Abstract

Despite widespread use of multiphoton fluorescence microscopy, development of endoscopes for nonlinear optical imaging has been stymied by the degradation of ultrashort excitation pulses that occurs within optical fiber as a result of the combined effects of group-velocity dispersion and self-phase modulation. We introduce microendoscopes (3501000 µm in diameter) based on gradient-index microlenses that effectively eliminate self-phase modulation within the endoscope. Laser-scanning multiphoton fluorescence endoscopy exhibits micrometer-scale resolution. We used multiphoton endoscopes to image fluorescently labeled neurons and dendrites.

© 2003 Optical Society of America

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References

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2002

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S. W. Clark, F. O. Ilday, and F. W. Wise, Opt. Lett. 26, 1320 (2001).
[CrossRef]

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

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

2000

1997

R. Juskaitis, T. Wilson, and T. F. Watson, Scanning 19, 15 (1997).
[CrossRef]

W. Denk and S. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

1996

1995

1990

C. J. R. Sheppard and M. Gu, Optik (Stuttgart) 86, 104 (1990).

1983

1965

H. Kogelnik, Bell Syst. Tech. J. 44, 455 (1965).
[CrossRef]

Bird, D.

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

Bouma, B. E.

Braun, A.

Buess, G.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

Clark, S. W.

Denk, W.

F. Helmchen, D. W. Tank, and W. Denk, Appl. Opt. 41, 2930 (2002).
[CrossRef] [PubMed]

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

W. Denk and S. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

Dickensheets, D. L.

Dlugan, A. L. P.

Fee, M. S.

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

Foster, M. A.

Gaeta, A. L.

Gmitro, A. F.

Goering, R.

Gu, M.

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

C. J. R. Sheppard and M. Gu, Optik (Stuttgart) 86, 104 (1990).

Helmchen, F.

F. Helmchen, D. W. Tank, and W. Denk, Appl. Opt. 41, 2930 (2002).
[CrossRef] [PubMed]

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

Ilday, F. O.

Juskaitis, R.

R. Juskaitis, T. Wilson, and T. F. Watson, Scanning 19, 15 (1997).
[CrossRef]

Kino, G. S.

Knittel, J.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

Kogelnik, H.

H. Kogelnik, Bell Syst. Tech. J. 44, 455 (1965).
[CrossRef]

Lane, P. M.

Leiner, D. C.

Lin, C. P.

MacAulay, C. E.

Messerschmidt, B.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

B. Messerschmidt, T. Possner, and R. Goering, Appl. Opt. 34, 7825 (1995).
[CrossRef] [PubMed]

Moll, K. D.

Myaing, M. T.

Norris, T. B.

Ouzounov, D. G.

Possner, T.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

B. Messerschmidt, T. Possner, and R. Goering, Appl. Opt. 34, 7825 (1995).
[CrossRef] [PubMed]

Prescott, R.

Richards-Kortum, R.

Rouse, A. R.

Schneider, L.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard and M. Gu, Optik (Stuttgart) 86, 104 (1990).

Shishkov, M.

Siegman, A. E.

A. E. Siegman, Lasers (University Science, Mill Valley, Calif., 1986).

Svoboda, S.

W. Denk and S. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

Tank, D. W.

F. Helmchen, D. W. Tank, and W. Denk, Appl. Opt. 41, 2930 (2002).
[CrossRef] [PubMed]

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

Tearney, G. J.

Urayama, J.

Watson, T. F.

R. Juskaitis, T. Wilson, and T. F. Watson, Scanning 19, 15 (1997).
[CrossRef]

Webb, R. H.

Webb, W. W.

Wilson, T.

R. Juskaitis, T. Wilson, and T. F. Watson, Scanning 19, 15 (1997).
[CrossRef]

Wise, F. W.

Zipfel, W. R.

Appl. Opt.

Bell Syst. Tech. J.

H. Kogelnik, Bell Syst. Tech. J. 44, 455 (1965).
[CrossRef]

Neuron

W. Denk and S. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

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

Opt. Lett.

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

Opt. Commun.

J. Knittel, L. Schneider, G. Buess, B. Messerschmidt, and T. Possner, Opt. Commun. 188, 267 (2001).
[CrossRef]

Opt. Express

Opt. Lett.

Optik (Stuttgart)

C. J. R. Sheppard and M. Gu, Optik (Stuttgart) 86, 104 (1990).

Scanning

R. Juskaitis, T. Wilson, and T. F. Watson, Scanning 19, 15 (1997).
[CrossRef]

Other

A. E. Siegman, Lasers (University Science, Mill Valley, Calif., 1986).

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Figures (3)

Fig. 1
Fig. 1

a, Photograph of three multiphoton endoscopes, of 1.0-, 0.5-, and 0.35-mm diameter, oriented with the coupling lens at the top of the figure. A minor tick on the scale equals 1.0 mm. b, Optical layout used for the endoscopic measurements of Figs. 2 and 3. The focal plane in the sample may be adjusted by movement of either the endoscope itself or the microscope objective that couples the excitation beam. PMT, photomultiplier tube. Custom software controlled a pair of galvanometers (Cambridge Technologies) for xy scanning. c, Optical schematic of the excitation beam in the endoscope GRIN triplet. Red lines are normal to the local wave fronts.

Fig. 2
Fig. 2

a, Plot of the fluorescence signal generated by a fluorescein solution as a function of incident pulse energy and average power at 810 nm delivered by a 500µm-diameter endoscope. Black circles, data; red curve, pure quadratic fit without a linear term, yx=a+bx2. On a double-log plot, fluorescence rises with a slope of 1.96±0.01. b, Line images of single 100-nm beads (Polysciences, Inc.), acquired in an axial plane through the bead center with 810-nm excitation. Intensity values are normalized to maximum intensity at the bead center. Red circles, data for a 1.0-mm-diameter endoscope with a 0.46 NA and 300µm WD. Blue circles, data for a 500µm-diameter endoscope with a 0.42 NA and a 300µm WD. Black circles, data for a 500µm-diameter endoscope with a 0.26 NA and an 800µm WD. Solid curves, parametric fits. Fits to a Gaussian or to the square of the Airy disc (Ref. 2) are nearly indistinguishable, and both yield average FWHM of 1.26±0.1 µm, 1.84±0.1 µm, and 2.86±0.2 µm.

Fig. 3
Fig. 3

Fluorescence images of a neuron and its dendrites in a zebra finch brain slice stained with Alexa-488 injected into brain area Hvc. Incident power at the sample was 35 mW. A Nikon M-Plan 40× microscope objective with a 0.5 NA was used to match the 0.5 NA at the center of the endoscope’s coupling lens. Angular and lateral misalignments lowered the optical transmission through the endoscope probe to 5565%. The 40× microscope objective magnification multiplies that of the endoscope probe. In a, the neuron was imaged with a 500µm-diameter endoscope (0.54× magnification), with an endoscopic objective lens of 800µm WD, 0.26 NA, and 122µm field of view. The scale bar is 10 µm. In b, the identical neuron was imaged with a 1.0-mm-diameter endoscope (0.95×), with an endoscopic objective lens of 300µm WD, 0.46 NA, and 211µm field of view.

Equations (3)

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B2πλn2IzIzdz,
w=w0cos2gz+C2 sin2gz1/2
B4n2IEgτλw020π/21C2-1sin2z+1dz=2π2En0n2Iτλ2.

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