Abstract

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by 4.8μm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner. The fabricated endoscope has an outer diameter of 3 mm, a rigid length of 4 cm, and acquires images at 4frames/s per focal plane, with lateral and axial resolutions for two-photon imaging of 0.8 and 10 μm, respectively.

© 2012 Optical Society of America

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2011

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

2010

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

2009

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

2008

2007

2003

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, Biomed. Eng. 2, 13 (2003).
[CrossRef]

2001

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

A. Hopt and E. Neher, Biophys J. 80, 2029 (2001).
[CrossRef]

1998

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

A. Schonle and S. W. Hell, Opt. Lett. 23, 325 (1998).
[CrossRef]

1996

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

1990

W. Cheong, S. A. Prahl, and A. J. Welsch, IEEE J. Quantum Electron. 26, 2166 (1990).
[CrossRef]

Akhtar, M.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Allen, J.

Bahlmann, K.

Bao, H.

Barzda, V.

Bewersdorf, J.

J. Bewersdorf, A. Egner, and S. W. Hell, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer Science+Business Media, 2006).

Brown, C. M.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

Buehler, C.

Carriles, R.

Cheong, W.

W. Cheong, S. A. Prahl, and A. J. Welsch, IEEE J. Quantum Electron. 26, 2166 (1990).
[CrossRef]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Denk, W.

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

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Egner, A.

J. Bewersdorf, A. Egner, and S. W. Hell, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer Science+Business Media, 2006).

Engelbrecht, C. J.

Fantini, S.

Fee, M. S.

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

Gu, M.

Heffer, E. L.

Hell, S. W.

A. Schonle and S. W. Hell, Opt. Lett. 23, 325 (1998).
[CrossRef]

J. Bewersdorf, A. Egner, and S. W. Hell, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer Science+Business Media, 2006).

Helmchen, F.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, Opt. Express 16, 5556 (2008).
[CrossRef]

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

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Hoover, E. E.

Hopt, A.

A. Hopt and E. Neher, Biophys J. 80, 2029 (2001).
[CrossRef]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Johnston, R. S.

Kim, K. H.

Kleinfeld, D.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Kobat, D.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

Lee, M. M.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Lee, W. A.

Li, M.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Li, X.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Maxfield, F. R.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Mitra, P. P.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Mukherjee, S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Nedivi, E.

Neher, E.

A. Hopt and E. Neher, Biophys J. 80, 2029 (2001).
[CrossRef]

Ng, C. K.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Ouzounov, D. G.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

Pattie, R.

Pavlova, I.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

Perner, S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Pologruto, T. A.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, Biomed. Eng. 2, 13 (2003).
[CrossRef]

Prahl, S. A.

W. Cheong, S. A. Prahl, and A. J. Welsch, IEEE J. Quantum Electron. 26, 2166 (1990).
[CrossRef]

Ragan, T.

Rivera, D. R.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

Rubin, M. A.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Sabatini, B. L.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, Biomed. Eng. 2, 13 (2003).
[CrossRef]

Scherr, D. S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Schonle, A.

Seibel, E. J.

Shear, J. B.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

Sheetz, K. E.

So, P. T. C.

Squier, J. A.

Svoboda, K.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, Biomed. Eng. 2, 13 (2003).
[CrossRef]

Tank, D. W.

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

Vance, R.

Webb, W. W.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

Welsch, A. J.

W. Cheong, S. A. Prahl, and A. J. Welsch, IEEE J. Quantum Electron. 26, 2166 (1990).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

Wu, Y.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Wysock, J. S.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Xi, J.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Xu, C.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

Zhang, Y.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Biomed. Eng.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, Biomed. Eng. 2, 13 (2003).
[CrossRef]

Biophys J.

A. Hopt and E. Neher, Biophys J. 80, 2029 (2001).
[CrossRef]

IEEE J. Quantum Electron.

W. Cheong, S. A. Prahl, and A. J. Welsch, IEEE J. Quantum Electron. 26, 2166 (1990).
[CrossRef]

J. Biomed. Opt.

Y. Wu, Y. Zhang, J. Xi, M. Li, and X. Li, J. Biomed. Opt. 15, 060506-1 (2010).

Neuron

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

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. USA

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, Proc. Natl. Acad. Sci. USA 108, 17598 (2011).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10763 (1996).
[CrossRef]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Proc. SPIE

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, Proc. SPIE 7161, 716117-1 (2009).

Other

J. Bewersdorf, A. Egner, and S. W. Hell, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer Science+Business Media, 2006).

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Figures (3)

Fig. 1.
Fig. 1.

(a) MME components. Inset: Close-up of the axially offset DCFs. (b) MME imaging setup. (c) Axial intensity profile of the thin film response from each DCF. The “Front” DCF is focused the furthest distance in front of the lens, followed by the “Mid” and “Back” DCF, which is focused the shortest distance.

Fig. 2.
Fig. 2.

(a) Approximately 25 μm lateral shift in FOV: Transmission images of a U.S. Air Force test target acquired from the illumination of each of the three DCFs. The FOV100μm×100μm (b) Simultaneous axial-sectioning: Intrinsic fluorescence images of ex vivo mouse lung (2 frames averaged) obtained from each fiber at different stage depths into the tissue. The indicated features come into focus initially in the image obtained from the “Front” DCF (z=30μm) and then come into focus 10 μm deeper (z=40μm) for the image obtained from the “Back” DCF, but are shifted 50μm laterally. Images are acquired at 4frames/s with an FOV130μm×100μm. The power from each fiber focused at the sample is 50mW. Scale bars are 10 μm for all images in (a) and (b).

Fig. 3.
Fig. 3.

Channel crosstalk imaging comparison. MPM images of mouse lung (80μm below surface) obtained via illumination solely from the “Front” DCF and collection from all fibers. The inset image obtained from the “Mid” DCF is a magnified image of the indicated central feature. The inset image’s intensity values are multiplied by an additional factor of 5 from the original image to better display the “ghost” feature. The power from each fiber focused at the sample is 50mW. Scale bars are 10 μm.

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