Abstract

We report a non-axial-scanning second harmonic imaging technique, in which the chromatic aberration of a Fresnel lens is exploited to focus different wavelengths of a fundamental beam into different axial positions to effectively realize axial scanning. Since the second harmonic signals at different axial positions are generated by different fundamental wavelengths and hence accordingly have different center wavelengths, they can be resolved and detected in parallel by using a spectrometer without axial mechanical scanning. We have demonstrated a system capable of achieving about 8 μm effective axial scanning range. Proof-of-concept imaging results are also presented.

© 2010 OSA

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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
  4. 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]
  5. E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  8. Wikipedia, http://en.wikipedia.org/wiki/Second_harmonic_imaging_microscopy .
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  14. Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
    [CrossRef] [PubMed]
  15. K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
    [CrossRef] [PubMed]
  16. F. T. S. Yu, Introduction to diffraction, information processing, and holography (MIT Press, Cambridge, MA, 1973).
  17. K. Shi, Supercontinuum imaging and spectroscopy, Penn State Doctoral Dissertation (2007).
  18. E. Hecht, Optics (Addison Wesley, 2001).
    [PubMed]
  19. S. C. H. O. T. T. Optical Glass Data Sheets, (2010). http://www.us.schott.com/advanced_optics/english/download/schott_optical_glass_june_2010_us.pdf?PHPSESSID=cf27dsjed4tvup6riaj7up87k4 .

2009 (2)

2006 (2)

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

2004 (1)

2003 (3)

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003).
[CrossRef] [PubMed]

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]

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

2000 (1)

1997 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1984 (1)

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

1978 (1)

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. Quantum Electron. 10(5), 435–439 (1978).
[CrossRef]

Boucher, Y.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Brown, E.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Campagnola, P. J.

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003).
[CrossRef] [PubMed]

Christie, R.

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]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

diTomaso, E.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Dobson, S. L.

Fainman, Y.

Gannaway, J. N.

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. Quantum Electron. 10(5), 435–439 (1978).
[CrossRef]

Grange, R.

Hsieh, C. L.

Hyman, B. T.

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]

Jain, R. K.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Li, P.

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12(10), 2096–2101 (2004).
[CrossRef] [PubMed]

Liu, Z.

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
[CrossRef] [PubMed]

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12(10), 2096–2101 (2004).
[CrossRef] [PubMed]

Loew, L. M.

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003).
[CrossRef] [PubMed]

McKee, T.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Mertz, J.

Molesini, G.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

Moreaux, L.

Nam, S. H.

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

Nikitin, A. Y.

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]

Pedrini, G.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

Pluen, A.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Poggi, P.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

Psaltis, D.

Pu, Y.

Quercioli, F.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

Sandre, O.

Seed, B.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Sheppard, C. J. R.

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. Quantum Electron. 10(5), 435–439 (1978).
[CrossRef]

Shi, K.

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
[CrossRef] [PubMed]

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12(10), 2096–2101 (2004).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Sun, P. C.

Webb, W. W.

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]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

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]

Xu, Q.

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
[CrossRef] [PubMed]

Yin, S.

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
[CrossRef] [PubMed]

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

K. Shi, P. Li, S. Yin, and Z. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express 12(10), 2096–2101 (2004).
[CrossRef] [PubMed]

Zipfel, W. R.

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]

Appl. Opt. (1)

J. Microsc. (2)

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc. 235(1), 79–83 (2009).
[CrossRef] [PubMed]

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

Nat. Biotechnol. (1)

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003).
[CrossRef] [PubMed]

Nat. Med. (1)

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Opt. Commun. (2)

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[CrossRef]

K. Shi, S. H. Nam, P. Li, S. Yin, and Z. Liu, “Wavelength division multiplexed confocal microscopy using supercontinuum,” Opt. Commun. 263(2), 156–162 (2006).
[CrossRef]

Opt. Express (2)

Opt. Quantum Electron. (1)

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. Quantum Electron. 10(5), 435–439 (1978).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (1)

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]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (6)

Wikipedia, http://en.wikipedia.org/wiki/Second_harmonic_imaging_microscopy .

R. W. Boyd, Nonlinear Optics (Elsevier, San Diego, CA, 2003).

F. T. S. Yu, Introduction to diffraction, information processing, and holography (MIT Press, Cambridge, MA, 1973).

K. Shi, Supercontinuum imaging and spectroscopy, Penn State Doctoral Dissertation (2007).

E. Hecht, Optics (Addison Wesley, 2001).
[PubMed]

S. C. H. O. T. T. Optical Glass Data Sheets, (2010). http://www.us.schott.com/advanced_optics/english/download/schott_optical_glass_june_2010_us.pdf?PHPSESSID=cf27dsjed4tvup6riaj7up87k4 .

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Figures (5)

Fig. 1
Fig. 1

Schematic diagram of the chromatic second harmonic imaging system.

Fig. 2
Fig. 2

(a) Second harmonic spectrum generated at different depth positions; A single cluster of nanocrystals (shown in the upper-left inset) was placed near the focal point of the objective lens L2 (c.f. Fig. 1). A sequence of spectra of the generated second harmonic signal was measured as the nanocrystal cluster was mechanically scanned. (b) Mapping relationship between the depth position and the second harmonic signal wavelength; in the equation, λ represents the signal wavelength in nm while z is the depth position in µm.

Fig. 3
Fig. 3

(a) Microscope image of a LiNbO3 crystal scratched by silicon carbide sandpaper showing an “X” shaped scratch on its surface (b) Chromatic second harmonic imaging results; the crystal was scanned in two lateral dimensions (30 μm × 30 μm). A series of second harmonic images at wavelengths from 404 nm to 413 nm with an interval of 0.52 nm were obtained and shown here. (c) Side cross-sectional imaging results; three images correspond to y = 20 μm, 26 μm, and 30 μm respectively.

Fig. 4
Fig. 4

(a) Microscope image of a LiNbO3 micro-crystal; (b) Chromatic second harmonic imaging results; the sample was scanned in two lateral dimensions (40 μm × 40 μm). The second harmonic images correspond to wavelengths from 404.2 nm to 410.0nm with a step of 0.52 nm; (c) Side cross-sectional imaging results; three images correspond to y = 10 μm, 26 μm, and 28 μm respectively.

Fig. 5
Fig. 5

(a) Schematic diagram of a chromatic SHG microscope with an improved signal collection efficiency (b) an example showing that the material dispersion of a singlet lens (Material: N-SF11 glass, f/F = 3.37) can potentially help improve the SHG signal collection efficiency; the blue curve shows negative of the term on the right hand side of Eq. (4) as a function of the second harmonic wavelength while the green curve shows the term on the left hand side of Eq. (4); the red curve shows the sum of the two.

Equations (4)

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

λ F = λ 0 F 0
Δ F F = Δ λ f λ 0 = Δ λ S H G λ 0 / 2 = Δ λ S H G λ S H G
Δ f f = Δ n ( n 1 )
f F Δ n ( n 1 ) Δ λ S H G λ S H G

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