Abstract

We demonstrate two complementary types of microscopy using an identical setup for single-pulse coherent anti-Stokes Raman scattering (CARS) imaging, which employs an ultrabroadband laser pulse with a spectral bandwidth of 4800 cm-1 and enables the suppression of nonresonant CARS signals. One is a novel type of microscopy that uses spectral phase modulation for the selective excitation of a single Raman mode. The selective excitation is achieved by the modulated pulse focusing its difference-frequency spectrum into a narrow spectral region. Another type is Fourier-transform CARS (FT-CARS) microspectroscopy based on the measurement of the CARS spectrum obtained from the Fourier-transform of the interferometric autocorrelation (IAC) signal. Vibrational spectral imaging of chemical and biological samples is demonstrated using the two types of microscopy.

© 2006 OSA

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References

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  1. M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett. 7(8), 350–352 (1982).
    [CrossRef]
  2. A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
    [CrossRef]
  3. E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers,” Opt. Lett. 27(13), 1168–1170 (2002).
    [CrossRef]
  4. J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
    [CrossRef]
  5. A. Volkmer, J. Cheng, and X. S. Xie, “Vibrational Imaging with High Sensitivity via Epidetected Coherent Anti-Stokes Raman Scattering Microscopy,” Phys. Rev. Lett. 87, 023901/1–4 (2001).
    [CrossRef]
  6. E. O. Potma, C. L. Evans, and X. S. Xie, “Heterodyne coherent anti-Stokes Raman scattering (CARS) imaging,” Opt. Lett. 31(2), 241–243 (2006).
    [CrossRef]
  7. C. L. Evans, E. O. Potma, and X. S. Xie, “Coherent anti-stokes raman scattering spectral interferometry: determination of the real and imaginary components of nonlinear susceptibility χ(3) for vibrational microscopy,” Opt. Lett. 29(24), 2923–2925 (2004).
    [CrossRef]
  8. J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
    [CrossRef]
  9. J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26(17), 1341–1343 (2001).
    [CrossRef]
  10. A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
    [CrossRef]
  11. N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
    [CrossRef]
  12. D. Oron, N. Dudovich, and Y. Silberberg, “Single-Pulse Phase-Contrast Nonlinear Raman Spectroscopy,” Phys. Rev. Lett. 89, 273001/1–4 (2002).
    [CrossRef]
  13. N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
    [CrossRef]
  14. H. Kano and H. Hamaguchi, “Vibrationally resonant imaging of a single living cell by supercontinuum-based multiplex coherent anti-Stokes Raman scattering microspectroscopy,” Opt. Express 13(4), 1322–1327 (2005).
    [CrossRef]
  15. T. Hellerer, A. M. K. Enejder, and A. Zumbuscha, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulse,” Appl. Phys. Lett. 85(1), 25–27 (2004).
    [CrossRef]
  16. J. P. Ogilvie, E. Beaurepaire, A. Alexandrou, and M. Joffre, “Fourier-transform coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 31(4), 480–482 (2006).
    [CrossRef]
  17. K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A. 77, 063832/1–13 (2008).
    [CrossRef]
  18. K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
    [CrossRef]
  19. K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses,” submitted.
  20. M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
    [CrossRef]

2006 (2)

2005 (2)

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

H. Kano and H. Hamaguchi, “Vibrationally resonant imaging of a single living cell by supercontinuum-based multiplex coherent anti-Stokes Raman scattering microspectroscopy,” Opt. Express 13(4), 1322–1327 (2005).
[CrossRef]

2004 (2)

2003 (1)

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
[CrossRef]

2002 (3)

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
[CrossRef]

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers,” Opt. Lett. 27(13), 1168–1170 (2002).
[CrossRef]

2001 (2)

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26(17), 1341–1343 (2001).
[CrossRef]

1999 (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

1989 (1)

K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
[CrossRef]

1982 (1)

1979 (1)

J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
[CrossRef]

Alexandrou, A.

Baum, P.

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Beaurepaire, E.

Bodermann, B.

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Book, L. D.

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
[CrossRef]

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26(17), 1341–1343 (2001).
[CrossRef]

Cheng, J.

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

Cheng, J. X.

Dudovich, N.

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
[CrossRef]

Duncan, M. D.

Enejder, A. M. K.

T. Hellerer, A. M. K. Enejder, and A. Zumbuscha, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulse,” Appl. Phys. Lett. 85(1), 25–27 (2004).
[CrossRef]

Evans, C. L.

Greve, M.

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Hamaguchi, H.

Hellerer, T.

T. Hellerer, A. M. K. Enejder, and A. Zumbuscha, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulse,” Appl. Phys. Lett. 85(1), 25–27 (2004).
[CrossRef]

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

Joffre, M.

Jones, D. J.

Kano, H.

Manuccia, T. J.

Mogi, K.

K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
[CrossRef]

Naganuma, K.

K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
[CrossRef]

Ogilvie, J. P.

Oron, D.

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
[CrossRef]

Oudar, J. L.

J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
[CrossRef]

Potma, E. O.

Reintjes, J.

Riedle, E.

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Shen, Y. R.

J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
[CrossRef]

Silberberg, Y.

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
[CrossRef]

Smith, R. W.

J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
[CrossRef]

Telle, H. R.

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Volkmer, A.

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
[CrossRef]

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

Xie, X. S.

E. O. Potma, C. L. Evans, and X. S. Xie, “Heterodyne coherent anti-Stokes Raman scattering (CARS) imaging,” Opt. Lett. 31(2), 241–243 (2006).
[CrossRef]

C. L. Evans, E. O. Potma, and X. S. Xie, “Coherent anti-stokes raman scattering spectral interferometry: determination of the real and imaginary components of nonlinear susceptibility χ(3) for vibrational microscopy,” Opt. Lett. 29(24), 2923–2925 (2004).
[CrossRef]

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers,” Opt. Lett. 27(13), 1168–1170 (2002).
[CrossRef]

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
[CrossRef]

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

J. X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26(17), 1341–1343 (2001).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

Yamada, H.

K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
[CrossRef]

Ye, J.

Zumbusch, A.

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

Zumbuscha, A.

T. Hellerer, A. M. K. Enejder, and A. Zumbuscha, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulse,” Appl. Phys. Lett. 85(1), 25–27 (2004).
[CrossRef]

Appl. Phys. B (1)

M. Greve, B. Bodermann, H. R. Telle, P. Baum, and E. Riedle, “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy,” Appl. Phys. B 81(7), 875–879 (2005).
[CrossRef]

Appl. Phys. Lett. (3)

J. L. Oudar, R. W. Smith, and Y. R. Shen, “Polarization-sensitive coherent anti-Stokes Raman spectroscopy,” Appl. Phys. Lett. 34(11), 758–760 (1979).
[CrossRef]

A. Volkmer, L. D. Book, and X. S. Xie, “Time-resolved coherent anti-Stokes Raman scattering microscopy: Imaging based on Raman free induction decay,” Appl. Phys. Lett. 80(9), 1505–1507 (2002).
[CrossRef]

T. Hellerer, A. M. K. Enejder, and A. Zumbuscha, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulse,” Appl. Phys. Lett. 85(1), 25–27 (2004).
[CrossRef]

IEEE J. Quantum Electron. (1)

K. Naganuma, K. Mogi, and H. Yamada, “General method for ultrashort light pulse chirp measurement,” IEEE J. Quantum Electron. 25(6), 1225–1233 (1989).
[CrossRef]

J. Chem. Phys. (1)

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherent anti-Stokes Raman spectroscopy in the fingerprint spectral region,” J. Chem. Phys. 118(20), 9208–9215 (2003).
[CrossRef]

J. Phys. Chem. B (1)

J. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An Epi-Detected Coherent Anti-Stokes Raman Scattering (E-CARS) Microscope with High Spectral Resolution and High Sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

Nature (1)

N. Dudovich, D. Oron, and Y. Silberberg, “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature 418(6897), 512–514 (2002).
[CrossRef]

Opt. Express (1)

Opt. Lett. (6)

Phys. Rev. Lett. (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

Other (4)

A. Volkmer, J. Cheng, and X. S. Xie, “Vibrational Imaging with High Sensitivity via Epidetected Coherent Anti-Stokes Raman Scattering Microscopy,” Phys. Rev. Lett. 87, 023901/1–4 (2001).
[CrossRef]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A. 77, 063832/1–13 (2008).
[CrossRef]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses,” submitted.

D. Oron, N. Dudovich, and Y. Silberberg, “Single-Pulse Phase-Contrast Nonlinear Raman Spectroscopy,” Phys. Rev. Lett. 89, 273001/1–4 (2002).
[CrossRef]

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Figures (7)

Fig. 1.
Fig. 1.

Time-spectral distribution of excitation pulse for selective excitation of a single Raman mode.

Fig. 2.
Fig. 2.

Experimental setup. BS: beam splitter, OB: objective lens, DM: dichroic mirror, PMT1: photomultiplier tube for forward-detection, PMT2: photomultiplier tube for epi-detection, SPF: short-pass filter, SLM: spatial light modulator.

Fig. 3.
Fig. 3.

(a) GDD dependence of the DF spectrum. (b) Focusing the DF spectrum into various narrow spectral regions.

Fig. 4.
Fig. 4.

Intensity cross sections of acetone, sandwiched between a hole-slide glass and a cover slip, by selective excitation at 2930 cm-1 (red) and 3400 cm-1 (green).

Fig. 5.
Fig. 5.

CARS images of an unstained HeLa cell by selective excitation at 2930 cm-1 (a), 3200 cm-1 (b) and 3400 cm-1 (c). Scale bar is 6 µm.

Fig. 6.
Fig. 6.

(a) Fourier spectrum of CARS-IAC signal from acetone. (b-d) CARS images of a polystyrene bead constructed from the integration in spectral bands of 0–100 cm-1(b), 2800–2950 cm-1(c), and 3000–3100 cm-1(d). (e–f) CARS images of a polystyrene bead in acetone reconstructed after the NSC process using the CARS spectrum from acetone (e) and the polystyrene bead (f). (g) Combined image of the two CARS images of (e) and (f). Scale bar is 2 µm.

Fig. 7.
Fig. 7.

(a, b) CARS images of an unstained HeLa cell constructed from integration in the spectral bands of 2800–2900 cm-1(a), and 2900–3000 cm-1(b). (c) Combined image of three CARS images of an unstained HeLa cell reconstructed after the NSC process using the CARS spectrum from mitochondria or endoplasmic reticulum (blue), the nucleus (blue) and water (red). Scale bar is 8 µm.

Equations (8)

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ϕ(ω)={ϕ"2(ωω0)2+ϕ"ΩR(ωωb)(ω<ωb=ωmaxΩR)ϕ"2(ωω0)2(ωωb).
ISH(τ){E(t)+E(tτ)}22dt
=1+2G2(τ)+4Re[F1(τ)exp(iω0τ)]+Re[F2(τ)exp(i2ω0τ)],
G2(τ)=A(t)2A(tτ)2dt,
F1(τ)={A(t)2+A(tτ)2}A(t)A*(tτ)dt,
F2(τ)=A2(t)A*2(tτ)dt
FT[G2(τ)]=A˜(ω)A˜*(ωΩ)dω2 .
CSP=1nΣi=0nSs(λi)Sr(λi)1nΣi=0nSs2(λi)1nΣi=0nSr2(λi),

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