Abstract

We describe a versatile, catheter-type two-photon probe, designed for in vivo and ex vivo imaging of the aqueous outflow pathway in the eye. The device consists of a silica double cladding fiber used for laser delivery and fluorescence collection, a spiral fiber scanner driven by a miniature piezoelectric tube, and an assembly of three micro-size doublet achromatic lenses used for focusing the laser and collecting the two-photon excitation signal. All the components have a maximum diameter of 2 mm and are enclosed in a length of 12-gauge stainless steel hypodermic tubing having an outer diameter of 2.8 mm. The lateral and axial resolutions of the probe are measured to be 1.5 μm and 9.2 μm, respectively. Different lens configurations and fibers are evaluated by comparing their spatial resolutions and fluorescence signal collection efficiencies. Doublet achromatic lenses and a double cladding fiber with a high inner cladding numerical aperture are found to produce a high signal collection efficiency, which is essential for imaging live tissues. Simple methods for reducing image distortions are demonstrated. Images of human trabecular meshwork tissue are successfully obtained with this miniature two-photon microscope.

© 2010 OSA

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2010 (2)

2009 (5)

2008 (4)

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, “Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo,” Opt. Express 16(8), 5556–5564 (2008).
[CrossRef] [PubMed]

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Y. C. Chang, J. Y. Ye, T. Thomas, Y. Chen, J. R. Baker, and T. B. Norris, “Two-photon fluorescence correlation spectroscopy through a dual-clad optical fiber,” Opt. Express 16(17), 12640–12649 (2008).
[PubMed]

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

2007 (2)

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

L. Fu and M. Gu, “Fibre-optic nonlinear optical microscopy and endoscopy,” J. Microsc. 226(3), 195–206 (2007).
[CrossRef] [PubMed]

2006 (6)

2005 (6)

L. Fu, X. S. Gan, and M. Gu, “Characterization of gradient-index lens-fiber spacing toward applications in two-photon fluorescence endoscopy,” Appl. Opt. 44(34), 7270–7274 (2005).
[CrossRef] [PubMed]

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

L. Fu, X. S. Gan, and M. Gu, “Nonlinear optical microscopy based on double-clad photonic crystal fibers,” Opt. Express 13(14), 5528–5534 (2005).
[CrossRef] [PubMed]

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

2004 (3)

2003 (4)

2002 (2)

2001 (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals,” Neuron 31(6), 903–912 (2001).
[CrossRef] [PubMed]

2000 (1)

A. M. Weiner, “Femtosecond pulse shaping using spatial light modulators,” Rev. Sci. Instrum. 71(5), 1929–1960 (2000).
[CrossRef]

1997 (2)

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

1996 (1)

H. Y. Gong, R. C. Tripathi, and B. J. Tripathi, “Morphology of the aqueous outflow pathway,” Microsc. Res. Tech. 33(4), 336–367 (1996).
[CrossRef] [PubMed]

1995 (1)

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1986 (1)

G. Binnig and D. P. E. Smith, “Single-tube three-dimensional scanner for scanning tunneling microscopy,” Rev. Sci. Instrum. 57(8), 1688–1689 (1986).
[CrossRef]

1985 (1)

1983 (1)

1980 (1)

Aksay, E.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

Ammar, D. A.

D. A. Ammar, T. C. Lei, E. A. Gibson, and M. Y. Kahook, “Two-photon imaging of the trabecular meshwork,” Mol. Vis. 16, 935–944 (2010).
[PubMed]

Andegeko, Y.

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

Anderson, E. P.

Baker, J. R.

Binnig, G.

G. Binnig and D. P. E. Smith, “Single-tube three-dimensional scanner for scanning tunneling microscopy,” Rev. Sci. Instrum. 57(8), 1688–1689 (1986).
[CrossRef]

Bird, D.

Boudoux, C.

Bouwmans, G.

Brown, C. M.

C. M. Brown, P. G. Reinhall, S. Karasawa, and E. J. Seibel, “Optomechanical design and fabrication of resonant microscanners for a scanning fiber endoscope,” Opt. Eng. 45(4), 043001 (2006).
[CrossRef]

Bückle, R.

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Buckup, T.

Chang, Y. C.

Chen, Y.

Chen, Y. C.

Cheung, E. L. M.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

Chirico, G.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Cho, M. R.

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Cobb, M. J.

Cocker, E. D.

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

Collini, M.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Coloma, F. M.

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

Côté, D.

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Cranfield, C.

Dantus, M.

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

Daxhelet, X.

Denk, W.

F. Helmchen, D. W. Tank, and W. Denk, “Enhanced two-photon excitation through optical fiber by single-mode propagation in a large core,” Appl. Opt. 41(15), 2930–2934 (2002).
[CrossRef] [PubMed]

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals,” Neuron 31(6), 903–912 (2001).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Diaspro, A.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Edward, D. P.

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Ehlers, A.

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Engelbrecht, C. J.

Ethier, C. R.

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

Fee, M. S.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals,” Neuron 31(6), 903–912 (2001).
[CrossRef] [PubMed]

Flusberg, B. A.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

Fu, L.

Gan, X. S.

Gibson, E. A.

D. A. Ammar, T. C. Lei, E. A. Gibson, and M. Y. Kahook, “Two-photon imaging of the trabecular meshwork,” Mol. Vis. 16, 935–944 (2010).
[PubMed]

Göbel, W.

Godbout, N.

Gong, H. Y.

H. Y. Gong, R. C. Tripathi, and B. J. Tripathi, “Morphology of the aqueous outflow pathway,” Microsc. Res. Tech. 33(4), 336–367 (1996).
[CrossRef] [PubMed]

Gordon, R. J.

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Gu, M.

Hauchi, K.

Helmchen, F.

Jain, A.

Johnson, M.

M. Johnson, “What controls aqueous humour outflow resistance?” Exp. Eye Res. 82(4), 545–557 (2006).
[CrossRef] [PubMed]

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

Johnston, R. S.

Jung, J. C.

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28(11), 902–904 (2003).
[CrossRef] [PubMed]

Kaatz, M.

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Kahook, M. Y.

D. A. Ammar, T. C. Lei, E. A. Gibson, and M. Y. Kahook, “Two-photon imaging of the trabecular meshwork,” Mol. Vis. 16, 935–944 (2010).
[PubMed]

Karasawa, S.

C. M. Brown, P. G. Reinhall, S. Karasawa, and E. J. Seibel, “Optomechanical design and fabrication of resonant microscanners for a scanning fiber endoscope,” Opt. Eng. 45(4), 043001 (2006).
[CrossRef]

Kerr, J. N. D.

Kim, P.

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Kimmey, M. B.

Knight, J. C.

König, K.

R. Le Harzic, I. Riemann, M. Weinigel, K. König, and B. Messerschmidt, “Rigid and high-numerical-aperture two-photon fluorescence endoscope,” Appl. Opt. 48(18), 3396–3400 (2009).
[CrossRef] [PubMed]

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Kusumi, A.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Le Harzic, R.

Lei, T. C.

D. A. Ammar, T. C. Lei, E. A. Gibson, and M. Y. Kahook, “Two-photon imaging of the trabecular meshwork,” Mol. Vis. 16, 935–944 (2010).
[PubMed]

Leiner, D. C.

Lemire-Renaud, S.

Leng, Y. X.

Li, X. D.

Lin, C. P.

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Liu, X. M.

Liu, Y. M.

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Lovozoy, V. V.

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

MacDonald, D. J.

Masters, B. R.

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

Mehta, A. D.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

Messerschmidt, B.

Morneau, D.

Motzkus, M.

Myaing, M. T.

Nakamura, H.

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

Nimmerjahn, A.

Nishizawa, K.

Norris, T. B.

Ozaki, K.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Pestov, D.

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

Piston, D. W.

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

Piyawattanametha, W.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

Prescott, R.

Puoris’haag, M.

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Reinhall, P. G.

C. M. Brown, P. G. Reinhall, S. Karasawa, and E. J. Seibel, “Optomechanical design and fabrication of resonant microscanners for a scanning fiber endoscope,” Opt. Eng. 45(4), 043001 (2006).
[CrossRef]

Riemann, I.

R. Le Harzic, I. Riemann, M. Weinigel, K. König, and B. Messerschmidt, “Rigid and high-numerical-aperture two-photon fluorescence endoscope,” Appl. Opt. 48(18), 3396–3400 (2009).
[CrossRef] [PubMed]

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Rivard, M.

Russell, P. S. J.

Saga, N.

Sako, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Schenkl, S.

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Schnitzer, M. J.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28(11), 902–904 (2003).
[CrossRef] [PubMed]

Seibel, E. J.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, “Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo,” Opt. Express 16(8), 5556–5564 (2008).
[CrossRef] [PubMed]

C. M. Brown, P. G. Reinhall, S. Karasawa, and E. J. Seibel, “Optomechanical design and fabrication of resonant microscanners for a scanning fiber endoscope,” Opt. Eng. 45(4), 043001 (2006).
[CrossRef]

Sekihata, A.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Shan, S.

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Shimada, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Singha, S.

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Sit, A. J.

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

Smith, D. P. E.

G. Binnig and D. P. E. Smith, “Single-tube three-dimensional scanner for scanning tunneling microscopy,” Rev. Sci. Instrum. 57(8), 1688–1689 (1986).
[CrossRef]

Stepnoski, R.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Strupler, M.

Tamm, E. R.

E. R. Tamm, “The trabecular meshwork outflow pathways: structural and functional aspects,” Exp. Eye Res. 88(4), 648–655 (2009).
[CrossRef] [PubMed]

Tanaka, K.

Tank, D. W.

F. Helmchen, D. W. Tank, and W. Denk, “Enhanced two-photon excitation through optical fiber by single-mode propagation in a large core,” Appl. Opt. 41(15), 2930–2934 (2002).
[CrossRef] [PubMed]

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals,” Neuron 31(6), 903–912 (2001).
[CrossRef] [PubMed]

Thomas, T.

Toyran, S.

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Tripathi, B. J.

H. Y. Gong, R. C. Tripathi, and B. J. Tripathi, “Morphology of the aqueous outflow pathway,” Microsc. Res. Tech. 33(4), 336–367 (1996).
[CrossRef] [PubMed]

Tripathi, R. C.

H. Y. Gong, R. C. Tripathi, and B. J. Tripathi, “Morphology of the aqueous outflow pathway,” Microsc. Res. Tech. 33(4), 336–367 (1996).
[CrossRef] [PubMed]

Verpillat, F.

von Vacano, B.

Wadsworth, W. J.

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Weiner, A. M.

A. M. Weiner, “Femtosecond pulse shaping using spatial light modulators,” Rev. Sci. Instrum. 71(5), 1929–1960 (2000).
[CrossRef]

Weinigel, M.

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Witt, T. E.

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

Wu, Y. C.

Xi, J. F.

Xi, P.

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

Xie, H. K.

Yamamoto, M.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Yanagisawa, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

Ye, J. Y.

Yun, S. H.

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Appl. Opt. (6)

Exp. Eye Res. (3)

M. Johnson, “What controls aqueous humour outflow resistance?” Exp. Eye Res. 82(4), 545–557 (2006).
[CrossRef] [PubMed]

E. R. Tamm, “The trabecular meshwork outflow pathways: structural and functional aspects,” Exp. Eye Res. 88(4), 648–655 (2009).
[CrossRef] [PubMed]

S. Toyran, Y. M. Liu, S. Singha, S. Shan, M. R. Cho, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study,” Exp. Eye Res. 81(3), 298–305 (2005).
[PubMed]

Invest. Ophthalmol. Vis. Sci. (2)

H. Nakamura, Y. M. Liu, T. E. Witt, R. J. Gordon, and D. P. Edward, “Femtosecond laser photodisruption of primate trabecular meshwork: an ex vivo study,” Invest. Ophthalmol. Vis. Sci. 50(3), 1198–1204 (2008).
[CrossRef] [PubMed]

A. J. Sit, F. M. Coloma, C. R. Ethier, and M. Johnson, “Factors affecting the pores of the inner wall endothelium of Schlemm’s canal,” Invest. Ophthalmol. Vis. Sci. 38(8), 1517–1525 (1997).
[PubMed]

J. Biomed. Opt. (2)

P. Xi, Y. Andegeko, D. Pestov, V. V. Lovozoy, and M. Dantus, “Two-photon imaging using adaptive phase compensated ultrashort laser pulses,” J. Biomed. Opt. 14(1), 014002 (2009).
[CrossRef] [PubMed]

P. Kim, M. Puoris’haag, D. Côté, C. P. Lin, and S. H. Yun, “In vivo confocal and multiphoton microendoscopy,” J. Biomed. Opt. 13(1), 010501 (2008).
[CrossRef] [PubMed]

J. Microsc. (3)

L. Fu and M. Gu, “Fibre-optic nonlinear optical microscopy and endoscopy,” J. Microsc. 226(3), 195–206 (2007).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(1), 9–20 (1997).
[CrossRef] [PubMed]

J. Neurophysiol. (1)

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiol. 92(5), 3121–3133 (2004).
[CrossRef] [PubMed]

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

Microsc. Res. Tech. (2)

H. Y. Gong, R. C. Tripathi, and B. J. Tripathi, “Morphology of the aqueous outflow pathway,” Microsc. Res. Tech. 33(4), 336–367 (1996).
[CrossRef] [PubMed]

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007).
[CrossRef] [PubMed]

Mol. Vis. (1)

D. A. Ammar, T. C. Lei, E. A. Gibson, and M. Y. Kahook, “Two-photon imaging of the trabecular meshwork,” Mol. Vis. 16, 935–944 (2010).
[PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods (1)

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[CrossRef] [PubMed]

Neuron (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, “A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals,” Neuron 31(6), 903–912 (2001).
[CrossRef] [PubMed]

Opt. Eng. (1)

C. M. Brown, P. G. Reinhall, S. Karasawa, and E. J. Seibel, “Optomechanical design and fabrication of resonant microscanners for a scanning fiber endoscope,” Opt. Eng. 45(4), 043001 (2006).
[CrossRef]

Opt. Express (6)

Opt. Lett. (10)

L. Fu and M. Gu, “Double-clad photonic crystal fiber coupler for compact nonlinear optical microscopy imaging,” Opt. Lett. 31(10), 1471–1473 (2006).
[CrossRef] [PubMed]

Y. C. Wu, J. F. Xi, M. J. Cobb, and X. D. Li, “Scanning fiber-optic nonlinear endomicroscopy with miniature aspherical compound lens and multimode fiber collector,” Opt. Lett. 34(7), 953–955 (2009).
[CrossRef] [PubMed]

D. Bird and M. Gu, “Compact two-photon fluorescence microscope based on a single-mode fiber coupler,” Opt. Lett. 27(12), 1031–1033 (2002).
[CrossRef] [PubMed]

M. T. Myaing, D. J. MacDonald, and X. D. Li, “Fiber-optic scanning two-photon fluorescence endoscope,” Opt. Lett. 31(8), 1076–1078 (2006).
[CrossRef] [PubMed]

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005).
[CrossRef] [PubMed]

W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, “Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective,” Opt. Lett. 29(21), 2521–2523 (2004).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28(11), 902–904 (2003).
[CrossRef] [PubMed]

X. M. Liu, M. J. Cobb, Y. C. Chen, M. B. Kimmey, and X. D. Li, “Rapid-scanning forward-imaging miniature endoscope for real-time optical coherence tomography,” Opt. Lett. 29(15), 1763–1765 (2004).
[CrossRef] [PubMed]

M. T. Myaing, J. Y. Ye, T. B. Norris, T. Thomas, J. R. Baker, W. J. Wadsworth, G. Bouwmans, J. C. Knight, and P. S. J. Russell, “Enhanced two-photon biosensing with double-clad photonic crystal fibers,” Opt. Lett. 28(14), 1224–1226 (2003).
[CrossRef] [PubMed]

B. von Vacano, T. Buckup, and M. Motzkus, “In situ broadband pulse compression for multiphoton microscopy using a shaper-assisted collinear SPIDER,” Opt. Lett. 31(8), 1154–1156 (2006).
[CrossRef] [PubMed]

Q. Rev. Biophys. (1)

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (2)

G. Binnig and D. P. E. Smith, “Single-tube three-dimensional scanner for scanning tunneling microscopy,” Rev. Sci. Instrum. 57(8), 1688–1689 (1986).
[CrossRef]

A. M. Weiner, “Femtosecond pulse shaping using spatial light modulators,” Rev. Sci. Instrum. 71(5), 1929–1960 (2000).
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (1)

M. W. Davidson, and M. Abramowitz, “Optical microscopy,” in Encyclopedia of imaging science and technology, J. P. Hornak, (J. Wiley, New York, 2002).

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Figures (8)

Fig. 1
Fig. 1

Schematic setup of the two-photon excitation fluorescence endoscope (a), structure of the piezoelectric actuator (b), drive waveform for a spiral scan of the fiber distal tip (c), and photograph of the assembled 2P microscope probe (d).

Fig. 2
Fig. 2

Schematic drawing of the three lens configurations. O: Objective plane (fiber end surface); P1: objective principle plane; P2: image principle plane; I: image plane. (a), L: 0.25 pitch GRIN lens; (b), L1: 0.15 NA aspheric lens, L2: 0.23 pitch GRIN lens; (c), L1: doublet lens with focal length of 6 mm; L2, L3: doublet lenses with focal length of 3 mm.

Fig. 3
Fig. 3

Two-photon images of fluorescent spheres with diameters of 10 μm (a), 3.0-3.4μm (b), and 1μm (c), obtained with the endoscope. Scale bars are 20 μm.

Fig. 4
Fig. 4

Fluorescence intensity distributions (open circles) of a 1μm fluorescent bead along the x (panel a) and z (panel b) directions, measured with the endoscope. The full width at half maximum (FWHM) values of the fitted Gaussian curves (solid red traces passing through the points) are 1.5 μm and 9.2 μm, which indicate the lateral and axial resolutions of the system, respectively. The computed point spread functions (solid blue curves), and the convolutions (black dashes) of the point spread functions and the beads profiles show the theoretical simulations.

Fig. 5
Fig. 5

Calculated chromatic effects of different lens configurations: (a) shift in the focal point with respect to the fiber tip and (b) change in the fluorescence collection efficiency as functions of fluorescence wavelength. The solid, dashed, and dotted curves correspond to the three doublet lens, single GRIN lens, and GRIN + aspheric lens configurations, respectively.

Fig. 6
Fig. 6

Scanning pattern of the laser focus obtained by applying constant amplitude sinusoidal waveforms to the two channels of the PZT. The relative phases between the two drive signals are (a) 90° and (b) 50°.

Fig. 7
Fig. 7

Two-photon images of 3.0-3.4 μm fluorescent beads obtained with the endoscope before (a) and after (b) removal of the center distortions. (c) and (d) are the waveforms used to drive the PZT to generate images (a) and (b), respectively.

Fig. 8
Fig. 8

Two-photon endoscopic images from a sequence of human TM z-sections. The depth interval of successive images is 20 μm. Scale bars are 20 μm. Top images show irregularly arranged strands in the uveal meshwork, and bottom images display flat and interacting beams or plates in the corneoscleral meshwork. Nuclei of TM cells were stained with DAPI.

Tables (1)

Tables Icon

Table 1 Comparison of the parameters of different double cladding fibers

Equations (3)

Equations on this page are rendered with MathJax. Learn more.

D l = M D f c ,
M = f 2 / f 1 ,
D F O V = M D s c a n ,

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