Abstract

We implement switching laser mode coherent anti-Stokes Raman-scattering (SLAM-CARS) microscopy to enhance the spatial resolution and contrast in label-free vibrational microscopy. The method, based on the intensity difference between two images obtained with Gaussian and doughnut-shaped modes, does not depend on the specimen and relies on minimal modifications of the typical CARS setup. We demonstrate subdiffraction resolution imaging of myelin sheaths in a mouse brainstem. A lateral resolution of 0.36λp is achieved.

© 2013 Optical Society of America

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2013

H. Dehez, M. Piché, and Y. D. Koninck, Opt. Express 21, 15912 (2013).
[CrossRef]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

2007

S. W. Hell, Science 316, 1153 (2007).
[CrossRef]

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

2006

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

2005

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

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

2003

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

2002

1994

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Camarillo, I. G.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Cheng, J. X.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

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

J. X. Cheng, A. Volkmer, and X. S. Xie, J. Opt. Soc. Am. B 19, 1363 (2002).
[CrossRef]

Côté, D.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Dehez, H.

Evans, C. L.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Fu, Y.

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

Ge, J.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Gu, Z.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Hanley, Q. S.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Hao, X.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Heintzmann, R.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Hell, S. W.

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Huff, T. B.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Jovin, T. M.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Koninck, Y. D.

Kuang, C.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Le, T. T.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Li, H.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Li, S.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Lin, C. P.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Liu, W.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Liu, X.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Munroe, P.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Nailon, J.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Nichols, M. B.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Piché, M.

Potma, E. O.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Puorishaag, M.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Rehrer, C. W.

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Sarafis, V.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Shi, R.

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

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Volkmer, A.

Wang, H.

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

Wang, Y.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Wichmann, J.

Xie, X. S.

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

J. X. Cheng, A. Volkmer, and X. S. Xie, J. Opt. Soc. Am. B 19, 1363 (2002).
[CrossRef]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Zickmund, P.

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

Biophys. J.

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

J. Opt. Soc. Am. B

Micron

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Mol. Imaging

T. T. Le, C. W. Rehrer, T. B. Huff, M. B. Nichols, I. G. Camarillo, and J. X. Cheng, Mol. Imaging 6, 205 (2007).

Nat. Methods

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. USA

C. L. Evans, E. O. Potma, M. Puorishaag, D. Côté, C. P. Lin, and X. S. Xie, Proc. Natl. Acad. Sci. USA 102, 16807 (2005).
[CrossRef]

Sci. Rep.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, Sci. Rep. 3, 1441 (2013).
[CrossRef]

Science

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

S. W. Hell, Science 316, 1153 (2007).
[CrossRef]

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Figures (3)

Fig. 1.
Fig. 1.

(a) Conventional CARS setup (gray) and SLAM-CARS setup modification (red). The fundamental Nd:YVO4 and OPO signal-pulse trains are used as the Stokes ωs and pump ωp beams, respectively. The pump beam is split in two and is either converted to a doughnut-shaped mode (red) using a VPP or is left unchanged. PBS, Polarization beam splitter; λ/4, quarter-wave plate; DM, dichroic mirror; M, mirror,; L, lens; O, objective lens; S, sample; F, anti-Stokes filter; PMT, photomultiplier tube. Experimental CARS microscopy images of 150 nm polystyrene beads embedded in 2% agarose gel for (b) Gaussian mode, (c) doughnut mode, and (d) SLAM-CARS. (e) Intensity profiles for Gaussian mode (black line), doughnut mode (blue line), and SLAM-CARS (green line). The profiles shown represent an average of the CARS signal from 10 beads. FWHM for Gaussian beam: 330 nm, FWHM for SLAM: 180 nm. Scale bars 500 nm.

Fig. 2.
Fig. 2.

Resolution and contrast enhancement realized by SLAM-CARS microscopy. CARS images of four point sources obtained by convolving experimentally obtained PSFs with adjacent radiating points separated by (a) 400 nm (the resolution limit of conventional CARS imaging) and (b) 300 nm. Plots show corresponding normalized intensity profiles evaluated along two arrowheads aligned in the vertical direction in the CARS (white arrowheads) and the SLAM-CARS image (green arrowheads). The resolution limit is defined by a 73% contrast between the peaks and the valley between the peaks. Scale bars: 500 nm. Experimental demonstration of resolution and contrast enhancement using (c) 350 nm and (d) 200 nm polystyrene beads. The images were acquired using an average pump power at the sample of 2 mW for the Gaussian pump beam, 4 mW for the doughnut-mode pump beam, and an average Stokes power of 5 mW. Images size: 4μm×4μm (180×180 pixels). The dwell time used was 3.1 μs, and 30 frames were accumulated and averaged per image. Normalized intensity profiles are evaluated for two beads in close proximity using CARS microscopy (white arrowheads) and SLAM-CARS microscopy (green arrowheads). Scale bars 500 nm.

Fig. 3.
Fig. 3.

Myelin sheaths in a mouse brainstem imaged with (a) standard CARS microscopy and (b) SLAM-CARS microscopy. The image was taken with an average pump power of 3 mW for Gaussian beam, 6 mW for doughnut-shaped beam, and an average Stokes power of 5 mW at the sample. Images size: 11μm×14μm (500×600 pixels). The dwell time was 3.1 μs, and 20 frames were accumulated and averaged per image. Scale bars 2.5 μm. (c) and (d) Magnified region [CARS (c) and SALM-CARS (d)] of mouse brainstem. Scale bars 500 nm. (e) Normalized intensity profiles of the regions between arrowheads of the CARS data (black line) and SLAM-CARS data (green line) for myelin image (upper panel) and magnified region (lower panel).

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