynamic radiusof �137 nm3 (Eq. (9)). The rotational time constant of mMDH(molecular weight �70 kDa [66]) is much slower than that of freeNADH (�665 Da), in agreement with Vishwasrao et al [9]. Belowbinding-site saturation, however, the mixture of free and en-zyme-bound NADH, with both mMDH (Fig. 7B) and LDH(Fig. 7C), exhibits associated anisotropy features that resemblethose of cellular autofluorescence (Fig. 7A). For example, the asso-ciated anisotropy decays of a mixture of NADH and mMDH([NADH]:[mMDH] � 16:1) is best described by s1 = 0.43 ns,a1 = 0.43, s2 = 0.80 ns, a2 = 0.48, u1 = 0.14 ns, b1 = 0.28,

Table 2Fitting parameters for time-resolved fluorescence associated anisotropy of intracellular NADH in breast normal (Hs578Bst) and cancer (Hs578T) cells using pseudo-single pointmeasurements. The results on free and enzyme-bound NADH in solution (PBS, pH 7.4) are also shown for comparative purpose as a function of the relative concentration ratio ofmMDH ([NADH] � 226 lM) and LDH ([NADH] � 183 lM).

a1 s2(ns) a2 s2(ns) b1 u1(ns) b2 u2(ns)

Cellular NADHHs578Bst (n = 9) 0.3(1)a 0.4(1) 0.7(1) 0.7(4) 0.42(5) 0.3(2) 0.36(1) 1Hs578T (n = 7) 0.4(1) 0.5(2) 0.6(1) 0.8(4) 0.36(6) 0.18(4) 0.40(3) 1

Free NADHIn PBS (pH7.4) – – – – 0.29 0.27 – –

[NADH]:[mMDH]16:1 0.52 0.43 0.48 0.80 0.28 0.14 0.41 29.58:1 0.33 0.43 0.67 0.80 0.26 0.12 0.43 30.04:1 0.15 0.43 0.85 0.80 0.10 0.03 0.45 27.92.6:1 0.08 0.43 0.93 0.80 0.05 0.04 0.46 26.82:1 – – – – – – 0.45 30.21:1 – – – – – – 0.44 30.0

[NADH]:[LDH]32:1 0.88 0.42 0.12 1.02 0.21 0.11 0.09 39.616:1 0.80 0.42 0.20 1.02 0.26 0.19 0.39 43.58:1 0.63 0.42 0.37 1.02 0.26 0.16 0.41 36.85:1 0.48 0.42 0.52 1.02 0.24 0.15 0.42 36.54:1 0.38 0.42 0.62 1.02 0.24 0.12 0.42 43.52:1 and 2:2 – – – – – – 0.414 39.6

a The numbers in parentheses represent the standard deviation of the last digit(s). The solution measurements were repeated two times and the standard deviation iswithin 10% of the stated fitting parameters.


u2 = 29.5 ns, and b2 = 0.41 (Fig. 7B, Table 2). Using extended obser-vation time and more data points on the titration curve, our asso-ciated 2P-anisotropy results of NADH, as a function of mMDHconcentration, agree with recent studies by Vishwasrao et al. [9].Similar measurements were carried out on NADH as a function ofLDH concentrations ([NADH]:[LDH] = 1:1–16:1). At a concentra-tion ratio of [NADH]:[LDH] = 8:1, for example, the associatedanisotropy resembles the cancer cell autofluorescence, withs1 = 0.37 ns, a1 = 0.62, s2 = 1.02 ns, a2 = 0.37, u1 = 0.16 ns,b1 = 0.26, u2 = 37 ns, and b2 = 0.41 (Fig. 7C, Table 2). Importantly,Deng et al. have identified four NADH binding sites in pig heartLDH with a dissociation constant of �6.8 lM [67], which we usedto calculate the corresponding fractions of free and LDH-boundNADH (see below).

4. Discussion

Intracellular NADH is a ubiquitous cofactor that participates inmany metabolic reactions, especially energy metabolism, ineukaryotic cells [3,18,24]. Changes in intracellular NADH concen-tration and the ratio of oxidized (NAD+) to reduced (NADH) cofac-tor are usually associated with cell transformation [10,13–15,24,68]. In most cancer cells, there is a reduced amount of NADHundergoing oxidation in the mitochondria due to the mutation ofenzyme complexes and subsequent uncoupling of the ETC [14].As a compensatory mechanism for ATP production, cancer cells ex-hibit an elevated glycolytic rate, i.e., the ‘‘Warburg effect” [5], lead-ing to larger pools of cytosolic NADH compared to normal cells. Asa result, preserving morphological context during energy metabo-lism studies in living cells is particularly important since oxida-tion–reduction reactions in living cells depend on the spatialdistribution of NADH [9].

DIC and confocal microscopy images of Rh123-stained Hs578Tand Hs578Bst cells exhibit apparent differences in cell morphologyand mitochondrial distribution (Fig. 1). The cancer cell line Hs578Tshows an increased nuclear-to-cytoplasmic ratio, which might beattributed to an increased proliferation rate in cancer cells[4,69,70]. The increased nuclear size and nucleo-cytoplasmic ratiohave also been reported in other cancer cell lines such as humangastric carcinoma [69], human liver cancer and the dysplastic liver

[71] cell lines. The observed peri-nuclear mitochondrial distribu-tions in the transformed breast cells (Hs578T) agree well with pre-vious reports on breast cancer (MCF-7) and human lung carcinoma(A549) cells [72]. These peri-nuclear mitochondrial features havebeen attributed to ATP demands for detoxification and high motil-ity in carcinoma cells [72].

The 2P-autofluorescence under our experimental conditions(740 nm excitation and 450 ± 50 nm detection) is attributed tointrinsic NADH following previous reports on a number of biolog-ical models [9,38,40,46,73]. The experimental evidence supportingsuch autofluorescence assignment to intracellular NADH includesthe cell response to hypoxia [9], sodium cyanide (NaCN) [38,46],and carbonyl cyanide 4-(trifluoromethoxy)phenyl hydrazone(FCCP) treatments [38,73]. In addition, intracellular NADH ismostly co-localized with the mitochondria. While it is possible thatintracellular flavin adenine dinucleotide (FAD) could be excited at740 nm [38], its contribution to the cellular NADH autofluores-cence was negligible under our detection conditions [9]. The func-tional response of normal breast cells to an ETC inhibitor (Fig. 4)supports such an argument [38]. As mentioned above, however,differentiating between intracellular NADH and NADPH is not pos-sible using our micro-spectroscopy techniques due to their similarphotophysical properties. In addition, the intracellular NADH levelis likely to be higher than that of NADPH [17,24].

Intracellular NADH autofluorescence lifetime imaging (Figs. 2and 3) reveals a heterogeneous environment in both breast normaland cancer cells. The multiexponential decays of cellular autofluo-rescence lifetime also indicate the presence of different excitedstates, likely due to the presence of multiple molecular conforma-tions (e.g., free and enzyme-bound NADH) [9,10,13] and a hetero-geneous cell environment. The intermediate (0.65 ± 0.08 ns,amplitude �48%) and slow (3.45 ± 0.07 ns, amplitude �14%) decaycomponents of cellular NADH autofluorescence using pseudo- sin-gle point measurements agree well with the spatially resolvedFLIM studies (0.7 ± 0.2 ns, �63% and 2.7 ± 0.3 ns, 38%) in normalcells. Using 2P-FLIM, these two autofluorescence lifetimes in breastcancer (MCF-7) cells have been assigned to free and enzyme-boundNADH [9,10,13]. However, different fluorescence lifetimes frommultiexponential decays may not necessarily correlate to specificmolecular conformations. For example, the fastest decay compo-


nent (e.g., 97 ± 38 ps in normal cells) in pseudo-single point, ma-gic-angle measurements is absent in FLIM analysis due to thelow temporal resolution. In addition, the 2P-fluorescence of NADH(PBS, pH 7.4) decays as a multiexponential as a function of mMDHand LDH concentrations (Table 1, Supplementary Fig. S1). Thefolded conformation of free NADH, mitochondrial swelling, and ex-cited-state processes such as charge transfer may contribute tosuch a fast lifetime component [9,56]. The 2P-autofluorescencelifetime distribution and pixel-to-pixel analysis in FLIM imagesindicate a significantly shorter lifetime in cancer cells as comparedwith normal cells (p < 0.05). These FLIM parameters generallyagree with other biochemical and spectroscopic studies of NADHin transformed (MCF-7) and non-transformed (MCF-10A) cells[10,60,74]. The cellular environment, however, is rather complexwith variable local pH, microviscosity, and refractive indices[54,55], which may influence the autofluorescence lifetime ofintracellular NADH. As a result, care must be taken in strictlyassigning the observed heterogeneity in intracellular NADH life-time to molecular conformations (free versus enzyme-bound). Itis also not clear how minor photobleaching during intrinsic NADHin living cells or tissues [46] may contribute to the observed fluo-rescence lifetime changes.

Using 2P-FLIM imaging of intracellular NADH in living cells in acalibrated microscope, we were able to convert fluorescence inten-sity into concentration images (Fig. 5A) after accounting for thelifetime (i.e., quantum yield) heterogeneity in living cells. It isworth mentioning that, in a given cell, the relative concentrationof intracellular NADH can be as high as a few 100s lM in some pix-els (Fig. 5A). Despite the large cell-to-cell variation (n = 7), theaverage concentration of cellular NADH in breast cancer(168 ± 49 lM) cells is about 1.8 times as high as that in normalcells (99 ± 37 lM) (Fig. 5B; Student’s t-test of p < 0.05). These ob-served trends are consistent with previous studies on other normaland malignant cell lines and tissues using biochemical and spectro-scopic methods [11,60,68]. For example, Uppal and Gupta have re-ported approximately 2-fold higher NADH fluorescence inmalignant human breast cancer tissues than in their normal coun-terpart [11]. Using UV micro-spectrofluorimetry and biochemicalcycling assays, Villette et al. have also reported enhanced NADHfluorescence intensity in normal and cancerous esophageal epithe-lium cells [68].

The observed response of intracellular NADH autofluorescenceto KCN treatment (Fig. 4) is consistent with the ETC inhibition,which interrupts the oxidation of NADH to NAD+. Since the intra-cellular level of NADH (fluorescent) and NAD+ (not fluorescent)are equilibrated under resting conditions, one may assume thatthe observed increase in NADH concentration (i.e., d[NADH]), un-der physiological manipulation of the same cell, would corre-spond to a decrease in [NAD+] by the same amount. Underrespiration chain inhibition using KCN, the relative changes ofcytosolic and mitochondrial autofluorescence intensity are43 ± 8% and 39 ± 12%, respectively (Fig. 4F). We may also assumethat the intracellular NADH concentration in normal breast cells(�99 lM) is increased by the same amount (�140 lM). Accord-ingly, the intracellular NAD+ is likely decreased by �41 lM,which indicates about �41% changes in the redox state of normalbreast cells upon ETC inhibition. One may reach a similar conclu-sion using KCN effects on the pixel-to-pixel lifetime analysis. Inthis case, we would assign the fast and slow decay componentscorrespond to free and enzyme-bound intracellular NADH,respectively. These rough approximations, however, should beconsidered as such due to other physiological effects of KCNtreatment. This argument is supported by the observed changeson mitochondrial and cytosolic NADH, which suggest that KCNeffect on cellular function may extend beyond the respirationchain inhibition.

For a mixture of fluorophores, time-resolved fluorescenceanisotropy is sensitive to both the hydrodynamic volume, confor-mations, and surrounding environment [56]. As a result, theseanisotropy measurements are one of the most direct approachesfor quantifying the fluorescence fractions of free and enzyme-bound NADH [9]. Intracellular NADH autofluorescence, in bothbreast normal and cancer cells, reveals an associated anisotropyat the single-cell level (Fig. 7A). These results provide direct evi-dence that the cellular autofluorescence is collectively emitted bya mixed population of NADH with different hydrodynamic vol-umes (e.g., free and enzyme-bound) and fluorescence properties.The observed associated anisotropy of cellular autofluorescencecan be described satisfactorily using only two species of distinctfluorescence lifetime and rotational time constants (Table 2),which we assign as free and enzyme-bound NADH. Conceptually,one would think of these associated anisotropy features as freeNADH dominating at early times due to its rapid rotation and theenzyme-bound cofactor appearing at longer rotational times dueto its relatively larger size. Based on these associated anisotropyanalyses [9,56], the population fraction of intracellular free NADHis ffree = 0.18 ± 0.08 (n = 9) in normal breast cells, which is signifi-cantly smaller than the enzyme-bound fraction (fbound =0.82 ± 0.08). These fractions are statistically different (Student’s t-test, p < 0.05) from those in breast cancer cells whereffree = 0.25 ± 0.08 (n = 7) and fbound = 0.75 ± 0.07. These results pro-vide direct quantitative evidence of free and enzyme-bound NADHat the single-cell level, where cancer Hs578T cells reveal a statisti-cally significantly larger fraction of free NADH than normal cells.Such an enhancement in intracellular NADH levels in transformedcells is attributed to compromised enzymatic activities in the ETCof mitochondria and, perhaps, an increase in the glycolytic rate[1,5,7,8,13–15,24]. These single-cell studies also support the find-ings by Vishwasrao et al. [9] in brain tissues, regardless of theinherent biocomplexity of the latter model. Since intracellularNADH is a necessary coenzyme for a range of reduction–oxidationreactions, a number of NADH–enzyme complexes are likely to exist[9,75] under a given physiological condition. Using our autofluo-rescence dynamics assay alone, however, we are unable to specu-late about the nature of an enzyme-bound species of intrinsicNADH.

In concentration image analyses (Fig. 5), we used NADH (PBS,pH 7.4) of known concentration as a reference to calibrate ourmicroscope under the same experimental conditions. We have alsoassumed similar two-photon excitation cross-sections of free andcellular NADH, which may not be accurate (Eqs. (2) and (3)). Re-cently, Kasischke et al. [40] reported an enhanced (by a factor often) two-photon excitation cross-section of mMDH-bound NADH.Assuming 82% of the intracellular NADH population is enzyme-bound, the weighted average concentration in breast cancer(Hs578T) cells will be equivalent to �22 lM. The correspondingintracellular NADH, weighted by 75% enzyme-bound cofactor, is�12 lM in breast normal (Hs578Bst) cells.

Our control measurements on NADH (pH 7.4), as a function ofmMDH and LDH concentrations, complement our cellular auto-fluorescence studies and enable us to examine the structural con-formations and 2P-excited-state lifetime enhancement uponenzyme binding. The multiexponential 2P-fluorescence decay ofNADH (PBS, pH 7.4), as a function of mMDH and LDH concentration(Supplementary Fig. S2A and B), suggest the presence of multiplestructural conformations, as was proposed previously [67,76]. Inaddition, the fluorescence intensity (data not shown) and averagelifetime of NADH–mMDH is twice (Supplementary Fig. S2C) thatof the unbound cofactor [77,78]. At fully occupied LDH sites[67,79], the average fluorescence lifetime is increased by 3-foldas compared with free NADH (Supplementary Fig. S1C). The ob-served enhancement of NADH fluorescence in the protein environ-


ment is due to the unfolded conformation of adenine and nicotin-amide ring [9,59,76]. It is worth mentioning that the slow compo-nent (1.57 ns, 6%) is significantly faster than that of cellularautofluorescence decays (�3.3 ns, �15%). Further, the average life-time of cellular autofluorescence (0.75–0.83 ns), using pseudo-sin-gle point measurements, is slower than the average lifetime(0.55 ns) of NADH in solution ([NADH]:[mMDH] = 16:1). Collec-tively, these comparative studies suggest a complex environmentand conformation of intracellular NADH as compared with solutionstudies presented here.

In controlled NADH titrations with mMDH and LDH, time-re-solved fluorescence anisotropy on a mixture of free and fullybound NADH–mMDH (i.e., below saturation) reveals an associatedanisotropy behavior that mimics cellular autofluorescence. Twofluorescence anisotropy decay components are also resolved insolution studies. Based on the pre-exponential parameters, the freeand mMDH-bound NADH fractions are ffree � 0.4 and fbound � 0.6,respectively (at [NADH]:[mMDH] = 16:1). Using the equilibriumconstant for NADH dissociation (Ke � 3.8 lM [80]), this molar frac-tion of [NADH]:[mMDH] in solution corresponds to a free-to-bound fraction of �13:1, which could be considered as an upperlimit for our cellular studies. The corresponding free and LDH-bound NADH populations are 0.7 and 0.3, respectively, at[NADH]:[LDH] = 8:1. The accuracy of these numbers is limited bythe number of fitting parameters, as well as the fast excited-statelifetime compared with the rotational diffusion of the slow, en-zyme-bound species. However, the observed associated anisotropyof cellular autofluorescence and solution studies of NADH–enzymemixing provides support to our conclusions.

In summary, our autofluorescence dynamics imaging assayindicates that the level of intracellular NADH and its conformationare sensitive to cell physiology to pathology. Using breast normal(Hs578Bst) and cancer (Hs578T) cells as a model system, our re-sults indicate that the cellular autofluorescence lifetime is largerthan that of free NADH in solution, which is attributed mainly toenzyme binding. The observed heterogeneity of the two-photonautofluorescence lifetime (i.e., quantum yield) throughout livingcells was used in intensity-to-concentration image conversion.The estimated intracellular NADH level in the breast cancer cellmodel is almost twice that in the non-transformed counterpart.For the first time, the two-photon cellular autofluorescence exhib-its associated anisotropy features, at the single-cell level, that di-rectly indicate the presence of two NADH species of differingconformations (i.e., free and enzyme-bound). These findings andtheir interpretation in living cells are examined using free NADH(PBS, pH 7.4) under controlled mixing with mitochondrial malatedehydrogenase (mMDH) and lactate dehydrogenase (LDH) in solu-tion. These studies demonstrate the sensitivity of intrinsic NADHdynamics imaging to cell physiology, as well as its potential appli-cation for sensing cellular respiration, apoptosis, and health prob-lems associated with mitochondrial anomalies such as cancer,aging, and neurodegenerative diseases. Our analytical assay, whichcan be applied to other labeled biomolecules, provides a comple-mentary, non-invasive approach to conventional biochemical tech-niques that require cell lysates and, therefore, the loss ofmorphological context.

Acknowledgements

We thank Dr. Yuexin Liu, Florly Ariola, and Ronn Walvick fortheir help during the early stages of this project. We are also grate-ful to Angel Davey (Chemistry) for her editorial comments on thismanuscript. This work was supported, in part, by the NationalInstitute of Health (AG030949), Johnson & Johnson (PSU, HuckInstitutes of the Life Sciences, Innovative Research Grant), the PennState Materials Research Institute, and the Center for Optical Tech-

nologies (NSF/Lehigh/Penn State). We thank Coherent Lasers, Inc.for their loan of a pulse picker (MIRA9200; Coherent) that was usedin this work.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jphotobiol.2008.12.010.

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