Abstract

A miniature objective lens with a tip diameter of 1.3mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86μm, respectively, by two-photon excitation fluorescence imaging of 200nm beads at a 735nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2μm polystyrene beads 5mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.

© 2007 Optical Society of America

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References

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2007

T. B. Huff and J. X. Cheng, J. Microsc. 225, 175 (2007).
[CrossRef] [PubMed]

2005

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, Biophys. J. 89, 581 (2005).
[CrossRef] [PubMed]

2004

W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, Opt. Lett. 29, 2521 (2004).
[CrossRef] [PubMed]

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1980 (2004).
[CrossRef]

2002

Biophys. J.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, Biophys. J. 89, 581 (2005).
[CrossRef] [PubMed]

J. Microsc.

T. B. Huff and J. X. Cheng, J. Microsc. 225, 175 (2007).
[CrossRef] [PubMed]

J. Neurophysiol.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1980 (2004).
[CrossRef]

J. Opt. Soc. Am. B

Nat. Methods

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.A.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 102, 16807 (2005).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

Diagram of CARS and TPEF imaging with the MPO lens. D, dichroic mirror; F, flipper mirror.

Fig. 2
Fig. 2

Resolution and sensitivity of the MPO lens. (a) X Z TPEF image of a 200 nm fluorescent bead in 0.5% agarose gel. Excitation, 200 fs , 735 nm , repetition rate 78 MHz , average power 30 mW at the sample. (b), (c) Axial and lateral TPEF intensity profiles along the vertical ( Z ) and horizontal ( X ) lines in (a). The FWHMs are shown. (d), (e) X Y E-CARS images of 2 μ m polystyrene beads in 0.5% agarose gel taken by the MPO lens and the 60 × objective. The pump and Stokes laser wavenumbers were 14,229 and 11,184 cm 1 , respectively, corresponding to 3045 cm 1 for ( ω p ω s ). The repetition rate was 3.9 MHz . For both objectives, the pump and Stokes laser powers at the sample were 3.6 and 1.26 mW , respectively. (f) Line intensity profiles along the lines in (d) and (e).

Fig. 3
Fig. 3

Penetration capability of the MPO lens. (a), (b) CARS images of 2 μ m polystyrene beads at different depths in 0.5% agarose gel. The depths were marked above the images. The intensity in (a) was reduced by 2 times from the actual intensity. The laser parameters were the same as for Fig. 2. (c), (d) E-CARS intensity versus depth for samples with (c) 5.7 × 10 7   beads cm 3 and (d) 5.7 × 10 8   beads cm 3 .

Fig. 4
Fig. 4

E-CARS image of axonal myelin in a rat spinal cord acquired with the MPO lens. Shown below the image is the CARS intensity profile along the line defined by the arrows. The pump and Stokes laser wavenumbers were 14,174 and 11,331 cm 1 , respectively. The pump and Stokes laser powers at the sample were 4.14 and 1.26 mW , respectively, at the repetition rate of 3.9 MHz .

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