223

Abstract Reduced nicotinamide adenine dinucleotide (NADH) and fl avin adenine dinucleotide (FADH

2 ) are electron carriers that play important roles in a wide range

of metabolic activities and mitochondrial functions in eukaryotic cells. NADH and FAD are naturally fl uorescent with distinct illumination/emission wavelengths for selective detection. Their autofl uorescence is also sensitive to protein binding and local environment. As a result, these intracellular coenzymes have potential as intrinsic biomarkers for a noninvasive imaging of metabolic activities and oxidation–reduction reactions in living cells either in vitro, ex vivo, or in vivo. This chapter highlights recent fi ndings of these coenzymes as natural biomarkers of metabolic and mitochondrial activities with an emphasis on a multiparametric imaging approach.

1 Introduction

Cellular coenzymes, such as reduced nicotinamide adenine dinucleotide (NADH) and fl avin adenine dinucleotide (FADH

2 ), play an important role in metabolic activ-

ities, oxidation–reduction reactions, and mitochondrial function in living cells [ 1 ] . Mitochondria support the energy demands for all cellular processes and therefore are essential to the life of all eukaryotic cells as well as human health [ 2 ] . In addi-tion, mitochondrial dysfunction is widely linked to a range of diseases and health problems such as cancer, Alzheimer’s disease, and other neurodegenerative diseases [ 3– 6 ] . As a result, there is a resurgent interest in developing intracellular NADH and

A. A. Heikal (*) Department of Chemistry and Biochemistry , Swenson College of Science and Engineering, The University of Minnesota Duluth , Duluth , MN , USA

Department of Pharmacy Practice and Pharmaceutical Sciences , College of Pharmacy, The University of Minnesota Duluth , 246 Chem, 1039 University Drive , Duluth , MN 55812 , USA e-mail: aaheikal@d.umn.edu

A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic and Mitochondrial Activities

Ahmed A. Heikal

C.D. Geddes (ed.), Reviews in Fluorescence 2010, Reviews in Fluorescence, DOI 10.1007/978-1-4419-9828-6_10,© Springer Science+Business Media, LLC 2012

224 A.A. Heikal

fl avin as natural biomarkers for monitoring metabolic activities and mitochondrial anomalies under pathophysiological conditions [ 1 ] , following the pioneering work of Britton Chance and his colleagues [ 7– 15 ] .

NADH is involved in mitochondrial function, Ca 2+ homeostasis, gene expres-sion, oxidative stress, aging, and apoptosis. Reduced nicotinamide adenine dinucle-otide phosphate (NADPH) is also involved in the reductive biosynthesis of fatty acids and steroids, antioxidation, and oxidative stress [ 16, 17 ] . Some evidence sug-gests that intracellular NADH concentration is greater (up to tenfold by some esti-mates) than that of NADPH [ 17, 18 ] . However, the correlation between these pyridine nucleotides’ autofl uorescence and cell pathology remains inconsistent [ 19, 20 ] possibly due to variation in used techniques and model systems. FADH

2 is

another coenzyme that is associated with respiration in all eukaryotic cells. In addi-tion to FAD, fl avoproteins such as lipoamide dehydrogenase (LipDH) and electron transfer fl avoprotein contribute signifi cantly to the cellular autofl uorescence [ 21– 24 ] . The autofl uorescence of fl avin and fl avoproteins is sensitive to cell treatment with the mitochondrial inhibitor cyanide in pancreatic islet [ 25 ] , rat cardiac myocytes [ 22, 26 ] , and guinea pig ventricular myocytes [ 27 ] . Importantly, NAD(P)H, FADH

2 ,

and their oxidized counterparts are critical for a broad array of redox reactions in living cells [ 16, 28, 29 ] . As a result, the ratiometric measure of energy respiration and the redox state of a cell can be carried out using mitochondrial NADH and FAD autofl uorescence under physiological conditions [ 22 ] .

Biochemical techniques are routinely used for estimating the cellular levels of NADH [ 30, 31 ] and fl avin [ 32 ] . For example, enzymatic methods [ 33 ] , cycling assays [ 34, 35 ] , and high-performance liquid chromatography (HPLC) analyses [ 18 ] have been used to determine the concentration of NADH in cells and tissues. A commercial NAD + /NADH assay kit, which is based on alcohol dehydrogenase cycling reaction, has a sensitivity of ~0.2 m M. Another single extraction procedure and a spectrophotometric assay have been developed to determine the concentration of oxidized and reduced NAD in cell monolayers [ 36 ] . A capillary electrophoresis method is based on an enzymatic cycling reaction to determine the concentration of NAD + and NADH at the single-cell level and with a single run [ 37 ] . Bacterial luciferase with a liquid scintillation spectrometer has also been used to determine subpicomolar concentration of NADH and fl avin mononucleotide (FMN) [ 38 ] . These conventional biochemical assays provide an accurate measure of the intrinsic metabolic cofactors. However, most of these methods also require destroying the cells or tissues and, therefore, provide only snapshots of the metabolic activities in the absence of any morphological context.

What makes NADH and FAD (see Fig. 1a, b for their chemical structures) so interesting is that they are naturally fl uorescent and, therefore, a genuine nonin-vasive imaging of metabolic activities can be carried out in living cells and tissues. The autofl uorescence properties of NADH and fl avin eliminate potential toxicity, nonspecifi c binding, and interference with biomolecular functions that are associated with the use of exogenous dyes. In addition, the autofl uorescence of these coenzymes can be excited using distinct illumination wavelengths, ranging from the ultraviolet to infrared regions, which facilitates complementary imaging using one-photon (1P) and two-photon (2P) fl uorescence microscopy, respectively.

225A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

Finally, the NADH and fl avin autofl uorescence are sensitive to protein binding and their local environment. The nicotinamide ring of NADH constitutes the reactive moiety that accepts a hydrogen ion and two electrons. The isoalloxazine ring is the reactive part of FAD that is responsible for light absorption in the ultraviolet region with visible emission. Based on these chemical structures (Fig. 1a, b ), NADH and FAD are fl uorescent while NAD + and FADH

2 are not.

Fig. 1 The chemical structure and spectroscopy of NADH and FAD. ( a ) The chemical structure of NADH with the nicotinamide ring as the reactive moiety that accepts a hydrogen ion and two electrons. ( b ) The chemical structure of FAD with the isoalloxazine ring that is responsible for light absorption in the ultraviolet region. ( c ) One-photon ( black : NADH, gray : FAD) and two-photon ( dark squares : NADH, gray circles : FAD) spectroscopy of NADH and FAD in PBS (pH 7.5). The fl uorescence spectrum of NADH ( dark , dotted line ) peaks at ~458 nm as compared with ~528 nm emission of FAD ( gray , dotted line ) in a buffered solution. 2P-excitation cross-section spectra of NADH ( dark squares ) and FAD ( gray circles ) are shown in GM units (1 GM = 10 −50 cm 4 s/photon molecule) [ 22 ] . These results indicate that both NADH and FAD can be excited nonlinearly using 730 nm, but only FAD can be excited at ~850 nm £ l £ 950 nm.

226 A.A. Heikal

The potential of these coenzymes as natural biomarkers for metabolic activities, mitochondrial function, and related health conditions is limitless. For example, mutated mitochondrial DNA in cancer alters the structure/function of NADH dehy-drogenase and cytochrome C of the electron transport chain (ETC) in the inner membrane of mitochondria. This would lead to the generation of free radicals, apop-tosis [ 39 ] , and changes in the mitochondrial membrane potential. Mitochondria have also been a target for developing therapeutic drugs due to their role in cell survival and health-related conditions [ 40 ] . For example, some anticancer agents are designed to stimulate proapoptotic mitochondrial events in tumor cells [ 41– 43 ] . Mitochondrial anomalies and oxidative stress are associated with neurodegenerative diseases and therefore are targets for new therapeutic drugs aimed at either preventing or slowing down the progression of those neurodegenerative disorders [ 44 ] . Uncoupling of the mitochondrial ETC has also been a pharmacological target for treating obesity [ 45 ] . As a result, mitochondrial NADH and fl avin autofl uorescence could serve as natural probes for mitochondrial activities under different pathophysiological conditions. For example, an elevated autofl uorescence of intracellular NADH has been reported in different cancer cell lines under UV illumination [ 33, 46 ] . Skala et al. investi-gated the sensitivity of intracellular NADH in hamster cheek pouch epithelial cells to the treatment with dimethylbenz[ a ]anthracene as a model of oral carcinogenesis [ 47 ] . The decrease of protein-bound NADH lifetime with dysplasia was attributed to a shift from oxidative phosphorylation to glycolysis [ 48 ] . The low-grade precancers and high-grade precancers could also be discriminated from normal tissues based on the lifetime component of protein-bound NADH. In another report [ 48 ] , the cellular NADH and FAD autofl uorescence lifetime was used to identify metabolic fi nger-prints of living cells at the earliest stages of cancer development.

In the cytoplasm, elevated glucose levels have been shown to affect glycolysis, sorbitol pathway activity, and the cytosolic redox state of NADH/NAD + [ 49 ] . The results support the hypothesis that metabolic imbalances, associated with a more reduced ratio of cytosolic NADH/NAD + , play an important role in mediating glu-cose- and diabetes-induced glomerular dysfunction in isolated blood-free glomeruli from Sprague–Dawley rat’s kidney [ 49 ] . The increased level of cytosolic ATP in pancreatic b -cells triggers insulin secretion by causing blockade of ATP-dependent K + channels [ 49 ] . Glucose metabolism is pivotal to glucose-induced insulin secre-tion from pancreatic b -cells [ 50– 52 ] . Duke et al. investigated NADH production during glycolysis and its role in b -cell glucose signaling [ 50 ] . The mitochondrial membrane depolarization leads to an elevation in cytosolic Ca 2+ and insulin secre-tion. NADH shuttle system plays an important role in glucose-induced activation of mitochondrial metabolism and insulin secretion [ 53 ] . By inhibiting the NADH shut-tles, for example, glucose-induced increases in NADH autofl uorescence were observed while the mitochondrial membrane potential, ATP content, and glucose-induced insulin secretion were reduced. In animal diabetic models, the increased ratio of cytosolic-free NADH/NAD + is considered one of the earliest metabolic imbalances linked to increased blood fl ow in tissues [ 51 ] . Piston and coworkers have studied the redox signal, as a function of pyruvate, in intact pancreatic islets using fl avoproteins and NAD(P)H autofl uorescence microscopy [ 25, 52 ] .

227A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

This chapter highlights the spectroscopic characteristics of NADH and fl avin, multiparametric fl uorescence approach, and recent fi ndings related to these natural biomarkers for metabolic and mitochondrial activities. Finally, the potential and challenges associated with these coenzymes as biomarkers are discussed.

2 NADH and FAD Spectroscopy Allows for Selective Excitation/Detection

One-photon and two-photon spectroscopy of NADH and FAD is shown in Fig. 1c [ 54 ] . The absorption properties of NADH and NADPH are similar, which is disad-vantageous for spectroscopy-based assays used in differentiating between metabolic activities associated with either coenzyme. The one-photon emission of NADH and FAD are distinct, which should allow for ratiometric measurements [ 22 ] . A double-beam spectrophotometer was fi rst used to monitor intracellular NADH absorption spectrum [ 55 ] . However, such UV–visible absorption spectra are usually broad with some degree of spectral overlap, which may undermine the specifi city of the absorb-ing species. The emission of free NADH (pH 7.4) is maximal at ~458 nm as com-pared with 528 nm for FAD. The emission peaks are sensitive to both viscosity and enzyme binding [ 56 ] . The 2P-excitation cross-section spectra of NADH [ 22, 57, 58 ] and fl avin [ 22 ] , in PBS buffer (pH 7.5), are shown in Fig. 1c [ 22, 59 ] . The two-photon microscopy of NADH (Fig. 1c , dark squares) and FAD (Fig. 1c , gray spheres) provides a spectral window for selective excitation/detection of these coenzymes [ 22 ] , which is advantageous due to the inherently high-spatial resolution, enhanced penetration depth, reduced light scattering, and low overall photodamage [ 60– 64 ] . Based on these spectral profi les, selective 2P-excitation of NAD(P)H and FAD becomes possible using 730 nm (NADH) [ 22, 59 ] and 900 nm (FAD), respectively [ 22 ] . Emission fi lters for two-photon excited NADH and FAD should be selected carefully to minimize spectral overlap while avoiding second-harmonic imaging at half the excitation wavelength.

The NADH autofl uorescence spectra in intact cells were reported using fl uores-cence spectrophotometry [ 65 ] . Similar measurements followed on various biologi-cal systems such as human blood sample [ 66 ] , human breast normal and cancer cell lines [ 19 ] , Chinese hamster ovary cells, rat neurons, normal and SV-40-transformed human keratinocytes [ 67 ] , bovine oligodendroglia, mink fi broblast and murine fi broblast [ 23 ] , and isolated rat hepatocytes [ 68 ] under different metabolic condi-tions. In vivo autofl uorescence spectrophotometry was used to detect intrinsic NADH and/or fl avoprotein signals in rabbit models [ 69 ] , brain and kidney of rats [ 8 ] , murine muscle tissues [ 70 ] , as well as squamous cell carcinoma [ 46 ] . The fl uo-rescence spectrophotometry is cost-effective and easy to perform on intact live cells and tissues. Determining the absolute NADH concentration from cell ensembles in a cuvette, however, can be a challenge and lacks the spatial resolution needed for cellular studies.

228 A.A. Heikal

Based on structure, spectroscopy, and the heterogeneity of cell environment, a multiparametric approach is being proposed (Fig. 2 ) for investigating different aspects of intracellular coenzymes. In this approach, differential interference con-trast (DIC) is used to image the cell morphology. The spatial distribution of cellular NADH and FAD is imaged using two-photon fl uorescence microscopy with the inherent high-spatial resolution that allows for separating the mitochondrial and

Fig. 2 Cellular autofl uorescence assignment to NADH at 730 nm (excitation) and 450 nm (detec-tion). The two-photon NADH autofl uorescence of in vitro glial cells ( a ) co-localizes with the mitochondria of the same cells after being stained with rhodamine-123 ( b ). These control experi-ments suggest that the majority of cell NADH exist in mitochondria, which is consistent with dominant role of oxidative phosphorylation in ATP production. This conclusion is also supported by the functional response of mitochondria to the respiratory chain manipulation using either cya-nide or FCCP ( c ) [ 22, 72, 91 ] . Interrupting the ETC enhances the mitochondrial NADH concentra-tion (by inhibiting NADH oxidation), which is opposite to the uncoupling of mitochondria using FCCP. This fi gure is a courtesy of Dr. Karl Kasischke.

229A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

cytosolic coenzymes. Fluorescence lifetime imaging (FLIM) reports on the spatial and conformational heterogeneity of NADH and FAD in their natural cell environ-ment. These FLIM studies could be used to convert intensity images into concentra-tion distribution when used in a calibrated microscope [ 71, 72 ] . Due to the interest in quantifying the free and enzyme-bound coenzymes, time-resolved anisotropy imaging provides a direct measure of the molar fraction of these distinct conforma-tions of NADH. From the sample perspective, the proposed multiparametric approach must also include a means for controlling pathophysiological parameters [ 73 ] for the realization of NADH and FAD potential as natural biomarkers for in vitro, ex vivo, and in vivo metabolic studies. Recent developments in multipho-ton microscopy [ 63, 74– 78 ] and endoscopy [ 77, 79 ] are very promising for in vivo applications.

3 Autofl uorescence Imaging of Intracellular Coenzymes in Living Systems

Cells and tissues are complex biological systems with other autofl uorescence emit-ters such as tryptophan, collagen, elastin, and porphyrins also contribute to the over-all autofl uorescence in cells/tissues [ 59 ] . As a result, careful experimental design and control experiments are needed to target and detect a specifi c biomolecule of interest. Based on the absorption–emission spectra, for example, the excitation laser wavelength and the emission detection fi lters could be selected to target a given fl uorophore. Second, the spatial distribution (localization) of the target biomolecule, as refl ected in autofl uorescence image, can be compared with our current knowl-edge of its compartmentalization in cells or tissues. Finally, the biological function of a target biomolecule would also dictate a specifi c response to the manipulation of cell physiology, which would enable us to predict how the autofl uorescence inten-sity (or concentration) may change accordingly. Figure 3 demonstrates some of these controls with respect to NADH autofl uorescence in glial cells under two-photon excitation. The NADH autofl uorescence (Fig. 3a ) is co-localized with rhod-amine-labeled mitochondria (Fig. 3b ) in the same cell. In addition, the integrated NADH autofl uorescence increases after cell treatment with cyanide (Fig. 3c ), which is a respiratory chain inhibitor. In contrast, the NADH autofl uorescence decreases upon mitochondrial uncoupling using FCCP (carbonyl cyanide p -(trifl uoromethoxy) phenyl hydrazone) (Fig. 3c ).

In conventional one-photon microscopy for intracellular coenzymes imaging, the cells are illuminated with a UV light to excite either NADH or fl avin and the autofl uorescence images are recorded under a given physiological condition. The fl uorescence intensity per pixel 1P ( , )F x y depends on the concentration of the fl uo-rophore ( , )C x y , fl uorescence quantum yield ( )f , the detection effi ciency ( )h , and the illumination intensity ( I ) such that [ 72 ] :

λ λ=1P 1P( , ) ( ) ( , ) ( )fl xF x y C x y Ifh s (1)

230 A.A. Heikal

where 1Ps is the one-photon excitation cross-section of a given fl uorophore. This approach has been used in a variety of biological systems such as contracting Xenopus skeletal muscle fi bers [ 80 ] , hippocampal slices [ 81 ] , and pancreatic islet b -cells [ 25 ] . The NAD(P)H and FAD autofl uorescence in neuronal CA1 of murine hippocampal slices are sensitive to postsynaptic neuronal activation using different electrical, glutamate iontophoresis, and bath-applied kainate stimuli [ 81 ] . Reinert et al. used NADH and fl avoproteins autofl uorescence to directly monitor the in vivo

Fig. 3 Multiparametric fl uorescence approach for noninvasive imaging of intracellular coenzymes in living cells or tissues. The biological context and intracellular coenzyme will dictate the experi-mental design such as excitation wavelength ( l

x ), readout fl uorescence ( l

fl ) variables and the physi-

ological constraints on the biological system. Confocal (one-photon, blue ) or two-photon ( red ) microscopy is used to selectively excite NADH and FAD with high spatial and temporal resolution of their autofl uorescence intensity. Spectral resolution of the autofl uorescence emission, using a microscope-compatible spectrofl uorimeter, can be used for molecular identifi cation. Autofl uorescence lifetime imaging enables us to assess the conformation (free versus protein-bound) and environmen-tal heterogeneity of intracellular NADH and fl avin. In addition, these fl uorescence lifetime and intensity images, recorded simultaneously on a calibrated microscope, can be used to construct concentration images of endogenous NADH and fl avin at the single cell level [ 72, 106 ] . Time-resolved fl uorescence anisotropy directly reports on the size, conformation and environmental restriction of these coenzymes at the single-cell level [ 72 ] or tissues [ 91 ] . Differential interference contrast (DIC) imaging also allows for monitoring changes in cell morphology [adapted from Biomarkers in Medicine (2010) 4(2), 241–263 [ 1 ] with permission from Future Medicine Ltd].

231A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

neuronal activity in the cerebellar cortex of ketamine/xylazine-anesthetized mouse under electrical surface stimulation [ 82, 83 ] . Blocking mitochondrial respiration with sodium cyanide, or inactivation of fl avoproteins with diphenyleneiodonium, substantially reduced the autofl uorescence signal, which was attributed to the oxi-dation–reduction of fl avoproteins. UV-based microscopy of NADH and fl avopro-teins, however, suffers from possible scattering, extended photobleaching, and smaller penetration depth in thick samples [ 84, 85 ] . The UV light exposure during imaging may also cause photodamage to cellular DNA [ 86 ] . In contrast, two-photon excitation microscopy provides an alternative approach to overcome some of these challenges [ 59, 62– 64, 74 ] .

In two-photon microscopy, the fl uorescence intensity at a give pixel, 2P ( , )F x y , is given by [ 57 ] :

λ λ λ= (2) 2

2P 2P( , ) ( ) ( ) ( , ) ( )2 x xF x y C x y g If

h sfl (2)

where (2)g is the second-order temporal coherence of the two-photon excitation laser pulses. Representative two-photon fl uorescence intensity image of intracellu-lar NADH in HTB125 epithelial cells is shown in Fig. 4a . Care must be taken, however, to avoid photoinduced stress or damage of living cells during two-photon imaging. The 2P-induced photodamage and its effect on DNA synthesis were reported in rat basophilic leukemia cells during NADH imaging [ 87 ] . The authors concluded that the two-photon-absorbed dose, rather than the laser power used in two-photon microscopy, is a better indicator of photodamage. A temporary reduction of DNA synthesis in RBL cells occurs above a threshold dose of ~2 × 10 53 photon 2 cm −4 s −1 [ 87 ] . Two-photon autofl uorescence microscopy of intrinsic NADH has been used to monitor energy metabolism in macrophages, pancreatic islet cells, skeletal muscle cells [ 88– 90 ] , brain slices [ 58, 91 ] , cardiomyocytes [ 22 ] , human breast normal and cancer cells [ 72 ] , cochlea [ 92 ] , and murine skeletal muscle in vivo [ 93 ] . For exam-ple, Vishwasrao et al. [ 91 ] demonstrated the sensitivity of NADH autofl uorescence, in the stratum radiatum of the CA1 layer of rat hippocampal slices, to the metabolic transition from normoxia to hypoxia. Following a 10-min hypoxic episode, the inte-grated autofl uorescence increased by ~48% followed by a decline to the baseline level when the hypoxic media was replaced with an oxygenated one. Kasischke et al. used 2P-autofl uorescence functional imaging of NADH in brain tissues to investigate the metabolic signatures in processes of astrocytes and neurons [ 58 ] . The results indicate neurometabolic coupling in which early oxidative metabolism in neurons is eventually sustained by late activation of the astrocyte–neuron lactate shuttle. Christie et al. also developed a thinned skull preparation in a Tg2576 trans-genic mouse model of Alzheimer’s disease for imaging of senile plaques over sev-eral months [ 94 ] . Their 2P-microscopy imaging of thiofl avine S-stained senile plaques in this mouse model support the dynamic feedback hypothesis of plaque growth.

Figure 4c shows a typical two-photon fluorescence intensity image of endo-genous fl avins in HTB125 epithelial cells. The two-photon autofl uorescence of

232 A.A. Heikal

single cardiac cells has been assigned to intrinsic fl avoproteins based on excitation wavelength (750, 800, and 900 nm), inhibition of oxidative phosphorylation [ 22 ] , and a spectral unmixing algorithm [ 26 ] . Huang et al. have shown that NADH and fl avin can be excited and detected separately toward ratiometric measurements of

Fig. 4 Two-photon autofl uorescence intensity and lifetime of intracellular NADH and fl avin in living cells. The 2P-autofl uorescence intensity imaging of natural NADH ( a ) and FAD ( c ) in HTB125 cells were recorded using 740 and 900 nm excitations, respectively, under different mag-nifi cations. The corresponding 2P-autofl uorescence lifetime image reveals that the average auto-fl uorescence lifetime of NADH ( b ) is relatively faster than that of FAD ( d ) as shown in the pixel-lifetime histogram ( e ). These intensity and lifetime images of cellular autofl uorescence indi-cate a heterogeneous concentration, conformation and surrounding environment of these coen-zymes throughout the cell [ 72 ].

233A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

metabolic state activities in isolated cardiomyocytes from a dog’s heart [ 22 ] . At 750-nm excitation, the NAD(P)H autofl uorescence increased following cyanide (oxidative phosphorylation inhibitor) treatment with a concurrent reduction in the fl avin signal. Opposite trends were also obtained using a mitochondrial uncoupler (FCCP) [ 22 ] . The 2P-microscopy imaging of cellular fl avin (Fig. 4c ) can be a bit challenging due to the low concentration, excitation cross-section, and fl uorescence quantum yield [ 95 ] , which is attributed to dynamic quenching by adenine [ 95, 96 ] , van der Waals interactions with tryptophan residues and/or the sulfur atoms in cysteine residues [ 97, 98 ] . Using fl uorescence assays, Brolin et al. reported the concentration of FAD (113–291 m M/kg DW) and FMN (9–139 m M/kg DW) in pancreatic islets and other organs from obese-hyperglycemic mice [ 99 ] . The ratio-metric FAD and NADH autofl uorescence also provide a measure of the redox and metabolic states of living cells [ 22, 48 ] . Rocheleau et al. have used the autofl uo-rescence of NADH:LipDH ratio as a measure of the b -cell redox potential in pancreatic islets [ 25 ] .

4 Fluorescence Lifetime as a Contrasting Agent for Molecular Conformation and Surrounding Environment

The excited-state fl uorescence lifetime of a given fl uorophore is sensitive to both its structural conformation and surrounding environment. Importantly, the fl uorescence lifetime is usually independent of both the fl uorophore concentration and the excita-tion laser intensity under typical experimental conditions. Time-correlated single-photon counting (TCSPC) technique is routinely used for time-domain measurements of fl uorescence lifetime [ 100, 101 ] . The fl uorescence of free NADH in a buffer (pH 7.4) decays as a biexponential with an average lifetime of 395 ± 20 ps at room tem-perature [ 72, 91 ] . The enzyme-bound NADH fl uorescence is also multiexponential with an average lifetime that is at least twice that of its free counterpart [ 72, 91 ] . By comparison, the fl uorescence of free FAD (pH 7.4) decays as a biexponential with t

1 = 2.57 ns ( a

3 = 0.71), t

2 = 4.42 ns ( a

2 = 0.29) with an average lifetime of 3.13 ns.

The LipDH fl uorescence decays as triple exponential ( t 1 = 268 ps, a

1 = 0.34,

t 2 = 2.17 ns, a

2 = 0.26, t

3 = 5.3 ns, and a

3 = 0.39), with an estimate average lifetime of

2.75 ns (Yu and Heikal, unpublished results). In principle, these distinctive fl uores-cence decay parameters of NADH and fl avin allow for the separation between free and enzyme-bound conformation in cell environment.

FLIM microscopy, which combines TCSPC with imaging [ 102 ] , is becoming a powerful tool for biological and biomedical studies. In FLIM, the fl uorescence life-time per pixel fl ( , )x yt can be generally described as follows:

-

=

= å / ( , )fl

1

( , ) ( , )e i

nt x y

ii

x y x y tt a

(3)

234 A.A. Heikal

where the number of decay components ( n = 1, 2, or 3) depends on the complexity of both the chemical structure and surrounding environment of the fl uorophore. In any given j th pixel of a FLIM image, the i th fl uorescence lifetime component, ( , )i x yt , and its corresponding amplitude, ( , )i x ya , can be estimated with an accuracy that depends on the total number of photons per pixel, the nature of the fl uorescence decay, and the signal-to-noise level. Importantly, the measured lifetime is directly related to the fl uorescence quantum yield r fl( )kf = τ , where rk is the radiative rate constant, and nonradiative rate ( nrk ) processes such as electron/proton transfer, isomerization, ionization, and dissociation that may compete with fl uorescence.

A typical 2P-FLIM image of intrinsic NADH in epithelial cells (HTB125) is shown in Fig. 4b along with its pixel-lifetime histogram analysis (Fig. 4e ). Pixel-to-pixel analysis of these FLIM images also indicate that the apparent mitochondrial NADH autofl uorescence decays as a biexponential with 600 ± 50 ps (75 ± 5%), 3.2 ± 0.2 ns (25 ± 5%), and an estimated average lifetime of 1.2 ± 0.2 ns. Complementary pseudo single-point 2P-autofl uorescence measurements (i.e., laser is being scanned over the whole cell [ 103 ] ) of NADH yield a triple exponential with 380 ± 20 ps (60 ± 5%), 1.2 ± 0.1 ns (29 ± 2%), 3.7 ± 0.4 ns (13 ± 2%), and an estimated average lifetime of 1.1 ± 0.2 ns [ 72 ] . FLIM studies of intracellular NAD(P)H autofl uorescence have been reported on a range of cell lines such as dermal fi broblasts from artifi cial skin constructs [ 104 ] .

For cellular fl avins, however, the autofl uorescence lifetime (Fig. 4c, e ) is rela-tively longer than that of NADH in the same cell line. In contrast to NADH, fl avin exists predominantly in mitochondria with negligible concentration in the cytosol and nucleus. Pixel-to-pixel analysis of FLIM images of intracellular fl avins indi-cates a biexponential decay with 1.3 ± 0.1 ns (68 ± 2%), 2.7 ± 0.1 ns (32 ± 2%), and an estimated average lifetime of 1.7 ± 0.2 ns. The complementary pseudo single-point autofl uorescence decays as a triple exponential with 350 ± 20 ps (42 ± 5%), 1.6 ± 0.1 ns (47 ± 3%), 4.6 ± 0.5 ns (13 ± 4%), and an estimated average lifetime of 1.4 ± 0.3 ns, which is signifi cantly faster than that of free FAD in a PBS buffer (pH 7.4) with 3.3 ± 0.1 ns (65 ± 2%), 5.7 ± 0.1 ns (35 ± 2%), and an estimated average lifetime of 4.2 ± 0.1 ns.

The intracellular reduction–oxidation ratio in normal (MCF10A) human breast cells is sensitive to confl uence, serum starvation, and potassium cyanide (KCN) poisoning [ 105 ] . Using 2P-FLIM, Bird et al. reported a signifi cant decrease in the fl uorescence lifetime of both free and protein-bound NADH as the cells became confl uent as well as with KCN treatment or serum starvation. In brain tissues, the total concentration of NADH upon hypoxia increased by 81 ± 39%, which is signifi cantly greater than the observed enhancement (48 ± 26%) of the total fl uores-cence [ 91 ] . Two-photon FLIM of FAD, in normal and precancerous epithelial mucosa tissues in hamsters under 890-nm excitation, suggests free (with a long lifetime) and protein-bound FAD conformations [ 48 ] . A decreased contribution of protein-bound FAD was also observed in the high-grade precancer epithelia based on changes in the measured autofl uorescence lifetime [ 48 ] .

Yu and Heikal also used 2P-FLIM to characterize the intracellular NADH auto-fl uorescence in breast cancer (HTB126) and normal (HTB125) cells [ 72 ] . Combining

235A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

2P-FLIM, the authors estimated an intracellular NADH concentration of 99 ± 37 m M in HTB125 and 168 ± 49 m M in HTB 126 [ 72 ] using an imaging processing algorithm [ 106 ] . Similar measurements on cellular fl avin also indicate a concentration of 144 ± 3.8 m M ( n = 6) (Yu and Heikal, unpublished results). Because the 2P-excitation cross-section of LipDH-bound FAD is ~10 times larger than that of free FAD [ 22 ] used as a reference, the average concentration of fl avin is ~14 m M in HTB125 cells.

Based on the notion that the slow decay component is assigned to enzyme-bound NADH in living cells, the majority of this coenzyme in living cells is free. Care must be taken, however, in FLIM data interpretation of cellular autofl uorescence due to the multiple exponential decays of free and enzyme-bound NADH autofl uo-rescence. A number of controlled experiments are required to confi dently assign different fl uorescence decay components in a FLIM image to specifi c molecular conformation (e.g., free versus enzyme-bound), excited state processes (e.g., charge transfer, energy transfer, isomerization) or environmental effects. In addition, the temporal resolution and signal-to-noise ratio per pixel in FLIM are inherently low as compared with single-point TCSPC modality. Since rotational diffusion of a mol-ecule depends directly on its size, time-resolved anisotropy of NADH and FAD (see below) would complement FLIM measurements in quantifying the free and enzyme-bound coenzyme [ 72, 91 ] .

5 Probing the Alignment, Conformations, and Relative Population Fractions of Coenzymes Using Anisotropy Imaging

Once excited using a polarized laser pulses, a fl uorophore will rotate (or tumble) during its excited state lifetime causing the emitted fl uorescence to be depolarized. The rotational time of a fl uorophore depends on its molecular size and the viscosity of surrounding environment [ 100 ] . The time-resolved fl uorescence, with parallel ( , , )I x y t and perpendicular ^ ( , , )I x y t polarizations (with respect to the excitation laser), is then recorded using TCSPC and used to calculate the anisotropy decay at a given pixel, ( , , )r x y t , such that [ 100 ] :

^

^

-=

+

( , , ) ( , , )( , , )

( , , ) 2 ( , , )

I x y t I x y tr x y t

I x y t I x y t

(4)

Depending on the chemical structure and surrounding environment of a given fl uorophore, time-resolved anisotropy can generally be described as multiexponen-tial decay [ 100 ] . For example, time-resolved anisotropy of FAD (PBS, pH 7.5) decays as a biexponential with time constants of ~138 ps (amplitude ~ 0.26) and ~526 ps (amplitude 0.06) (Fig. 5 , curve 1). Similar measurements on NADH in a buffer (data not shown) also indicate a biexponential with time constants of ~120 ps (amplitude ~ 0.20) and ~510 ps (amplitude 0.13).

236 A.A. Heikal

The anisotropy decay of a fl uorophore, either partitioned in different cellular envi-ronments or mixed with other molecules of different sizes, could be rather complex [ 100 ] . For example, mixed fl uorophores of different sizes and fl uorescence properties (e.g., excited-state lifetime) exhibit an associated anisotropy decay [ 107 ] . Time-resolved associated anisotropy of intracellular NADH was observed in cultured epithelial cells (Fig. 5 , curve 2) [ 72 ] as well as in ex vivo neural tissues [ 91 ] . The associated anisotropy of cellular NADH, a mixture of free (subscript 1) and enzyme-bound (subscript 2) coenzyme, can be described as [ 72, 91 ] :

- - - -

- -

+=

+

1 1 2 2

1 2

/ / / /1 1 2 2

/ /1 2

e . e e . e( , , ) ,

e e

t t t t

t tr x y t

φ φt t

t t

a b a ba a

(5)

where 1 2and t t and 1 2andf f are the fl uorescence lifetime and rotational time of species 1 and 2, respectively. The corresponding amplitude fractions are 1 2anda a (for fl uorescence decay components) and 1 2andb b (for anisotropy decay components). In this model, one species (subscript 1) is assumed to have both fast fl uorescence lifetime ( 1t ) and fast rotational time ( 1f ), which are distinguishable from the second species. As a result, anisotropy-based approach offers the most direct and sensitive discrimination of free and enzyme-bound NADH [ 72, 91 ] .

Time-resolved anisotropy of intrinsic NADH in brain hippocampal tissue, iso-lated from Sprague–Dawley rats, reveals an associated anisotropy decay that is

Fig. 5 Time-resolved autofl uorescence anisotropy of intracellular NADH and FAD. Time-resolved anisotropy of free FAD ( curve 1 ) in a buffer (PBS, pH 7.4) decays as a single exponential with 120 ps rotational time. A similar anisotropy decay was observed for free NADH in a buffer (data not shown). The intracellular NADH autofl uorescence, excited at 730 nm, exhibits associated anisot-ropy ( curve 2 ), which is a direct evidence of the presence of two populations of free and enzyme-bound populations at equilibrium [ 72, 91 ] . The associate anisotropy decay also indicates that the two emitting species have distinctive sizes and autofl uorescence properties (e.g., quantum yield or lifetime). In contrast, time-resolved autofl uorescence anisotropy of intracellular FAD ( curve 3 ), excited at 900 nm, indicates mostly enzyme bound with a much slower rotational time than free FAD in a buffer ( curve 1 ). These results combined demonstrate the potential of an integrated experi-mental approach toward conducting biochemical analyses on living cells and tissues [ 1 ].

237A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic…

sensitive to oxygen content [ 91 ] . Using a global analysis of the observed associated anisotropy, Vishwasrao et al. determined noninvasively the population fractions of free and enzyme-bound NADH in neural tissue. For example, the free/bound ratio of cellular NADH was estimated to be 0.78 ± 0.05. It is likely that NADH is distributed among various enzyme-bound species with their own chemical potential and binding constants. The results also indicate that the NADH response to hypoxic inhibition of energy metabolism is more complicated than a simple increase in NADH concentra-tion [ 91 ] . The underlying mechanism of oxygen sensitivity may also involve changes in the conformation and concentration of NADH as well as osmotic swelling of mitochondria [ 91 ] . Recently, Yu and Heikal also reported that cellular NADH auto-fl uorescence in cultured epithelial cells exhibits an associated anisotropy at the sin-gle-cell level [ 72 ] . For the fi rst time, these cultured cell [ 72 ] or brain tissue [ 91 ] studies provide a direct evidence for the equilibrated existence of free and enzyme-bound populations (with free-to-bound ratio of ~0.78) of cellular NADH.

In contrast to intrinsic NADH, time-resolved anisotropy of intracellular fl avin exhibits biexponential anisotropy (Fig. 5 , curve 3) with a dominant slow rotational component that indicates a mostly enzyme-bound coenzyme (Yu and Heikal, unpub-lished results). The rotational time of cellular fl avin is much slower than that of free FAD (PBS, pH 7.5) and at room temperature (Fig. 5 , curve 1). The distinct nature of NADH and FAD rotational diffusion indicates that the associated autofl uorescence signals originate from different emitters. Importantly, time-resolved anisotropy decays of these intracellular coenzymes also suggest that the multiexponential fl uo-rescence decays (observed in FLIM measurements) are likely to originate from structural and environmental heterogeneity rather than fast excited-state dynamics.

6 Concluding Remarks

Intrinsic NAD(P)H and FAD coenzymes are integral part of a complex network of metabolic pathways that are essential for the survival and function of mammalian cells. Conventional biochemical methods are very useful in quantifying the cellular contents of biomolecules. However, they are inherently invasive and require the destruction of the cells, which yield snapshots of the metabolic state of cells but without the morphological context. Traditional cuvette-based studies of these cel-lular coenzymes also lack the spatial information associated with these coenzymes.

NADH and fl avin are naturally fl uorescent and, therefore, allow for a true nonin-vasive imaging of metabolic activities in living cells and tissues. The concentrations and distributions of these naturally fl uorescent coenzymes correlate with the meta-bolic and physiological states of cells and tissues, both in vitro as well as in vivo. Unlike extrinsic fl uorescent markers which must be added to cells or tissues, cellu-lar coenzymes are nontoxic and unlikely to interfere with the diagnostic readout because they are natural participants in redox reactions and cellular metabolic activ-ities. In contrast, exogenous fl uorescence dyes can be toxic and may suffer from nonspecifi c binding as well as interference with biomolecular functions. In addition,

238 A.A. Heikal

the fl uorescence of these coenzymes can be excited using different illumination wavelengths, ranging from ultraviolet to infrared regions, for complementary imaging using one- and two-photon fl uorescence microscopy, respectively. Importantly, the autofl uorescence of NADH is sensitive to protein binding and local environment. Collectively, the fl uorescence properties and functional role of NADH and FAD are the trademarks of a good biomarker.

Inherent challenges, however, remain in using NADH and FAD autofl uorescence as natural biomarkers for metabolic activities and mitochondrial function. First, the spatial distribution of NADH concentration requires noninvasive, quantitative tech-niques with high-spatial resolution to differentiate between cytosolic (glycolysis), mitochondrial (oxidative phosphorylation and TCA cycle), and nuclear (transcrip-tion) coenzymes. Second, an ability to selectively monitor the concentration of these coenzymes is essential for a meaningful readout without bias due to possible photo-damage. Third, the fact that these coenzymes are involved in a range of metabolic activities and biochemical reactions can complicate data interpretation. Fourth, the concentration of these coenzymes is sensitive to many physiological conditions such as oxygen content in vivo blood fl ow, enzymatic mutations, the presence of other metabolites, and chemical stimulation. Fifth, in vivo imaging [ 108, 109 ] is essential for clinical diagnostics to realize the full potential of these coenzymes as natural biomarkers for numerous health problems such as cancer, neurodegenerative diseases, diabetes, and aging. To overcome some of these challenges, a multipara-metric approach is described (Fig. 2 ) for noninvasive and quantitative imaging of intracellular NADH and fl avin. The major advantages of this approach also include real-time monitoring of the metabolic activities using intrinsic NADH and fl avin within the morphological context of cells. Importantly, researchers are now in a position to start focusing on mechanistic studies of these coenzymes in cell biology, biomedicine, and pharmaceutics.

Acknowledgments The author thanks his former student, Dr. Qianru Yu, for her help in obtaining some of the data used in this chapter. Figure 3 is a courtesy of Dr. Karl Kasischke (University of Rochester, School of Medicine and Dentistry, Rochester, NY) and published here with permission. This work was partially supported by the National Institute of Health (AG030949) and the National Science Foundation (MCB0718741). The editorial comments by Dr. Shelley Smith (University of Minnesota-Duluth) are deeply appreciated.

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