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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 5 — May. 1, 2011
  • pp: 1030–1039
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In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy

Jonathan A. Palero, Arjen N. Bader, Henriëtte S. de Bruijn, Angélique van der Ploeg van den Heuvel, Henricus J. C. M. Sterenborg, and Hans C. Gerritsen  »View Author Affiliations


Biomedical Optics Express, Vol. 2, Issue 5, pp. 1030-1039 (2011)
http://dx.doi.org/10.1364/BOE.2.001030


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Abstract

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.

© 2011 OSA

1. Introduction

The autofluorescence of reduced nicotinamide adenine dinucleotides (NADH) can reveal the metabolic state of a cell. For decades now, NADH, the principal electron donor in glycolytic and oxidative energy metabolism, has been used as a convenient noninvasive probe of cellular metabolic state [1

1. B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962). [CrossRef] [PubMed]

]. Although it exists in an oxidized (NAD+) and a reduced (NADH) form, only NADH is intrinsically fluorescent, whereas its oxidized product (NAD+) is not. Chance et al. took advantage of this phenomenon and demonstrated that microfluorometry of NADH provides a means to probe the oxidation-reduction state of cells and tissues [1

1. B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962). [CrossRef] [PubMed]

]. This pioneering work opened the door to more studies that utilized probing NADH fluorescence to reveal the metabolic activity within the cell. The emergence of nonlinear-excited fluorescence microscopy paved the way to a new era of cellular imaging in thick samples as well as living animals [2

2. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]

4

4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]

] leading to key advancement in metabolic studies of cells from isolated samples to in vivo conditions [5

5. E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005). [CrossRef] [PubMed]

,6

6. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007). [CrossRef] [PubMed]

].

In recent years, there have been a growing number of reports on studies of NADH that were largely focused on time-resolved fluorescence and anisotropy measurements demonstrating capability to discriminate protein-bound and free NADH in the intracellular environment [7

7. H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem. 280(26), 25119–25126 (2005). [CrossRef] [PubMed]

13

13. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]

]. In principle, discrimination between protein-bound and free NADH is also achievable by fluorescence emission spectroscopy. However, high spectral resolution measurement is necessary to discriminate the two emission bands due to close proximity of their spectral peaks (λboundNADH ≈445 nm; λfreeNADH ≈460 nm) and their large spectral overlap (ΔλboundNADH ≈ΔλfreeNADH ≈90 nm). To address this issue, we developed high-resolution spatial and spectral nonlinear spectral imaging microscopy (NSIM) [14

14. J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]

,15

15. J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009). [CrossRef]

]. This technique combines spectral imaging microscopy [16

16. P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. Van Sark, D. J. Van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, “High sensitivity spectrograph for use in fluorescence microscopy,” Appl. Spectrosc. 55(8), 1005–1012 (2001). [CrossRef]

,17

17. A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. K. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011). [CrossRef] [PubMed]

] with nonlinear microscopy (multiphoton-excited fluorescence and second harmonic generation) to obtain spectrally-resolved optical sections of living tissues. Further implementation of linear spectral unmixing showed the potential of NSIM in obtaining biochemical information from in-vivo spectral images of mouse [3

3. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J. 93(3), 992–1007 (2007). [CrossRef] [PubMed]

,4

4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]

,18

18. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008). [CrossRef] [PubMed]

] and human skin [19

19. A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011). [CrossRef] [PubMed]

]. In this study, we demonstrate the capability of NSIM to discriminate in-vivo protein-bound and free NADH in mouse keratinocytes following total acute ischemia. Moreover, the use of low excitation power levels and the inherent localization of nonlinear excitation allowed us to acquire spectral images for up to 3.3 hr with minimal photobleaching.

2. Materials and method

2.1. Animal model

The Committee on Animal Research of the Erasmus University Rotterdam approved the experimental protocol. The skin of two female inbred albino hairless mice (SKH1 HR, Charles River, Someren, The Netherlands) was used in vivo. The mice were fed on a diet free of chlorophyll (Hope Farms BV, Woerden, The Netherlands) for a minimum of 2 weeks before starting the experiments to remove the autofluorescence emission from mouse skin centered at 675 nm attributed to pheophorbide-a [20

20. G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988). [CrossRef] [PubMed]

]. The mice were placed on a home-made temperature-controlled microscope stage. During pre-ischemia imaging, the mice were anaesthetized using Ketamine (80mg/kg, Janssen Pharmaceutica, Tilburg, The Netherlands) and Rompun (10mg/kg Janssen Pharmaceutica, Tilburg, The Netherlands), both were administered intraperitoneally. Acute ischemia was induced by euthanizing the anaesthetized animal. Blood oxygenation in the mouse tongue was measured using differential path-length spectroscopy [21

21. A. Amelink and H. J. Sterenborg, “Measurement of the local optical properties of turbid media by differential path-length spectroscopy,” Appl. Opt. 43(15), 3048–3054 (2004). [CrossRef] [PubMed]

]. Blood oxygenation levels of ~50% were found in the anaesthetized mouse; this value dropped to 0% immediately (< 1 min) after administration of the euthanasia solution. All experiments were limited to four hours after anesthesia administration.

2.2. Nonlinear spectral imaging microscope

The basic NSIM setup used for detecting autofluorescence signal from in vivo keratinocytes was similar to that described previously [14

14. J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]

]. A diagram of the experimental setup is shown in Fig. 1
Fig. 1 Schematic diagram of the spectral imaging setup. The intrinsic emission of a sample is epi-detected without a pinhole. The signal is spectrally dispersed by two prisms, focused with an achromat, and detected with a back-illuminated CCD array.
. The excitation light source (765 nm) was a mode-locked titanium:sapphire (Ti:Sa) laser (Tsunami, Spectra-Physics, Sunnyvale, CA), pumped by a 5W neodymium yttrium vanadate (Nd:YVO4) laser (Millennia, Spectra-Physics, Sunnyvale, CA). The laser light was scanned using a galvanometer mirror scanner (040EF, LSK, Stallikon, Switzerland). In addition to the beam-scanning mechanism, the microscope was also equipped with an XYZ piezo translation (sample) stage (Physik Instrumente, Karlsruhe/Palmbach, Germany). The laser light was focused by an objective lens on the sample and the fluorescence emission was collected by the same objective lens. The results reported here were acquired in the inverted geometry using an infinity-corrected oil-immersion objective lens (60× and NA = 1.40, Nikon, Japan).

The emission passed through a dichroic mirror and was filtered by a set of short-pass Schott® optical glass filters (total thickness = 7 mm; BG-40, Schott, Mainz, Germany). The filtered light was dispersed by two prisms and focused on the charge-coupled device (CCD) camera by a UV-VIS-IR achromat (diameter = 40 mm, focal length = 160 mm; Bernhard Halle Nachfl., Berlin, Germany). The spectrograph was equipped with a thermoelectrically cooled, back-illuminated CCD camera (Princeton Instruments, Spec-10:2KBUV, 16-bit, ST-133 controller, typical read noise 3 e- rms at 100-kHz digitization). In our experiments, the exposure time was limited to 2.1 ms per pixel and employed excitation powers of less than 5 mW. Photobleaching was observed to be minimal at this low power level. Based on the recorded images prior to the administration of euthanasia solution, we estimated a fluorescence (bleaching) decay rate (1/e) of 2.5 × 10−3/frame at 2 min/frame. Data processing and visualization were carried out using a program (SpecView) written in IDL 6.0 (Research Systems Inc., Boulder, CO).

Wavelength and instrument spectral response calibration of the instrument was carried out to ensure accurate spectral measurements. A white light source and a calibrated monochromator were used to calibrate the instrument wavelength and the calibration accuracy was better than 0.5 nm over the whole wavelength range. The spectral response correction was calculated by measuring the spectra of two standard fluorophores: tryptophan in water and quinine sulfate in perchloric acid [22

22. J. A. Gardecki and M. Maroncelli, “Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy,” Appl. Spectrosc. 52(9), 1179–1189 (1998). [CrossRef]

].

2.3. Visualization and analysis of spectral image data

The recorded spectral image data were converted to RGB images for visualization using a program written in IDL 6.0 (Research Systems Inc., Boulder, CO) as described in detail previously [4

4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]

]. Briefly, the conversion procedure involves down sampling the 100-channel (wavelength) spectral image to a 3-channel (RGB) color image. The full spectral image data set were analyzed using a separate program written in IDL 6.0. Pixel-averaged spectra of selected regions-of-interest (ROIs) were obtained for spectral analysis depicted in Fig. 2
Fig. 2 Representative time-lapse RGB spectral images of mouse keratinocytes in vivo before (t < 0 min) and during (t > 0 min) ischemia showing increase and decrease in autofluorescence (AF). Red arrows: hair follicles. Colors represent emission wavelength as indicated in color bar. All images are 224 × 224 pixels, 100 µm × 100 µm.
(yellow dashed box). The basis of selecting an area as an alternative to using the whole image is to ensure that only keratinocytes from a single tissue layer, in our case mainly the stratum basale were analyzed.

Linear spectral unmixing analysis was performed by means of iterative linear least squares fitting procedure (Gaussian multi-peaks, Origin® 6.1, OriginLab Corporation, Northampton, MA, USA). Three Gaussian curves were fitted globally using the difference spectra (see Fig. 3 D
Fig. 3 A: Color map of the temporal behavior of the autofluorescence spectra obtained from the pixel-averaged spectral data from a region of interest (ROI; yellow box in Fig. 2). White line indicates the average peak wavelength (λ = 456 nm) of the autofluorescence spectra before ischemia. Black dotted line indicates the autofluorescence peak wavelength depicting spectral blue-shift after ischemia (t > 0 min) and a red-shift at t > 50 min. B: Normalized integrated autofluorescence intensity (integrated between 400 nm and 600 nm). C: ROI pixel-averaged autofluorescence spectra at representative time points showing spectral variation during ischemia. D: Difference spectra (relative to AF spectrum at t = 0 min) at representative time points.
) from all time points relative to the time before acute ischemia was induced, thus removing static components from the analyses. The shared parameters for this procedure were the spectral peak positions and spectral widths while the amplitudes were allowed to vary. All fitting procedures were carried out in the wavenumber space. The global fit yielded the time-varying amplitudes of the spectral components attributed to the autofluorescence of protein-bound and free NADH and flavins as depicted in Fig. 4 A
Fig. 4 A: Three spectral components used for spectral unmixing with peak/ full-width-at-half-maximum (FWHM) values of: (1) 448 nm/ 91 nm; (2) 459 nm/ 91 nm; and (3) 528 nm /77 nm, attributed to protein-bound and free NADH and flavins, respectively. Curves are normalized to the peaks’ amplitude. B: Spectral unmixing analysis showing the spectral components (1 to 3) attributed to: (1) protein-bound NADH; (2) free NADH; and (3) flavins, and the sum of the fitted components. Also shown is the residual of the fit. Fitting r2 coefficient of determination is 0.996. C: Color map of the χ2 error obtained by global fitting ten arbitrary difference spectra as a function of the peak wavelengths of the first two components. The intersection point of the two dotted lines indicates the local minimum (λ1 = 448 nm, λ2 = 459 nm). D and E: Dependence of the global fitting error to the peak wavelength of spectral component 1 (at λ2 = 459 nm) and peak wavelength of spectral component 2 (at λ1 = 448 nm), respectively.
.

3. Results

3.1. Cellular autofluorescence modification following total acute ischemia

The capability of nonlinear microscopy to produce optical sections through thick tissues allowed us to record spectral images of living cells inside the epidermis. In addition, the high sensitivity of the spectral imaging system across a broad spectral range (350 nm to 600 nm) permitted acquisition of autofluorescence spectral images with relatively high signal-to-noise ratio (SNR) using low excitation power levels, typically around 5 mW [14

14. J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]

]. Long-term in vivo imaging of cells within tissues using the present setup is thus achieved with minimal photobleaching and low phototoxicity.

Time series of spectrally-resolved nonlinear-excited fluorescence optical sections were recorded from the skin epidermis of anaesthetized living hairless mouse (see Fig. 2). Because of the undulating characteristic of epidermal layers, the recorded spectral image sections consisted of keratinocytes associated to stratum basale and stratum spinosum. Note that only keratinocytes in the stratum basale were analyzed. We acquired images of basal cells since their keratin content is less than that of the other epidermal layers, thus minimizing keratin autofluorescence contribution to the overall emission spectrum. Bright green-fluorescing hair follicles were also observed in the spectral images (see Fig. 2, red arrows). The mice were subjected to total acute ischemia during which the intrinsic fluorescence of the keratinocytes increased in intensity by an average of 71 ± 5% (averaged from t = 50 min to t = 80 min) as depicted in Fig. 3, A and B. At t > 80 min, a decrease in autofluorescence intensity was observed to below pre-ischemic baseline or normoxic levels. The autofluorescence of the hair follicles remained fairly constant ( ± 4%) throughout the experiment. The variation in the cellular autofluorescence emission spectral shape after the onset of ischemia is depicted in Fig. 3, A and C. The spectra showed an initial blue-shift of the spectral peak from 456 nm (t = 0 min) to 450 nm (t = 20 min) and a subsequent red-shift to 455 nm (t = 80 min). At t = 150 min, the autofluorescence spectral peak was observed to be 460 nm, 5 nm longer than the normoxic (t = 0 min) autofluorescence spectrum. The variation in the spectral shape as demonstrated by the shifting of the spectral peaks strongly indicates metabolic response to ischemia of at least two spectral components. The difference spectra, that is the difference between the autofluorescence spectrum at any given time point (ischemic) and the baseline (normoxic) autofluorescence spectrum at t = 0 min, showed a more clear evidence that at least two components with different response to ischemia comprise the cellular autofluorescence spectrum (see Fig. 3 D). Here, it can be deduced that during the early condition after the onset of ischemia, the intensity of short-wavelength spectral component increased more than the long-wavelength spectral component. At ~80 min, both components have higher intensities relative to normoxic levels but of relatively equivalent ratio as that of the normoxic condition. At longer times, both components decreased in intensity below the normoxic level as depicted by negative values in the difference spectrum for t > 110 min (see Fig. 3 D).

3.2. Probing bound/free NADH in mouse keratinocytes

The time-dependence of the amplitudes of the three spectral components for two independent in vivo experiment sets are shown in Fig. 5, A and B
Fig. 5 A and B: Time-dependence of the relative amplitudes relative to the average component amplitudes at t < 0 min (normoxic) of each of the fitted components from two independent in vivo experiment sets. A minimum of 25 epidermal cells (within the region of interest as depicted in Fig. 2, yellow box), was measured for each time point, for each experiment. Reference (normoxic) condition (t ≤ 0 min), ischemia (t > 0 min).
. Spectral unmixing revealed that indeed the third spectral component with amplitude A3, attributed to flavin autofluorescence, has a minor contribution (~5%) to the overall spectral emission. Both experiments showed a significant difference between the amplitude variation of the two spectral components A1 and A2, attributed to protein-bound and free NADH fluorescence, respectively. Furthermore, similarities were found between the amplitude variations of corresponding spectral components. For instance, in both experiments, A2 showed a broad peak in time (FWHM, ~100 min) with maxima at ~100 min while A1 demonstrated a relatively narrower peak in time (FWHM, ~50 min) with maxima at ~50 min. These suggest that at early times following ischemia onset, the increase in fluorescence is mainly due to increase in protein-bound NADH (113%) and after t = 50 min, the protein-bound NADH autofluorescence decreases below normoxic levels (~70%) while free NADH remained increasing (up to ~85%). After ~100 min, free NADH gradually decreases below normoxic levels (~20%).

4. Discussion

The results presented in this work demonstrate the dynamic effect of total acute ischemia on the autofluorescence spectra of in vivo mouse skin keratinocytes. Linear spectral unmixing analysis suggests that the metabolically active fluorophores are protein-bound and free NADH. The contributions of protein-bound and free NADH show different dynamic reactions to hypoxia, anoxia and ischemia. NADH protein-bound to the complex I (NADH:ubiquinone oxidoreductase) of mitochondria is the major source of protein-bound NADH fluorescence. The temporal protein-bound NADH profile shows the dynamics of the transition of normoxia to hypoxia to anoxia. The ratio between protein-bound and free NADH change when the cells shift to anaerobic metabolism and finally the deprivation of glucose reduces the total NADH fluorescence. Our results, therefore, signify that in mouse keratinocytes all the major metabolic pathways (glycolysis, anaerobic fermentation and oxidative phosphorylation) respond to the loss of blood-supplied (via capillaries) oxygen and glucose. Another important implication of our results is the confirmation of the presence of metabolically functional mitochondria in epidermal keratinocytes, contrary to the conclusion of a recent study that keratinocytes are functionally anaerobic and that keratinocytic mitochondria are metabolically dysfunctional [27

27. G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003). [CrossRef] [PubMed]

]. Our observation showing significant response of mitochondrial-bound NADH to ischemia strongly suggests inhibition of mitochondrial complex I, hence, an evidence of mitochondrial oxidative phosphorylation of keratinocytes under normoxic conditions.

The results also demonstrate that although there is evidence supporting the hypothesis that cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of epidermis [28

28. M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002). [CrossRef] [PubMed]

], capillary oxygen supply remains to be the major source for epidermal metabolic function. Based on optical measurements of oxygen flux on the surface of human skin, measured values of oxygen diffusion in skin tissues, and assumption of homogeneity of skin, the thickness of the skin tissue that is supplied by atmospheric oxygen was estimated to be 266–375 μm [28

28. M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002). [CrossRef] [PubMed]

]. The measurements presented in the present study were carried out at depths of ~50 μm relative to the surface of the skin and the analysis of the results clearly showed metabolic response to complete deprivation of blood-supplied oxygen. On the other hand, the possibility that obstruction of blood supply establishes a hypoxic rather than an anoxic environment cannot be disregarded. In fact, it may even explain the slow metabolic response (~2 h) of the keratinocytes following the loss of blood-supplied oxygen.

Most imaging experiments on free and protein-bound NADH were carried out using two-photon excited fluorescence lifetime imaging (FLIM) [8

8. V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008). [CrossRef] [PubMed]

13

13. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]

]. Discrimination between the free and protein-bound NADH is achieved by taking advantage of the difference in their fluorescence lifetimes: the short lifetime component is attributed to the free NADH (τfreeNADH = 0.3 to 0.5 ns) while the long lifetime component is attributed to the protein-bound NADH (τboundNADH = 1.6 to 3.7 ns) [8

8. V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008). [CrossRef] [PubMed]

,9

9. V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009). [CrossRef]

,12

12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]

]. Comparison between FLIM and spectral imaging in terms of ability to discriminate between protein-bound and free NADH is not straightforward and depending on the type of experiment different aspects need to be taken into account. An important factor in the in-vivo imaging of NADH is the sensitivity and non-invasiveness of the applied method: autofluorescence levels are in general low and in in-vivo experiments only low excitation levels are tolerated. The high time resolution of FLIM dictates the use of photomultiplier tubes or avalanche photo diodes with peak quantum efficiencies in the range of 10-40% while the present spectral imaging setup is equipped with a detector with a >90% peak quantum efficiency. Moreover, FLIM requires the use of band-pass filters to select the emission of the NADH and suppress autofluorescence contributions from other components such as flavins. In contrast, spectral imaging detects a broad spectral range covering the entire (protein-bound and free) NADH emission band. In our experience both techniques require comparable amounts of signal for reliable analyses of the protein-bound and free NADH components. The higher sensitivity of spectral imaging results in discrimination between the two components at relatively short pixel dwell times of 2 ms per pixel compared to 11 to 17 ms per pixel [9

9. V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009). [CrossRef]

12

12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]

] in FLIM studies. Furthermore, the spectral imaging provides valuable additional information on morphology and the presence of other components besides NADH.

Finally, this study has demonstrated the capability of nonlinear spectral metabolic imaging in obtaining both morphological and biochemical information to unravel the dynamic metabolic response of living cells inside tissues for a long period of time following acute ischemia. Our results represent clear indication that spectral imaging effectively discriminates protein-bound and free NADH.

Acknowledgments

This work is part of the research programme of the Stichting voor Fundamenteel Onderzoek der Materie (FOM, financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)).

References and links

1.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962). [CrossRef] [PubMed]

2.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]

3.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J. 93(3), 992–1007 (2007). [CrossRef] [PubMed]

4.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]

5.

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005). [CrossRef] [PubMed]

6.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007). [CrossRef] [PubMed]

7.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem. 280(26), 25119–25126 (2005). [CrossRef] [PubMed]

8.

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008). [CrossRef] [PubMed]

9.

V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009). [CrossRef]

10.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007). [CrossRef] [PubMed]

11.

Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009). [CrossRef] [PubMed]

12.

D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]

13.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]

14.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]

15.

J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009). [CrossRef]

16.

P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. Van Sark, D. J. Van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, “High sensitivity spectrograph for use in fluorescence microscopy,” Appl. Spectrosc. 55(8), 1005–1012 (2001). [CrossRef]

17.

A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. K. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011). [CrossRef] [PubMed]

18.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008). [CrossRef] [PubMed]

19.

A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011). [CrossRef] [PubMed]

20.

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988). [CrossRef] [PubMed]

21.

A. Amelink and H. J. Sterenborg, “Measurement of the local optical properties of turbid media by differential path-length spectroscopy,” Appl. Opt. 43(15), 3048–3054 (2004). [CrossRef] [PubMed]

22.

J. A. Gardecki and M. Maroncelli, “Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy,” Appl. Spectrosc. 52(9), 1179–1189 (1998). [CrossRef]

23.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992). [CrossRef] [PubMed]

24.

R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996). [CrossRef] [PubMed]

25.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008). [CrossRef] [PubMed]

26.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005). [CrossRef] [PubMed]

27.

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003). [CrossRef] [PubMed]

28.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002). [CrossRef] [PubMed]

29.

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970). [CrossRef] [PubMed]

30.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007). [CrossRef] [PubMed]

OCIS Codes
(170.1470) Medical optics and biotechnology : Blood or tissue constituent monitoring
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Noninvasive Optical Diagnostics

History
Original Manuscript: February 25, 2011
Revised Manuscript: March 19, 2011
Manuscript Accepted: March 29, 2011
Published: April 1, 2011

Citation
Jonathan A. Palero, Arjen N. Bader, Henriëtte S. de Bruijn, Angélique van der Ploeg van den Heuvel, Henricus J. C. M. Sterenborg, and Hans C. Gerritsen, "In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy," Biomed. Opt. Express 2, 1030-1039 (2011)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-2-5-1030


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References

  1. B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962). [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]
  3. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J. 93(3), 992–1007 (2007). [CrossRef] [PubMed]
  4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]
  5. E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005). [CrossRef] [PubMed]
  6. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007). [CrossRef] [PubMed]
  7. H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem. 280(26), 25119–25126 (2005). [CrossRef] [PubMed]
  8. V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008). [CrossRef] [PubMed]
  9. V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009). [CrossRef]
  10. M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007). [CrossRef] [PubMed]
  11. Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009). [CrossRef] [PubMed]
  12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]
  13. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]
  14. J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]
  15. J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009). [CrossRef]
  16. P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. Van Sark, D. J. Van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, “High sensitivity spectrograph for use in fluorescence microscopy,” Appl. Spectrosc. 55(8), 1005–1012 (2001). [CrossRef]
  17. A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. K. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011). [CrossRef] [PubMed]
  18. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008). [CrossRef] [PubMed]
  19. A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011). [CrossRef] [PubMed]
  20. G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988). [CrossRef] [PubMed]
  21. A. Amelink and H. J. Sterenborg, “Measurement of the local optical properties of turbid media by differential path-length spectroscopy,” Appl. Opt. 43(15), 3048–3054 (2004). [CrossRef] [PubMed]
  22. J. A. Gardecki and M. Maroncelli, “Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy,” Appl. Spectrosc. 52(9), 1179–1189 (1998). [CrossRef]
  23. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992). [CrossRef] [PubMed]
  24. R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996). [CrossRef] [PubMed]
  25. K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008). [CrossRef] [PubMed]
  26. N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005). [CrossRef] [PubMed]
  27. G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003). [CrossRef] [PubMed]
  28. M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002). [CrossRef] [PubMed]
  29. T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970). [CrossRef] [PubMed]
  30. Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007). [CrossRef] [PubMed]

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