Abstract

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75mm×0.75mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of 16°. Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52kHz.

© 2006 Optical Society of America

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2006 (1)

2005 (3)

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, Opt. Lett. 30, 2272 (2005).
[CrossRef] [PubMed]

D. Kim, K. H. Kim, S. Yazdanfar, and P. T. C. So, in Proc. SPIE 5700, 14 (2005).
[CrossRef]

2004 (3)

2003 (3)

2001 (2)

Y. Pan, H. Xie, and G. K. Fedder, Opt. Lett. 26, 1966 (2001).
[CrossRef]

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

1996 (1)

1994 (1)

D. L. Dickensheets and G. S. Kino, in Proc. SPIE 2184, 39 (1994).
[CrossRef]

1991 (1)

L. Giniunas, R. Juskaitis, and S. V. Shatalin, J. Opt. Soc. Am. A 27, 724 (1991).

Aguirre, A. D.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Anderson, E. P.

Bird, D.

Born, M.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, 1999), p. 949.

Chen, Y.

X. Liu, M. J. Cobb, Y. Chen, M. B. Kimmey, and X. Li, Opt. Lett. 29, 1763 (2004).
[CrossRef] [PubMed]

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Cobb, M. J.

Cocker, E. D.

Cranfield, C.

Delaney, P. M.

P. M. Delaney and M. R. Harris, in Handbook of Biological Confocal Microscopy, 2nd ed., J.B.Pawley, ed. (Plenum, 1995), pp. 515-523.

Denk, W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Dickensheets, D. L.

D. L. Dickensheets and G. S. Kino, Opt. Lett. 21, 764 (1996).
[CrossRef] [PubMed]

D. L. Dickensheets and G. S. Kino, in Proc. SPIE 2184, 39 (1994).
[CrossRef]

Dombeck, D. A.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

Dorschel, K.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Fan, L.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Fedder, G. K.

Fee, M. S.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Flusberg, B. A.

Fu, L.

Fujimoto, J. G.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Fujino, M.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Giniunas, L.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, J. Opt. Soc. Am. A 27, 724 (1991).

Gobel, W.

Gu, M.

Hah, D.

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

Harris, M. R.

P. M. Delaney and M. R. Harris, in Handbook of Biological Confocal Microscopy, 2nd ed., J.B.Pawley, ed. (Plenum, 1995), pp. 515-523.

Helmchen, F.

Herz, P. R.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Hofmann, U.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Hsu, S.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Izatt, J. A.

Jain, A.

Jung, J. C.

Juskaitis, R.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, J. Opt. Soc. Am. A 27, 724 (1991).

Kasischke, K. A.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

Kerr, J. N.

Kim, D.

D. Kim, K. H. Kim, S. Yazdanfar, and P. T. C. So, in Proc. SPIE 5700, 14 (2005).
[CrossRef]

Kim, K. H.

D. Kim, K. H. Kim, S. Yazdanfar, and P. T. C. So, in Proc. SPIE 5700, 14 (2005).
[CrossRef]

Kimmey, M. B.

Kino, G. S.

D. L. Dickensheets and G. S. Kino, Opt. Lett. 21, 764 (1996).
[CrossRef] [PubMed]

D. L. Dickensheets and G. S. Kino, in Proc. SPIE 2184, 39 (1994).
[CrossRef]

LaCosse, J.

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, in Conference on Lasers and Electro-Optics (CLEO 2000), Vol. 39 of OSA Trends in Optics and Photonics (Optical Society of America, 2000), pp. 447-448.

Lee, D.

D. Lee and O. Solgaard, in Proceedings of the Solid-State Sensor and Actuator Workshop, Hilton Head, South Carolina, June 6-10, 2004, pp 352-355.

Levene, M. J.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

Li, X.

Liu, X.

Molloy, R. P.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

Muehlmann, S.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Nimmerjahn, A.

Pan, Y.

Patterson, P. R.

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

Piyawattanametha, W.

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, in Conference on Lasers and Electro-Optics (CLEO 2000), Vol. 39 of OSA Trends in Optics and Photonics (Optical Society of America, 2000), pp. 447-448.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Rao, K. D.

Schnitzer, M. J.

Schutz, R.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Shatalin, S. V.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, J. Opt. Soc. Am. A 27, 724 (1991).

Smith, S. W.

So, P. T. C.

D. Kim, K. H. Kim, S. Yazdanfar, and P. T. C. So, in Proc. SPIE 5700, 14 (2005).
[CrossRef]

Solgaard, O.

D. Lee and O. Solgaard, in Proceedings of the Solid-State Sensor and Actuator Workshop, Hilton Head, South Carolina, June 6-10, 2004, pp 352-355.

Tank, D. W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Toshiyoshi, H.

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, in Conference on Lasers and Electro-Optics (CLEO 2000), Vol. 39 of OSA Trends in Optics and Photonics (Optical Society of America, 2000), pp. 447-448.

Wagner, B.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Webb, W. W.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

Witt, M.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

Wolf, E.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, 1999), p. 949.

Wu, M. C.

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, in Conference on Lasers and Electro-Optics (CLEO 2000), Vol. 39 of OSA Trends in Optics and Photonics (Optical Society of America, 2000), pp. 447-448.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

Xie, H.

Yazdanfar, S.

Zara, J. M.

J. Microelectromech. Syst. (1)

W. Piyawattanametha, P. R. Patterson, D. Hah, H. Toshiyoshi, and M. C. Wu, J. Microelectromech. Syst. 14, 1329 (2005).
[CrossRef]

J. Neurophysiol. (1)

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
[CrossRef]

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

L. Giniunas, R. Juskaitis, and S. V. Shatalin, J. Opt. Soc. Am. A 27, 724 (1991).

Neuron (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (8)

Proc. SPIE (2)

D. L. Dickensheets and G. S. Kino, in Proc. SPIE 2184, 39 (1994).
[CrossRef]

D. Kim, K. H. Kim, S. Yazdanfar, and P. T. C. So, in Proc. SPIE 5700, 14 (2005).
[CrossRef]

Other (6)

P. M. Delaney and M. R. Harris, in Handbook of Biological Confocal Microscopy, 2nd ed., J.B.Pawley, ed. (Plenum, 1995), pp. 515-523.

W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, P. R. Herz, A. D. Aguirre, Y. Chen, and J. G. Fujimoto, in Conference on Lasers and Electro-Optics (CLEO) (Optical Society of America, 2004), paper CWS2.

W. Piyawattanametha, H. Toshiyoshi, J. LaCosse, and M. C. Wu, in Conference on Lasers and Electro-Optics (CLEO 2000), Vol. 39 of OSA Trends in Optics and Photonics (Optical Society of America, 2000), pp. 447-448.

U. Hofmann, S. Muehlmann, M. Witt, K. Dorschel, R. Schutz, and B. Wagner, presented at the Miniaturized Systems with Micro-Optics and MEMS meeting, Santa Clara, Calif., 1999.

D. Lee and O. Solgaard, in Proceedings of the Solid-State Sensor and Actuator Workshop, Hilton Head, South Carolina, June 6-10, 2004, pp 352-355.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, 1999), p. 949.

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Figures (3)

Fig. 1
Fig. 1

Electron micrographs of a two-dimensional MEMS scanner. a, 750 μ m × 750 μ m scanning mirror in a 3.2 mm × 3.0 mm die. Six banks of vertical comb actuators drive the mirror, which has a gimbal design. b, Inner axis torsional spring. c, Outer axis comb bank. Scale bars are 250 μ m .

Fig. 2
Fig. 2

Response characteristics of a 750 μ m × 750 μ m MEMS scanner. For both a and b, the voltage signal was applied to only one of the two opposing comb banks for each rotational axis. a, Optical deflection angle as a function of dc voltage. The maximum deflection angles are ± 7.6 ° and ± 3.0 ° for the inner (blue solid curve) and outer (red dashed curve) axes, respectively. b, Frequency response functions for the inner (blue solid curve) and outer (red dashed curve) axes, obtained by applying voltage signals of peak-to-peak amplitudes 45 and 58 V to the inner and outer axes, respectively.

Fig. 3
Fig. 3

Two-photon fluorescence images of pollen grains acquired using instrumentation based on a MEMS scanner and a Ti:sapphire laser tuned to 850 nm . a–c, Images acquired using a 40 × 0.8 NA water-immersion microscope objective. The fast-axis acquisition rate was 3.2 kHz for a and b and 3.0 kHz for c. Image b is a sum projection from a stack of 38 images acquired at 1 μ m increments. d and e, Images acquired using a doublet GRIN microendoscope probe of 0.47 NA and a 10 × 0.25 NA microscope objective to couple light into the probe.[3, 5] The fast axis was driven at resonance, allowing a double-sided acquisition rate of 3.52 kHz . Image e is a maximum intensity projection from a stack of 46 images acquired at 1 μ m increments. Laser power at the sample was 20 m W for a and b, 28 mW for c, and 40 mW for d and e. These power levels were needed because of the fast acquisition rate. Scale bars are 5 μ m .

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