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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 6 — May. 25, 2012
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Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation)

Hiroki Segawa, Masanari Okuno, Hideaki Kano, Philippe Leproux, Vincent Couderc, and Hiro-o Hamaguchi  »View Author Affiliations


Optics Express, Vol. 20, Issue 9, pp. 9551-9557 (2012)
http://dx.doi.org/10.1364/OE.20.009551


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Abstract

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

© 2012 OSA

1. Introduction

Multimodal nonlinear optical imaging is now widely used to become one of the most powerful methods to analyze biological systems such as living cells and tissues [1

1. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008). [CrossRef] [PubMed]

6

6. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008). [CrossRef] [PubMed]

]. Using various nonlinear optical processes, we can visualize the molecular compositions and their distribution in living cells with different contrast enhancements. One of the typical multimodal imaging techniques is the combination of two-photon excitation fluorescence (TPEF) with second harmonic generation (SHG) or third harmonic generation (THG) [1

1. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008). [CrossRef] [PubMed]

3

3. 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]

]. While TPEF visualizes labeled or intrinsic fluorophores, SH and TH complementarily probe non-labeled objects in cells and tissues. SH is sensitive to a non-centrosymmetric structure such as collagen fibers in a tissue and TH is sensitive to an interface where optical properties change [7

7. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999). [CrossRef] [PubMed]

]. Recently, multimodal nonlinear optical imaging has been applied to cancer diagnosis [8

8. S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011). [CrossRef]

].

A Ti:Sapphire laser oscillator is often employed for multimodal nonlinear optical imaging in order to obtain high photon density at the focal position [2

2. J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express 12(11), 2478–2486 (2004). [CrossRef] [PubMed]

4

4. H. Chen, H. Wang, M. N. Slipchenko, Y. Jung, Y. Shi, J. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009). [CrossRef] [PubMed]

,6

6. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008). [CrossRef] [PubMed]

]. With such a light source, however, we have to tune the laser wavelength to achieve resonances [6

6. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008). [CrossRef] [PubMed]

,9

9. A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002). [CrossRef] [PubMed]

]. This difficulty is bypassed with the use of the recently developed supercontinuum (or white-light laser source), since its broad spectral profile, typically spanning more than an octave, automatically fulfills multiple resonance conditions. Many resonances can thus be detected simultaneously in the form of a spectrum and full spectral information on molecules in a living cell is obtainable together with their detailed spatial distributions (spectral imaging) [10

10. H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007). [CrossRef] [PubMed]

,11

11. M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008). [CrossRef] [PubMed]

].

In the present study, we attempt tetra-modal molecular imaging using multiplex coherent anti-Stokes Raman scattering (CARS), SHG, THG and multiplex third-order sum frequency generation (TSFG). TSFG is a third order nonlinear optical process similar to THG. The TH signal is generated by single color excitation with an angular frequency of ω1 as ωTH = ω1 + ω1 + ω1. On the other hand, the TSFG process is expressed as ωTSF = ω1 + ω2 + ω3 (see Fig. 1(a)
Fig. 1 Diagrams of (a) the THG process, (b) the TSFG process and (c) the experimental setup for multiplex CARS, SHG, THG and multiplex TSFG multimodal imaging. PCF stands for photonic crystal fiber, SF short-pass filter, LF long-pass filter, NF notch filter, and DM dichroic mirror.
and 1(b)). Although second-order sum frequency generation microscopy is reported [12

12. N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006). [CrossRef] [PubMed]

], no report has been made on TSFG microscopy. In the present experimental condition, the TSF signal is generated from the combination of a narrow-band laser (ω1) and a broad-band white-light laser (ω2), with ωTSF = 2ω1 + ω2 and ω1 + 2ω2. Using this new multimodal imaging scheme, living cells are clearly visualized with different molecular contrasts through multiplex CARS, SHG, THG and multiplex TSFG simultaneously.

2. Experimental

The experimental setup used in the present study is schematically shown in Fig. 1(c). We use a Q-switched subnanosecond microchip Nd:YAG laser as the primary laser source. The fundamental output (1064 nm, 33 kHz) is equally divided by a beam splitter. One portion is used as the ω1 beam and the other is coupled into a photonic crystal fiber (PCF). The white-light continuum from the PCF (1.1 μm to 1.6 μm) is used as the ω2 radiation. The broadband ω2 beam passes through long-pass filters in order to eliminate the spectral component from 350 nm to 1064 nm. The ω1 and ω2 beams are superposed by a notch filter, which reflects only 1064-nm ω1 beam and transmits the other spectral components (ω2 beam), and then introduced into a modified inverted microscope (Nikon: ECLIPSE Ti). The ω1 and ω2 beams are tightly focused onto the sample by an objective (Nikon: Plan 100x / NA 1.25), and forward-propagating signals are collected by another objective (Nikon: Plan S Fluor 40x / NA 0.9). The sample is placed on a piezo stage (Mad City Lab: Nano-LP200), and is scanned in lateral direction in order to obtain spectra and images without moving the focal spots of the two laser beams. The signal beam is then divided by a dichroic mirror into the visible and near-infrared (NIR) spectral components, and they are detected separately by two sets of spectrometers and detectors. After eliminating the remnant of excitation laser beams by a short-pass (< 1050 nm) filter and a 1064-nm notch filter, we use a NIR-sensitive polychromator (Princeton Instruments: LS785) and a CCD camera (Princeton Instruments: PIXIS 100BR eXcelon) for the CARS signal detection in the NIR region. The CARS spectral coverage is over 3000 cm−1, so that we can measure both the fingerprint region and the X-H (C-H, N-H, and O-H) stretch region simultaneously. Visible spectral components containing SH, TH, and the multiplex TSF signals pass a short-pass (< 800 nm) filter for blocking the laser light, and are detected by another spectrometer (Princeton Instruments: SpectraPro 300i) and a CCD camera (Princeton Instruments: PIXIS 100BR eXcelon). Since two CCD cameras and the piezo stage are electronically synchronized, we can perform multiplex CARS, SHG, THG and multiplex TSFG spectral imaging simultaneously.

The sample was living budding yeast cell (a zygote of Saccharomyces cerevisiae and Saccharomyces bayanus) cultured in the YPD medium (a yeast complete medium containing yeast extract, glucose and polypeptone) except for those shown in the left side image of Figs. 3(a) and 3(b), which were cultured in the LA (lactic acid) medium. Before the measurement, yeast cells were sandwiched by two cover slips, and were sealed with Vaseline to prevent volatilization of the medium. Yeast cells were immobilized physically by a small sample volume between the two cover slips. The sample thickness was thus close to the diameter of budding yeast cells.

3. Results and discussion

The TH image in Fig. 2(d) is very similar to the TSF image in Fig. 2(e). It is known that, under a tightly focused condition, the TH signal is selectively generated at an interface [7

7. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999). [CrossRef] [PubMed]

,16

16. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1604–1610 (2002). [CrossRef]

]. This selectivity arises from the Gouy phase shift [17

17. S. Feng and H. G. Winful, “Physical origin of the Gouy phase shift,” Opt. Lett. 26(8), 485–487 (2001). [CrossRef] [PubMed]

] under a tightly focused condition that shortens the coherence length of the THG process. Thus, the TH signal vanishes for an isotropic material but becomes detectable for a system with optical inhomogeneity such as interfaces [7

7. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999). [CrossRef] [PubMed]

,16

16. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1604–1610 (2002). [CrossRef]

]. Since the TSFG process is described by the same framework with the THG process, it is not surprising if the TSF image is similar to the TH image. However, for multimodal imaging with CARS, there is a notable difference between THG and TSFG. The TSFG and CARS processes require both the ω1 and ω2 radiations. In contrast, the THG process requires only the ω1 radiation. Therefore, the CARS and TSF signals originate from the same position where the ω1 and ω2 radiations are spatially overlapped, while the CARS and TH signals do not. This is an advantage of CARS and TSFG simultaneous imaging. Moreover, the wavelengths of TH and TSF signals correspond to 355 nm and 359-457 nm, respectively. Therefore, we can also investigate the electronic resonance effect in the broad spectral range by simultaneous detection of TH and TSF.

4. Conclusion

We have developed a label-free tetra-modal molecular imaging system. The multiplex CARS, SHG, THG and multiplex TSFG signals are obtained simultaneously using a white-light laser source. This technique is applied to budding yeast cells. By the combination of SH-CARS and TSF-CARS, we visualized intracellular organelles such as SPB, lipid droplets and cell wall with their molecular information.

Acknowledgment

The authors gratefully acknowledge J. Ukon, HORIBA, Ltd. for assisting a fruitful collaboration between Japanese and French labs. This work is supported by a SENTAN-S from JST. H. Kano gratefully acknowledges financial support by Grand-Aid for Scientific Research on Priority Areas “Molecular Science for Supra Functional Systems” [477] from MEXT, and the Global COE Program for “Chemistry Innovation”. The authors thank LEUKOS Company for technical support with the dual-output supercontinuum laser source. The authors thank Suntory for supplying us the yeast cells.

References and links

1.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008). [CrossRef] [PubMed]

2.

J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express 12(11), 2478–2486 (2004). [CrossRef] [PubMed]

3.

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.

H. Chen, H. Wang, M. N. Slipchenko, Y. Jung, Y. Shi, J. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009). [CrossRef] [PubMed]

5.

J. W. Jhan, W. T. Chang, H. C. Chen, M. F. Wu, Y. T. Lee, C. H. Chen, and I. Liau, “Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing structure-constituent correlation of tissues,” Opt. Express 16(21), 16431–16441 (2008). [CrossRef] [PubMed]

6.

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008). [CrossRef] [PubMed]

7.

D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999). [CrossRef] [PubMed]

8.

S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011). [CrossRef]

9.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002). [CrossRef] [PubMed]

10.

H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007). [CrossRef] [PubMed]

11.

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008). [CrossRef] [PubMed]

12.

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006). [CrossRef] [PubMed]

13.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010). [CrossRef] [PubMed]

14.

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004). [CrossRef]

15.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005). [CrossRef] [PubMed]

16.

J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1604–1610 (2002). [CrossRef]

17.

S. Feng and H. G. Winful, “Physical origin of the Gouy phase shift,” Opt. Lett. 26(8), 485–487 (2001). [CrossRef] [PubMed]

18.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002). [CrossRef] [PubMed]

19.

B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975). [PubMed]

20.

S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol. 20(1), 1–28 (2004). [CrossRef] [PubMed]

21.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006). [CrossRef] [PubMed]

22.

V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13(20), 8263–8276 (2005). [CrossRef] [PubMed]

OCIS Codes
(190.4180) Nonlinear optics : Multiphoton processes
(300.6230) Spectroscopy : Spectroscopy, coherent anti-Stokes Raman scattering
(180.4315) Microscopy : Nonlinear microscopy
(180.5655) Microscopy : Raman microscopy

ToC Category:
Microscopy

History
Original Manuscript: January 30, 2012
Revised Manuscript: March 24, 2012
Manuscript Accepted: March 28, 2012
Published: April 11, 2012

Virtual Issues
Vol. 7, Iss. 6 Virtual Journal for Biomedical Optics

Citation
Hiroki Segawa, Masanari Okuno, Hideaki Kano, Philippe Leproux, Vincent Couderc, and Hiro-o Hamaguchi, "Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation)," Opt. Express 20, 9551-9557 (2012)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-20-9-9551


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References

  1. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc.229(1), 141–150 (2008). [CrossRef] [PubMed]
  2. J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express12(11), 2478–2486 (2004). [CrossRef] [PubMed]
  3. 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. H. Chen, H. Wang, M. N. Slipchenko, Y. Jung, Y. Shi, J. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express17(3), 1282–1290 (2009). [CrossRef] [PubMed]
  5. J. W. Jhan, W. T. Chang, H. C. Chen, M. F. Wu, Y. T. Lee, C. H. Chen, and I. Liau, “Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing structure-constituent correlation of tissues,” Opt. Express16(21), 16431–16441 (2008). [CrossRef] [PubMed]
  6. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol.164(1), 140–145 (2008). [CrossRef] [PubMed]
  7. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express5(8), 169–175 (1999). [CrossRef] [PubMed]
  8. S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev.5(4), 496–512 (2011). [CrossRef]
  9. A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A.99(17), 11014–11019 (2002). [CrossRef] [PubMed]
  10. H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem.79(23), 8967–8973 (2007). [CrossRef] [PubMed]
  11. M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett.33(9), 923–925 (2008). [CrossRef] [PubMed]
  12. N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc.128(11), 3482–3483 (2006). [CrossRef] [PubMed]
  13. M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl.49(38), 6773–6777 (2010). [CrossRef] [PubMed]
  14. H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B108(48), 18762–18771 (2004). [CrossRef]
  15. Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry44(30), 10009–10019 (2005). [CrossRef] [PubMed]
  16. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B19(7), 1604–1610 (2002). [CrossRef]
  17. S. Feng and H. G. Winful, “Physical origin of the Gouy phase shift,” Opt. Lett.26(8), 485–487 (2001). [CrossRef] [PubMed]
  18. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J.82(1), 493–508 (2002). [CrossRef] [PubMed]
  19. B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol.124(1), 511–523 (1975). [PubMed]
  20. S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol.20(1), 1–28 (2004). [CrossRef] [PubMed]
  21. D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006). [CrossRef] [PubMed]
  22. V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express13(20), 8263–8276 (2005). [CrossRef] [PubMed]

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