Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy with high sensitivity and high three- dimensional resolution has been developed for the vibrational imaging of chemical species. Due to the coherent nature of the CARS emission, it has been reported that the detection of epi-CARS and forward-CARS (F-CARS) signals depends on the size and shape of the sample. We investigate theoretically and experimentally the effects on the CARS signal of refractive index mismatches between the sample and its surroundings. Backward-CARS and F-CARS signals are measured for different polystyrene bead diameters embedded in different refractive index solvents. We show that index mismatches result in a backward-reflected F-CARS signal that generally dominates the experimentally backward-detected signal. Simulations based on geometrical and wave optics comparing forward- and backward-detected signals for polystyrene beads embedded in different index solvents confirm our findings. Furthermore, we demonstrate that the maxima of forward- and backward-detected signals are generated at different positions along the optical axis in the sample if refractive index mismatches are present between the sample and its surroundings.

© 2006 Optical Society of America

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  1. H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
    [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, 4142-4145 (1999).
    [CrossRef]
  3. A. Volkmer, "Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy," J. Phy. D 38, R59-R81 (2005).
    [CrossRef]
  4. G. C. Bjorklund, "Effects of focusing on third-order nonlinear processes in isotropic media," IEEE J. Quantum Electron. QE11, 287-296 (1975).
  5. E. O. Potma, W. P. Boeij, and D. A. Wiersma, "Nonlinear coherent four-wave mixing in optical microscopy," J. Opt. Soc. Am. B 17, 1678-1684 (2000).
    [CrossRef]
  6. A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (2001).
    [CrossRef]
  7. J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (2002).
    [CrossRef]
  8. T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).
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    [CrossRef]
  10. S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
    [CrossRef]
  11. H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
    [CrossRef]
  12. G. J. De Grauw, J. M. Vroom, H. T. M. Van der Voort, and H. C. Gerritsen, "Imaging properties in two-photon excitation microscopy and effects of refractive-index mismatch in thick specimens," Appl. Opt. 38, 5995-6003 (1999).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  15. 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, 1168-1170 (2002).
    [CrossRef]
  16. J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
    [CrossRef]

2005 (2)

A. Volkmer, "Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy," J. Phy. D 38, R59-R81 (2005).
[CrossRef]

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

2004 (1)

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

2002 (3)

2001 (1)

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

2000 (1)

1999 (2)

1994 (2)

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[CrossRef]

T. D. Visser and J. L. Oud, "Volume measurements in 3-dimensional microscopy," Scanning 16, 198-200 (1994).
[CrossRef]

1993 (1)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

1992 (1)

T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).

1976 (1)

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
[CrossRef]

1975 (1)

G. C. Bjorklund, "Effects of focusing on third-order nonlinear processes in isotropic media," IEEE J. Quantum Electron. QE11, 287-296 (1975).

Bjorklund, G. C.

G. C. Bjorklund, "Effects of focusing on third-order nonlinear processes in isotropic media," IEEE J. Quantum Electron. QE11, 287-296 (1975).

Bloembergen, N.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
[CrossRef]

Boeij, W. P.

Brakenhoff, G. J.

T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).

Cheng, J. X.

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (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, 1168-1170 (2002).
[CrossRef]

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

Cremer, C.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

De Grauw, G. J.

Decraemer, W. F.

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

Diaspro, A.

Dirckx, J. J. J.

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

Federici, F.

Gerritsen, H. C.

Hannien, P.

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[CrossRef]

Hell, S.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

Hell, S. W.

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[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, 4142-4145 (1999).
[CrossRef]

Jacobsen, H.

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[CrossRef]

Jones, D. J.

Kuypers, L. C.

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

Lotem, H.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
[CrossRef]

Lynch, R. T.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
[CrossRef]

Oud, J. L.

T. D. Visser and J. L. Oud, "Volume measurements in 3-dimensional microscopy," Scanning 16, 198-200 (1994).
[CrossRef]

T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).

Potma, E. O.

Reiner, G.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

Robello, M.

Soini, E.

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[CrossRef]

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

Timmermans, J. P.

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

Van der Voort, H. T. M.

Visser, T. D.

T. D. Visser and J. L. Oud, "Volume measurements in 3-dimensional microscopy," Scanning 16, 198-200 (1994).
[CrossRef]

T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).

Volkmer, A.

A. Volkmer, "Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy," J. Phy. D 38, R59-R81 (2005).
[CrossRef]

J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (2002).
[CrossRef]

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

Vroom, J. M.

Wiersma, D. A.

Xie, X. S.

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (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, 1168-1170 (2002).
[CrossRef]

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (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, 4142-4145 (1999).
[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, 4142-4145 (1999).
[CrossRef]

Appl. Opt. (2)

IEEE J. Quantum Electron. (1)

G. C. Bjorklund, "Effects of focusing on third-order nonlinear processes in isotropic media," IEEE J. Quantum Electron. QE11, 287-296 (1975).

J. Microsc. (3)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

H. Jacobsen, P. Hannien, E. Soini, and S. W. Hell, "Refractive-index-induced aberrations in 2-photon confocal fluorescence microscopy," J. Microsc. 176, 226-230 (1994).
[CrossRef]

L. C. Kuypers, W. F. Decraemer, J. J. J. Dirckx, and J. P. Timmermans, "A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch," J. Microsc. 218, 68-78 (2005).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (2)

J. Phy. D (1)

A. Volkmer, "Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy," J. Phy. D 38, R59-R81 (2005).
[CrossRef]

J. Phys. Chem. B (1)

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

Opt. Lett. (1)

Optik (1)

T. D. Visser, J. L. Oud, and G. J. Brakenhoff, "Refractive-index and axial distance measurements in 3D microscopy," Optik 90, 17-19 (1992).

Phys. Rev. A (1)

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14 (5), 1748-1755 (1976).
[CrossRef]

Phys. Rev. Lett. (2)

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

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy," Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

Scanning (1)

T. D. Visser and J. L. Oud, "Volume measurements in 3-dimensional microscopy," Scanning 16, 198-200 (1994).
[CrossRef]

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Figures (9)

Fig. 1
Fig. 1

Experimental setup used for collinear CARS microscopy (F, filter; BS, beam splitter; BC, beam combiner; LE and LF, lenses; C, condenser).

Fig. 2
Fig. 2

Radiation pattern of a spherical shaped sample with different diameter d, (d = 100 nm and d = 2 μm). Ratio E-CARS–F-CARS versus the diameter of the sample (λ P = 740 nm, λ S = 840 nm). Polarizations of the pump and Stokes beams are set along the x axis.

Fig. 3
Fig. 3

(a) F-CARS xy image of 2 μm polystyrene bead in air sitting on a glass cover slip (Raman shift 1600 cm−1 corresponding to C=C stretch). The average pump and Stokes powers are 200 and 100 μW, respectively, at 400 kHz repetition rate (gray scale in arbitrary units). (b) F-CARS (solid triangles) and B-CARS (open squares) z axis signal profiles (taking at the bead center in the xy plane). Gaussian fit curves (solid curves) for F-CARS (FWHM = 4.50 μm) and B-CARS (FWHM = 4.15 μm).

Fig. 4
Fig. 4

F-CARS and B-CARS profiles (markers) along z axis (taking at the bead center in the xy plane) for 2 μm beads embedded in different media of refractive index n (a) air, n = 1; (b) agarose gel, n = 1.33; (c) oil, n = 1.45. Gaussian fit curves (solid curves).

Fig. 5
Fig. 5

Refractive model system used in geometrical optics simulations. The bead stands on a glass cover slip and is embedded in a solvent of refractive index n. (Left) H-CARS signal is considered as a pure reflection of the forward-emitted CARS signal on the upper bead interface. (Right) F-CARS signal is refracted by the bead–solvent interface.

Fig. 6
Fig. 6

Calculated numerical apertures as a function of the emitted CARS position z in a 2 μm polystyrene bead. (a) Resulted NA F of forward-refracted beams. (b) Resulted NA B of backward-reflected beams.

Fig. 7
Fig. 7

Calculated collection functions CF (z) (solid triangles) and CB (z) (open squares) in a 2 μm diameter polystyrene bead (dotted curve).

Fig. 8
Fig. 8

FDTD calculation in a 2 μm polystyrene bead centered at z = 4 μm. (a) Excitation Poynting intensity mapping I ex(x, z) (λ = 800 nm, waist 0.5 μm) in the bead settled on a glass–air interface (z = 3 μm; white line). In the absence of bead the beam focuses at z laser = 4; 5 and 6 μm (dashed line). (b) z max versus z laser for the bead embedded in various media (oil, agarose, air).

Fig. 9
Fig. 9

Comparison between experimental and-theoretical CARS signals obtained when scanning along the z axis for a 2 μm bead diameter (z scan performed along the symmetry axis of the bead). Experimental F-CARS signal (solid triangle), B-CARS (open squares), I F-CARS and I B-CARS (continuous black curve). (a) Bead embedded in agarose, (b) bead embedded in air.

Equations (3)

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P NL ( ω AS ) = χ ( 3 ) E P ( ω P ) 2 E S ( ω S ) * ,
C F ( z ) = B ( z ) 1 [ 1 O N col F 2 ] 1 / 2 1 [ 1 O N F 2 ] 1 / 2 ,
I F-CARS ( z ) = K z C F ( z ) I ex         3 ( z z ) d z ,

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