Abstract

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.

© 2005 Optical Society of America

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References

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  1. J. C. Jung and M. J. Schnitzer, Opt. Lett. 28, 902 (2003).
    [CrossRef] [PubMed]
  2. J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
    [CrossRef] [PubMed]
  3. M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, J. Neurophysiol. 91, 1908 (2004).
    [CrossRef]
  4. A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
    [CrossRef] [PubMed]
  5. D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
    [CrossRef] [PubMed]
  6. W. Gobel, J. N. Kerr, A. Nimmerjahn, and F. Helmchen, Opt. Lett. 29, 2521 (2004).
    [CrossRef]
  7. D. G. Ouzounov, K. D. Moll, M. A. Foster, W. R. Zipfel, W. W. Webb, and A. L. Gaeta, Opt. Lett. 27, 1513 (2002).
    [CrossRef]
  8. F. Helmchen, D. W. Tank, and W. Denk, Appl. Opt. 41, 2930 (2002).
    [CrossRef] [PubMed]
  9. W. Gobel, A. Nimmerjahn, and F. Helmchen, Opt. Lett. 29, 1285 (2004).
    [CrossRef]
  10. F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
    [CrossRef] [PubMed]
  11. L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
    [CrossRef]
  12. D. L. Dickensheets and G. S. Kino, Proc. SPIE 2184, 39 (1994).
    [CrossRef]
  13. H. K. Kim, M. J. F. Digonnet, G. S. Kino, J. Shin, and S. Fan, Opt. Express 12, 3436 (2004).
    [CrossRef] [PubMed]
  14. G. P. Agrawal, Nonlinear Fiber Optics, 3rd ed. (Elsevier Science, San Diego, Calif., 2001).
  15. M. Gu and C. J. R. Sheppard, Optik (Stuttgart)  86, 104 (1990).

2004 (6)

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

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

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

W. Gobel, A. Nimmerjahn, and F. Helmchen, Opt. Lett. 29, 1285 (2004).
[CrossRef]

H. K. Kim, M. J. F. Digonnet, G. S. Kino, J. Shin, and S. Fan, Opt. Express 12, 3436 (2004).
[CrossRef] [PubMed]

W. Gobel, J. N. Kerr, A. Nimmerjahn, and F. Helmchen, Opt. Lett. 29, 2521 (2004).
[CrossRef]

2003 (2)

2002 (2)

2001 (1)

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

1994 (1)

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

1991 (1)

L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
[CrossRef]

1990 (1)

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

Agrawal, G. P.

G. P. Agrawal, Nonlinear Fiber Optics, 3rd ed. (Elsevier Science, San Diego, Calif., 2001).

Aksay, E.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Bird, D.

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]

Dickensheets, D. L.

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

Digonnet, M. J. F.

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]

Fan, S.

Fee, M. S.

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

Flusberg, B. A.

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

Foster, M. A.

Gaeta, A. L.

Giniunas, L.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
[CrossRef]

Gobel, W.

Gu, M.

D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
[CrossRef] [PubMed]

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

Helmchen, F.

Jung, J. C.

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, Opt. Lett. 28, 902 (2003).
[CrossRef] [PubMed]

Juskaitis, R.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
[CrossRef]

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, H. K.

Kino, G. S.

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]

Mehta, A. D.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

Moll, K. D.

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]

Nimmerjahn, A.

Ouzounov, D. G.

Schnitzer, M. J.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, Opt. Lett. 28, 902 (2003).
[CrossRef] [PubMed]

Shatalin, S. V.

L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
[CrossRef]

Sheppard, C. J. R.

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

Shin, J.

Stepnoski, R.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[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]

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]

D. G. Ouzounov, K. D. Moll, M. A. Foster, W. R. Zipfel, W. W. Webb, and A. L. Gaeta, Opt. Lett. 27, 1513 (2002).
[CrossRef]

Zipfel, W. R.

Appl. Opt. (1)

Curr. Opin. Neurobiol. (1)

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, Curr. Opin. Neurobiol. 14, 617 (2004).
[CrossRef] [PubMed]

Electron. Lett. (1)

L. Giniunas, R. Juskaitis, and S. V. Shatalin, Electron. Lett. 27, 724 (1991).
[CrossRef]

J. Neurophysiol. (2)

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

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

Neuron (1)

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

Opt. Express (1)

Opt. Lett. (5)

Optik (1)

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

Proc. SPIE (1)

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

Other (1)

G. P. Agrawal, Nonlinear Fiber Optics, 3rd ed. (Elsevier Science, San Diego, Calif., 2001).

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Figures (4)

Fig. 1
Fig. 1

a, Schematic of the imaging head. Arrows extending from the micromotor and scanner indicate directions of movement. b, Photographic overlay of near-field intensity distribution of infrared light in the lowest-order mode (red pseudocolor) exiting the bandgap fiber (green pseudocolor). The scale bar is 10 μ m . The mode appears off center from the 6.8 - μ m -diameter air core due to a small shift between image acquisitions. c, Endoscope probe (green pseudocolor) and a real image of the Lissajous scanning pattern (red pseudocolor) that arises from a reflection at the junction of the GRIN objective and relay lenses. The Lissajous driving frequencies were 538 and 698 Hz . Only a portion of the pattern appears due to an image acquisition time of 0.067 s . The scale bar is 200 μ m .

Fig. 2
Fig. 2

a, Computer-aided-design (CAD) model of device components mounted on the baseplate. White, baseplate (nylon); red, endoscope probe and bandgap fiber; light blue, piezo actuator; gold, piezo clip (titanium); gray, wedge mechanism for coarse focusing (acetal); pink, spring for wedge mechanism; orange, micromotor; blue, micromotor shuttle (acetal) for finer focusing; green, screws and rails (stainless steel). b, CAD model of the imaging head. White, baseplate and casing (nylon); blue, fluorescence collection fiber; green, screws (stainless steel). The bandgap fiber (pink), actuator leads (red and purple), and motor control lines (yellow) exit through a slot in the casing. c, Photograph of components assembled as in panel a. Scale bar is 1 cm . d, Photograph of the imaging head.

Fig. 3
Fig. 3

a, Second-order intensity autocorrelation of laser pulses with spectra centered at 791 nm . Measurements were performed just outside the laser (black circles; solid line, Gaussian fit with 1 e width τ 0 = 150 fs ), after propagation through 1.5 m of HC-800-02 (Crystal Fibre) bandgap fiber (red triangles), and after propagation through the fiber and a 1.0 - cm -long endoscope probe (open blue squares). b, Determination of lateral resolution using normalized line images of single 100 - nm -diameter fluorescent beads. Images were acquired in a plane through the axial center of the bead using 802 nm excitation, with the imaging system equipped with a 0.48 NA endoscope probe (red circles), or with the probe alone (black circles). For the latter measurements the excitation beam was expanded to overfill the probe and raster scanned using galvanometer mirrors.[1] Solid lines fits are to the square of an Airy disk.[15] Inset, Determination of axial resolution using amplitudes of fits to a stack of lateral line images, acquired in axial planes spaced either 0.5 or 0.8 μ m apart, with the entire imaging system (red circles) or the probe alone (black circles). Solid lines are Gaussian fits.

Fig. 4
Fig. 4

Images of cerebral blood vessels labeled with fluorescein in anesthetized mice. The frame rate was 2 Hz . a, b, Vessels near the neocortical surface. Excitation power was 15 mW at the specimen and the endoscope had a working distance (WD) of 250 μ m in air. Lissajous driving frequencies were 537 and 699 Hz . c–e, Hippocampal vessels imaged through a 152 μ m cover glass at the tip of a guide tube that was implanted just dorsal to hippocampus. The WD was 160 μ m in air. Driving frequencies were 538 and 698 Hz . c, Vessels 20 μ m below the dorsal hippocampal surface imaged with 30 mW power. d, Magnified image of the boxed region in c, obtained by decreasing the Lissajous driving voltages. e, Capillaries 80 μ m below the hippocampal surface imaged with 80 mW power. Images a–c are 128 × 128   pixels . Images d and e are 128 × 62 and 96 × 46   pixels , respectively. a and c display single image frames, while b, d, and e show averages over six frames. All scale bars are 10 μ m .

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