Abstract

We demonstrate a low-cost-stimulated Raman scattering (SRS) microscope using continuous-wave (cw) lasers as excitation sources. A dual modulation scheme is used to remove the electronic background. The cw-SRS imaging of lipids in fatty liver is demonstrated by excitation of CH stretch vibration.

© 2013 Optical Society of America

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References

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  1. J. X. Cheng and X. S. Xie, eds., Coherent Raman Scattering Microscopy (Taylor & Francis, 2012).
  2. W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
    [CrossRef]
  3. C. Y. Chung, J. Boik, and E. O. Potma, Annu. Rev. Phys. Chem.64, 77 (2013).
    [CrossRef]
  4. E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, Opt. Lett. 27, 1168 (2002).
    [CrossRef]
  5. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
    [CrossRef]
  6. D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
    [CrossRef]
  7. M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
    [CrossRef]
  8. K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
    [CrossRef]
  9. A. Owyoung and E. D. Jones, Opt. Lett. 1, 152 (1977).
    [CrossRef]
  10. I. Rajapaksa and H. Kumar Wickramasinghe, Appl. Phys. Lett. 99, 161103 (2011).
    [CrossRef]
  11. K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
    [CrossRef]
  12. Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
    [CrossRef]
  13. Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
    [CrossRef]
  14. A. Schonle and S. W. Hell, Opt. Lett. 23, 325 (1998).
    [CrossRef]
  15. A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
    [CrossRef]
  16. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
    [CrossRef]
  17. Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
    [CrossRef]

2011

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
[CrossRef]

I. Rajapaksa and H. Kumar Wickramasinghe, Appl. Phys. Lett. 99, 161103 (2011).
[CrossRef]

2008

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

2007

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

2005

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

2002

1999

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

1998

A. Schonle and S. W. Hell, Opt. Lett. 23, 325 (1998).
[CrossRef]

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef]

1996

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

1995

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

1977

Baur, D.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

Berns, M. W.

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Boik, J.

C. Y. Chung, J. Boik, and E. O. Potma, Annu. Rev. Phys. Chem.64, 77 (2013).
[CrossRef]

Booth, M. J.

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef]

Chapman, C. F.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

Cheng, D. K.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

Cheng, J. X.

D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
[CrossRef]

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, Opt. Lett. 27, 1168 (2002).
[CrossRef]

Chung, C. Y.

C. Y. Chung, J. Boik, and E. O. Potma, Annu. Rev. Phys. Chem.64, 77 (2013).
[CrossRef]

Freudiger, C. W.

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Harke, B.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

He, C.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Hell, S. W.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

A. Schonle and S. W. Hell, Opt. Lett. 23, 325 (1998).
[CrossRef]

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef]

Holtom, G. R.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Huttman, G.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Jones, D. J.

Jones, E. D.

Kang, J. X.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Koester, H. J.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

Konig, K.

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Kumar Wickramasinghe, H.

I. Rajapaksa and H. Kumar Wickramasinghe, Appl. Phys. Lett. 99, 161103 (2011).
[CrossRef]

Liang, H.

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Liu, Y.

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Lu, S.

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

Min, W.

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Noack, J.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Owyoung, A.

Paltauf, G.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Potma, E. O.

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, Opt. Lett. 27, 1168 (2002).
[CrossRef]

C. Y. Chung, J. Boik, and E. O. Potma, Annu. Rev. Phys. Chem.64, 77 (2013).
[CrossRef]

Rajapaksa, I.

I. Rajapaksa and H. Kumar Wickramasinghe, Appl. Phys. Lett. 99, 161103 (2011).
[CrossRef]

Saar, B. G.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Schonle, A.

Slipchenko, M. N.

D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
[CrossRef]

Sonek, G. J.

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

Tromberg, B. J.

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Tsai, J. C.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Uhl, R.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

Vogel, A.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

Xie, X. S.

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, Opt. Lett. 27, 1168 (2002).
[CrossRef]

Ye, J.

Zhang, D.

D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
[CrossRef]

Annu. Rev. Phys. Chem.

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, Annu. Rev. Phys. Chem. 62, 507 (2011).
[CrossRef]

Appl. Phys. B

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Appl. Phys. Lett.

I. Rajapaksa and H. Kumar Wickramasinghe, Appl. Phys. Lett. 99, 161103 (2011).
[CrossRef]

Biophys. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, Biophys. J. 71, 2158 (1996).
[CrossRef]

J. Microsc.

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef]

J. Phys. Chem. Lett.

D. Zhang, M. N. Slipchenko, and J. X. Cheng, J. Phys. Chem. Lett. 2, 1248 (2011).
[CrossRef]

Nat. Methods

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[CrossRef]

Nature

K. Konig, H. Liang, M. W. Berns, and B. J. Tromberg, Nature 377, 20 (1995).
[CrossRef]

Opt. Lett.

Science

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857 (2008).
[CrossRef]

Other

C. Y. Chung, J. Boik, and E. O. Potma, Annu. Rev. Phys. Chem.64, 77 (2013).
[CrossRef]

J. X. Cheng and X. S. Xie, eds., Coherent Raman Scattering Microscopy (Taylor & Francis, 2012).

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Figures (4)

Fig. 1.
Fig. 1.

Schematic of a cw-SRS microscope. The orange and blue lines represent the Stokes and pump beams, respectively. FG, function generator; LPF, low-pass filter; GSM, galvo scanning mirror; PD, photodiode; RA, resonant amplifier; LIA, lock-in amplifier.

Fig. 2.
Fig. 2.

cw SRS spectrum of cyclohexane liquid and shot noise detection. (a) A large offset was found in the single modulation scheme. The reference curve (black) was recorded by blocking both laser beams. (b) In the dual modulation scheme, the electronic offset was removed. The cw-SRS spectrum is consistent with the spontaneous Raman spectrum. The pump and Stokes powers were 20 and 108 mW, respectively. (c) Shot noise limit of the pump laser. Noise is square root dependent on the laser power, the fit r-square value is 0.99.

Fig. 3.
Fig. 3.

cw SRS imaging of the interface between air and an olive oil droplet. (a) cw SRS spectrum (single modulation; the pump and Stokes powers were 20 and 108 mW) of olive oil is identical with the spontaneous Raman spectrum in the CH2 stretch vibration region. (b) cw SRS image of oil at the CH2 stretch vibration mode at 2850cm1. (c) Off-resonance cw-SRS image recorded at 2765cm1. Pixel dwell time: 1 ms. Image size: 100×100 pixels. Frame averaged 100 times. The pump and Stokes powers were 6 and 35 mW, respectively. Scale bar: 10 μm. (d) SRS image recorded by a picosecond pulsed laser. Pixel dwell time: 4 μs. Image size: 512×512 pixels. The pump and Stokes powers were 6 and 35 mW, respectively. Scale bar: 10 μm.

Fig. 4.
Fig. 4.

cw SRS imaging of a fatty liver slice. (a) cw SRS image of a 75μm×75μm area of fatty liver. The cw-SRS signal generated a clear and identical contrast contour for lipid droplets. (b) Off-resonant imaging of fatty liver, which the Raman shift beat at 2765cm1. Pixel dwell time: 1 ms. Image size: 100×100 pixels. Frame averaged 100 times. The pump and Stokes powers were 6 and 35 mW, respectively. (c) The transmission image of the same area showed some lipid droplets inside. Scale bar: 20 μm.

Equations (5)

Equations on this page are rendered with MathJax. Learn more.

ΔIsigpulσIpISf×{exp[4ln2(t/τ)2]}2dt,
ΔIsigcwσIpcwIScw,
Ipcw=Ipf×exp[4ln2(t/τ)2]dt,
IScw=ISf×exp[4ln2(t/τ)2]dt.
ΔIsigpulΔIsigcw={exp[4ln2(t/τ)2]}2dtf×{exp[4ln2(t/τ)2]dt}2.

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