Supercontinuum Dynamically Visualizes a Dividing Single Cell

Oct 30, 2007 - Using this multiplex CARS technique, we have been successful in ..... 15GS0204) from the Ministry of Education, Culture, Sports, Scienc...
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Anal. Chem. 2007, 79, 8967-8973

Supercontinuum Dynamically Visualizes a Dividing Single Cell Hideaki Kano and Hiro-o Hamaguchi*

Department of Chemistry, School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan

During cell division, various organelles behave dynamically. Visualization of these dynamic behaviors of organelles is a promising one step forward for understanding life at the molecular level. One- or two-photon excited fluorescence microscopy has so far been used for visualizing these cell dynamics. The fluorescent probe introduced into a living cell can visualize the spatial distribution of a target molecule in real time, enabling the tracing of cell dynamics at the molecular level. Introducing a fluorescent probe into a cell, however, may alter the physical and chemical conditions of the cell. Here we show a new method for direct (no need for staining cells) visualization of living cell processes with coherent antiStokes Raman scattering (CARS) spectroscopy. A new light source, supercontinuum generated from a photonic crystal fiber, has facilitated ultrabroadband (>3500 cm-1) multiplex CARS spectroscopy and imaging with high molecular specificity. Using this multiplex CARS technique, we have been successful in tracing the whole cell division process, the splitting of a mother cell into two daughter cells, appearance and disappearance of septum, and dynamic distribution changes of organelles consisting of lipid membrane. The supercontinuum has also facilitated simultaneous measurement of the CARS and two-photon excited fluorescence (TPEF) spectra, enabling what we call multiple nonlinear spectral imaging. Three-dimensional image reconstruction of a living cell with high speed is now possible to elucidate more detailed molecular-level dynamics inside a dividing living cell. The supercontinuum (SC) light source has been receiving considerable attention as a cutting-edge tool for optical microscopy. In comparison with conventional incoherent light source or monochromatic laser source, the SC light source has the following two advantages.1 First, the SC spectrum spans several optical octaves.2 Second, the temporal duration of the SC is sufficiently short, and hence the peak power is sufficiently high to induce multiphoton processes. In the past few years, various kinds of new microscopic and microspectroscopic techniques * To whom correspondence should be addressed. Hiro-o Hamaguchi, Department of Chemistry, School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan. Phone: 81-3-5841-4327. Fax: 81-3-3818-4621. E-mail: [email protected]. (1) Russell, P. Science 2003, 299, 358-362. (2) Ranka, J. K.; Windeler, R. S.; Stentz, A. J. Opt. Lett. 2000, 25, 25-27. 10.1021/ac071416z CCC: $37.00 Published on Web 10/30/2007

© 2007 American Chemical Society

have been explored using the SC light source, such as coherent anti-Stokes Raman scattering (CARS) microscopy,3 CARS microspectroscopy,4-8 and two-photon excited fluorescence microscopy.9,10 Among them, CARS microspectroscopy is the most promising, because it enables us to perform simultaneous multivibrational mode imaging for unstained samples at the molecular level.11,12 CARS is one of the third-order nonlinear optical processes that provide vibrational spectra. In the CARS process, both pump (ωp) and Stokes (ωs) laser fields interact with a molecule. If the frequency difference (ωp- ωs) between those of the pump and Stokes lasers matches a particular vibrational frequency of the sample molecule, a strong CARS signal is generated at the frequency of 2ωp - ωs. Since molecules have many Raman active vibrational modes, the spectral profile of the CARS signal provides important information on molecular structure and dynamics in the form of vibrational spectra. Although CARS microscopy has made remarkable progress,13-18 CARS microspectroscopy is still under development. In order to obtain CARS spectrum under a microscope, a broadband Stokes laser19-21 and/or pulse shaping techniques22-24 have been exploited. However, the spectral cover(3) Paulsen, H. N.; Hilligsoe, K. M.; Thogersen, J.; Keiding, S. R.; Larsen, J. J. Opt. Lett. 2003, 28, 1123-1125. (4) Yakovlev, V. V. J. Raman Spectrosc. 2003, 34, 957-964. (5) Kee, T. W.; Cicerone, M. T. Opt. Lett. 2004, 29, 2701-2703. (6) Kano, H.; Hamaguchi, H. Appl. Phys. Lett. 2005, 86, 121113. (7) von Vacano, B.; Wohlleben, W.; Motzkus, M. Opt. Lett. 2006, 31, 413415. (8) Ivanov, A. A.; Podshivalov, A. A.; Zheltikov, A. M. Opt. Lett. 2006, 31, 33183320. (9) McConnell, G.; Riis, E. J. Biomed. Opt. 2004, 9, 922-927. (10) Isobe, K.; Watanabe, W.; Matsunaga, S.; Higashi, T.; Fukui, K.; Itoh, K. Jpn. J. Appl. Phys., Part 2 2005, 44, L167-L169. (11) Kano, H.; Hamaguchi, H. J. Phys. Chem. B 2006, 110, 3120-3126. (12) Kano, H.; Hamaguchi, H. Chem. Lett. 2006, 35, 1124-1125. (13) Duncan, M. D.; Reintjes, J.; Manuccia, T. J. Opt. Lett. 1982, 7, 350-352. (14) Zumbusch, A.; Holtom, G. R.; Xie, X. S. Phys. Rev. Lett. 1999, 82, 41424145. (15) Hashimoto, M.; Araki, T.; Kawata, S. Opt. Lett. 2000, 25, 1768-1770. (16) Cheng, J.-X.; Jia, Y. K.; Zheng, G.; Xie, X. S. Biophys. J. 2002, 83, 502509. (17) Ichimura, T.; Hayazawa, N.; Hashimoto, M.; Inouye, Y.; Kawata, S. Phys. Rev. Lett. 2004, 92, 220801. (18) Evans, C. L.; Potma, E. O.; Puoris’haag, M.; Cote, D.; Lin, C. P.; Xie, X. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16807-16812. (19) Otto, C.; Voroshilov, A.; Kruglik, S. G.; Greve, J. J. Raman Spectrosc. 2001, 32, 495-501. (20) Mueller, M.; Schins, J. M. J. Phys. Chem. B 2002, 106, 3715-3723. (21) Cheng, J.-X.; Xie, X. S. J. Phys. Chem. B 2004, 108, 827-840. (22) Dudovich, N.; Oron, D.; Silberberg, Y. Nature 2002, 418, 512-514. (23) Marks, D. L.; Boppart, S. A. Phys. Rev. Lett. 2004, 92, 123905. (24) von Vacano, B.; Buckup, T.; Motzkus, M. Opt. Lett. 2006, 31, 24952497.

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age was limited by the laser bandwidth. This problem has been solved by the SC provided by photonic crystal fibers (PCFs)4,6,25 or a tapered fiber.5 The spectral coverage of multiplex CARS microspectroscopy based on PCFs has been extended to be about 3000 cm-1,26 which is broad enough to cover not only the fingerprint region but also the C-H, N-H, and O-H stretch regions. In addition to the multiplex CARS process, two-photon excited fluorescence (TPEF)27 can also be measured using the SC. The combined method of multiplex CARS with TPEF, namely, multiple nonlinear spectral imaging, has proven useful particularly for life sciences.28 Nonlinear spectral imaging is highly useful for investigating dynamical behavior of living cells at the molecular level. In the present study, we focus on the cell cycle of fission yeast, Schizosaccharomyces pombe. Fission yeast is one of the simplest unicellular eukaryotes. The cell cycle of fission yeast has been extensively studied, because it has a lot of features similar to those of higher eukaryotes. Therefore, fission yeast is one of the most widely used model organism in various research fields of biochemistry and genetics. Previously, we reported the structure, transformation, and bioactivity of single living yeast cells by confocal Raman microspectroscopy29,30 and multiplex CARS microspectroscopy.28 We have successfully visualized not only the distributions of molecular species but also the cell activity of the growing and dying yeast cells. In particular, we have found a strong Raman band in the mitochondria of a living fission yeast cell, which sharply reflects the metabolic activity of mitochondria.29,30 We call it the “Raman spectroscopic signature of life”. In order to investigate the Raman spectroscopic signature of life further and to elucidate detailed dynamics in the course of cell division, nonlinear spectral imaging is one of the most promising methods because it provides full spectral information at each spatial point with high speed. This work demonstrates the capability of the nonlinear spectral imaging technique for investigating dynamical behaviors inside a living cell. EXPERIMENTAL SECTION Materials. The sample is living fission yeast Schizosaccharomyces pombe (S. pombe). In order to visualize the nuclei or mitochondria of S. pombe cells, a green fluorescent protein (GFP) was used. Details of the procedure have been described elsewhere.29 These cells were cultured in YE liquid medium supplemented with 100 mg/L Geneticin (Sigma). A small amount of the growing cells and the YE broth were spread on a slide-glass and sandwiched with a cover glass. The edge of the cover glass was sealed with Vaseline to prevent the volatilization of water. Because of a small quantity of the sample, yeast cells were immobilized between a slide-glass and a cover glass. All measurements were performed at room temperature. Methods. The nonlinear spectral imaging experiment was carried out by an ultrabroadband multiplex CARS microspectro(25) Petrov, I. G.; Yakovlev, V. V. Opt. Express 2005, 13, 1299. (26) Kano, H.; Hamaguchi, H. J. Raman Spectrosc. 2006, 37, 411-415. (27) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73-76. (28) Kano, H.; Hamaguchi, H. Opt. Express 2005, 13, 1322-1327. (29) Huang, Y.-S.; Karashima, T.; Yamamoto, M.; Hamaguchi, H. Biochemistry 2005, 44, 10009-10019. (30) Naito, Y.; Toh-e, A.; Hamaguchi, H. J. Raman Spectrosc. 2005, 36, 837839.

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Figure 1. (a) Multiplex CARS spectrum of a polystyrene bead with a diameter of 10 µm. The exposure time is 1 s. (b) Intensity-corrected CARS spectrum of a polystyrene bead. The CARS spectrum of the bead was corrected by that of the nonresonant background signal of an underneath cover glass under the same experimental conditions. A sharp structure at 3500 cm-1 is an artifact due to the intensity correction.

scopic system, which was developed by our group. Details of the system have been described elsewhere.6,26 Briefly, an unamplified mode-locked Ti:sapphire laser oscillator (Coherent, Vitesse-800) was used as the laser source. Typical wavelength, temporal duration, pulse energy, and repetition rate were 800 nm, 100 fs, 12 nJ, and 80 MHz, respectively. A portion of the output from the oscillator was seeded into a PCF (crystal fiber, NL-PM-750) for generating the SC. The fundamental of the Ti:sapphire laser oscillator and the SC were used for the pump (ω1) and Stokes (ω2) lasers, respectively. In order to obtain Raman spectrum with high spectral resolution, the pump laser pulse was spectrally filtered using a narrow band-pass filter. The bandwidth was about 20 cm-1. In the present setup, we used only the near infrared (NIR) spectral component of the SC. Two laser pulses were superimposed collinearly using an 800 nm Notch filter and then tightly focused onto the sample with a 40 × 0.9 NA microscope objective attached to an inverted microscope (Nikon, TE2000-S). A 40×, 0.6 NA microscope objective was used to collect the forward-propagating CARS signal. Finally, the CARS signal was guided to a polychromator (Acton, SpectraPro-300i) and was detected by a CCD camera (Roper Scientific, Spec-10:400BR/XTE or PIXIS 100B). In order to achieve high-speed imaging capability, a CCD camera (PIXIS 100B) was used with an optimal configuration. Multiplex CARS images were measured by a point-by-point acquisition of the CARS spectrum. The sample was moved using piezo-driven xyz translators (MadCity, NanoLP100). The pump and Stokes pulse energies were at most 100 and 100 pJ, respectively. The spatial resolution was estimated to be about 0.5 and 1.5 µm for the lateral and axial directions, respectively.

Figure 2. (a). CARS and TPEF spectra of a living yeast cell at G1 (gap-1) phase (uncorrected). Each spectrum is obtained at the position of septum (red), mitochondria (green), and surrounding water (black). All spectra are spatially averaged in the region of (0.3 µm2). Exposure time is 200 ms. Apparent peaks at the Raman shift of approximately 3200 cm-1 are due to the spectral profile of the Stokes laser; (b and c) nonlinear spectral imaging of a living yeast cell at the G1 phase. CARS at the C-H stretching vibrational mode (b) and TPEF (c) images are indicated. The scale bar corresponds to 2 µm. The CARS and TPEF spectra for septum, mitochondria, and surrounding water in Figure 2a are obtained at the position of a cross in Figure 2b, cross in Figure 2c, and open circle in Figure 2c, respectively.

RESULTS AND DISCUSSION Figure 1a shows a typical multiplex CARS spectrum of a polystyrene bead with a diameter of 10 µm. This result demonstrates ultrabroadband multiplex CARS detection capability by our apparatus. The spectral profile of the CARS signal is deformed mainly by that of the NIR Stokes laser (Figure 1a). The intensity correction is carried out using the nonresonant background signal of an underneath cover glass measured under the same experimental conditions. The corrected CARS spectrum is shown in Figure 1b. The CARS spectrum of a polystyrene bead shows many peaks due to various Raman resonances. The intense peaks around 3058, 2850, and 995 cm-1 originate from the aromatic C-H, aliphatic C-H, and ring stretch vibrational modes, respectively. The nonresonant background is also overlapped in the multiplex CARS spectrum, which gives rise to dispersive line shapes due to the interference effect. As shown in Figure 1b, our setup covers a wide range of vibrational resonances (>3500 cm-1) at a fixed pump-Stokes delay. The wavenumber region can be tuned by the pump-Stokes delay because the Stokes laser pulses have a PCF-length dependent temporal chirp.26

Figure 2a shows typical CARS and TPEF spectra of a living yeast cell at the G1 (gap-1) phase, when a septum is formed at the middle of the cell. We use a yeast cell whose mitochondria are tagged by GFP. Each spectrum is obtained at the position of the septum (red), mitochondria (green), and surrounding water (black). All spectra are spatially averaged in the region of 0.3 µm2. In order to avoid overlap between the CARS and TPEF signals, the spectral profile of the SC and the pump-Stokes delay were optimized. At the positions of the septum and mitochondria, a strong CARS signal is observed around the Raman shift of 2860 cm-1. From our previous Raman study,29 the septum and mitochondria are known to give strong Raman signals at the C-H stretch region. This strong Raman signal at 2860 cm-1 is assigned to the C-H stretch modes.6 It is noted that the CARS signal inside of the cell is less intense at the Raman shift of approximately 3000 cm-1 than that of surrounding water. It is caused by the destructive interference between the CARS and the nonresonant background, giving rise to dispersive line shapes in multiplex CARS spectra. In the present condition, vibrational resonance is not clearly Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 3. (a). XYZ pseudo-color image stack of the CARS signal at the Raman shift of the C-H stretching vibrational mode; (b) XYZ pseudocolor image stack of the TPEF signal. The sample is a living yeast cell, whose nucleus is labeled by GFP. Each lateral (XY) image was obtained by ∆Z ) 305 nm step. In total, 21 images are displayed from the top (upper left) to the bottom (lower right) part of the yeast cell. Exposure time at each spatial point is 50 ms; (c) three-dimensional volume rendering of datasets of the CARS and TPEF signals. Orange, brown, and green areas correspond to the strong CARS sinal intensity, moderate (about one-half in the intensity in comparison with the orange area) CARS signal intensity, and TPEF signal intensity, respectively.

observed in the fingerprint region. It is partly explained by weak and congested resonances in this region, which are overwhelmed by the nonresonant background. It is noted that an optimum delay 8970

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time is found to reduce the nonresonant background significantly, which enables us to investigate a detailed spectral profile in the fingerprint region.26,31,32 On the other hand, broad and structure-

Figure 4. CARS at the C-H stretching vibrational mode (a) and TPEF (b) images, respectively. The sample is a living yeast cell, whose nucleus is labeled by GFP. The scale bar corresponds to 2 µm. The number at the upper left shows the time course of the observation. Exposure time at each spatial point is 50 ms. Lateral (XY) images consist of 61 × 61 pixels and are measured in 3.8 min per one image.

less peaks are observed at around 510 nm. From the peak position, this band is assigned to TPEF due to GFP. Since mitochondria of the yeast cell are labeled by GFP, the TPEF signal should be strongly observed at the position of mitochondria. (31) Pestov, D.; Murawski, R. K.; Ariunbold, G. O.; Wang, X.; Zhi, M.; Sokolov, A. V.; Sautenkov, V. A.; Rostovtsev, Y. V.; Dogariu, A.; Huang, Y.; Scully, M. O. Science 2007, 316, 265-268. (32) Petrov, G. I.; Arora, R.; Yakovlev, V. V.; Wang, X.; Sokolov, A. V.; Scully, M. O. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 776-779.

Figure 2b,c shows the result of multiple nonlinear spectral imaging of a living yeast cell at the G1 phase. CARS at the C-H stretching vibrational mode and TPEF images are indicated in Figure 2b and Figure 2c, respectively. Since multiplex CARS microspectroscopy enables us to obtain full spectral information of the CARS signal, we can extract only the vibrationally resonant CARS signal at each spatial point by taking account of a dispersive line shape of the CARS spectrum.28 This process significantly Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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enhances the vibrational contrast without any real-space image data processing. The CARS and TPEF spectra for the septum, mitochondria, and surrounding water shown in Figure 2a are obtained at the position of a cross in Figure 2b, cross in Figure 2c, and open circle in Figure 2c, respectively. Since mitochondria in the yeast cell are labeled by GFP, Figure 2c indicates the distribution of mitochondria. It is clear that no GFP signal is observed at the position of septum. In order to examine both CARS and TPEF images in more detail, these images are merged (Figure 2d). The yellow color in Figure 2d shows that both CARS and TPEF signals are strong at that point. Although the CARS image is in accord with the TPEF image in several areas, they are not perfectly coincident with each other. It indicates that the CARS signal at the C-H stretch mode probes not only mitochondria but also other organelles having many C-H bonds. One of the candidates is the endoplasmic reticulum, which is also composed of lipid membrane and is widely found in eukaryotic cells. Further work such as CARS imaging at the fingerprint region will shed more light on these unidentified organelles. Owing to the high three-dimensional spatial resolution and large penetration depth of the NIR laser beams, the nonlinear spectral imaging technique provides optical sectioning of a thick sample. Figure 3a shows a XYZ pseudo-color image stack of the CARS signal at the Raman shift of the C-H stretch mode. The signal intensity at the red region is larger than that at the blue. Figure 3b shows a XYZ pseudo-color image stack of the TFEP signal. The sample is a living yeast cell whose nucleus is labeled by GFP. Each lateral (XY) image was obtained by ∆Z ) 305 nm step. In total, 21 images are displayed from the top (upper left) to the bottom (lower right) part of the yeast cell. Exposure time at each spatial point is 50 ms. It is clear that the CARS image gives strong vibrational contrast around the nucleus. As discussed in the previous subsection, this structure is partly due to membranous organelles such as mitochondria. On the other hand, the nucleus is clearly visualized by the TPEF image. Figure 3c shows the three-dimensional volume rendering of the data sets of the CARS and TPEF signals. Orange, brown, and green areas correspond to the strong CARS signal intensity, and moderate (about one-half in the intensity in comparison with the red area) CARS signal intensity, and the TPEF signal, respectively. A living yeast cell is well reconstructed as a three-dimensional object. The nonlinear spectral imaging technique allows us to obtain both multiplex CARS and TPEF images with high speed. We have applied this technique to the cell division process. Figure 4a and Figure b shows a CARS at the C-H stretch mode and TPEF pseudo-color images, respectively. The sample is a living yeast cell whose nucleus is labeled by GFP. The number at the upper left shows the time of observation. Exposure time at each spatial point is 50 ms. Each image is constructed from the bottom to the top part of the figure by scanning the sample. Lateral (XY) images consist of 61 × 61 pixels and are measured in 3.8 min per one image, which determines the temporal resolution in the present experiment. Two living cells are found at the G1 phase. First, both cells have septa at around the center of the cell. A strong CARS signal due to membranous organelles is also observed at each compartment. First, we focus on the yeast cell at the lower side 8972

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of the images. The CARS signal at the septum decreases in the cell division (from images a-1 to a-4). Finally, the cell splits into two daughter cells (a-4). One of the daughter cells moves (image a-8). For the other daughter cell, the CARS signal inside of the cell does not show significant distribution change from images a-10 to a-21 in comparison with that in the dividing process (from images a-1 to a-4). Second, the yeast cell at the upper side is discussed. The CARS signal intensity at the position of septum gradually increases from images a-1 to a-10. Next, it decreases slightly at around image a-13, and the cell splits into two daughter cells at image a-15. After the cell division, the daughter cell in the field of view still shows dynamic distribution change of the CARS intensity inside of the cell, which is in contrast to the cell at the lower part. It could be explained by the migration of organelles in the axial direction. As shown in Figure 4b, the relative positions of nuclei inside of the cell do not change significantly in the course of the cell cycle during the observation. It is also noted that the TPEF signal intensity becomes weaker and weaker in the course of the cell division. It is probably due to a photobleaching effect by laser irradiation. On the other hand, the CARS signal intensity does not deteriorate. This result manifests another advantage of the CARS imaging, which does not suffer inherently from the photobleaching effect. CONCLUSIONS In conclusion, we have developed the multiple nonlinear spectral imaging technique, which provides ultrabroadband (>3500 cm-1) CARS and TPEF spectra simultaneously. This technique is applied to investigate a living fission yeast cell. Two- and threedimensional images of a living cell are successfully obtained with high speed. Several organelles such as septum and membranous organelles are visualized by the CARS signal at the C-H stretch mode. Since we have full spectral information of the CARS signal, any vibrationally resonant signal can be extracted by spectral analysis without degradation of the spatial resolution. On the other hand, the TPEF image reveals distribution of an organelle labeled by GFP such as mitochondria or nucleus. Furthermore, we have studied the cell cycle by nonlinear spectral imaging. It clearly visualizes the splitting of a mother cell to two daughter cells, appearance and disappearance of septum, and dynamic distribution change of membranous organelles such as the mitochondria. The signal intensity of the CARS (and Raman) process in general depends on the concentration and cross section of the target molecule. For instance, nucleic acid in a yeast cell is weakly observed by spontaneous Raman microspectroscopy.29 In order to trace dynamical behavior of a living cell, the combination of CARS with the fluorescence technique, namely, nonlinear spectral imaging, is therefore one of the most promising methods. Since CARS microspectroscopy can trace unknown molecules which have not been tagged so far by fluorescence techniques, it can be used to explore dynamics of such molecules. Nonlinear spectral imaging opens up a new possibility for looking in more detail into the distribution of organelles and their dynamics during cell division. ACKNOWLEDGMENT This research is supported by a Grant-in-Aid for Creative Scientific Research (Grant No. 15GS0204) from the Ministry of

Education, Culture, Sports, Science, and Technology of Japan. H.K. is supported by a Grant-in-Aid for Young Scientists (B) (Grant No. 18750007) from Japan Society for the Promotion of Science and research grants from The Kurata Memorial Hitachi Science and Technology Foundation. The authors thank Mr. T. Nakatsuka

for his help in sample preparation and Dr. T. Karashima and Prof. M. Yamamoto for providing the S. pombe strain. Received for review July 3, 2007. Accepted August 23, 2007. AC071416Z

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