Abstract

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5μm depth resolution.

© 2005 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  2. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
    [CrossRef]
  3. D. Oron and Y. Silberberg, J. Opt. Soc. Am. B 21, 1964 (2004).
    [CrossRef]
  4. A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
    [CrossRef]
  5. J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
    [CrossRef]
  6. T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
    [CrossRef] [PubMed]
  7. D. Oron, E. Tal, and Y. Silberberg, Opt. Express 13, 1468 (2005).
    [CrossRef] [PubMed]
  8. O. E. Martinez, IEEE J. Quantum Electron. 23, 59 (1987).
    [CrossRef]
  9. A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
    [CrossRef] [PubMed]
  10. E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
    [CrossRef]
  11. G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
    [CrossRef]
  12. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
    [CrossRef]
  13. M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
    [CrossRef]
  14. M. D. Duncan, J. Reintjes, and T. J. Manuccia, Opt. Lett. 7, 350 (1982).
    [CrossRef] [PubMed]
  15. A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
    [CrossRef]

2005

2004

2001

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

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

1999

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

1998

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

1997

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

1996

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

1995

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

1994

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1987

O. E. Martinez, IEEE J. Quantum Electron. 23, 59 (1987).
[CrossRef]

1982

Andersen, P.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

Athey, B.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Bewersdorf, J.

Bliton, A. C.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Bouevitch, O.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Brakenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Duncan, M. D.

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

Hell, S.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Hell, S. W.

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Hopt, A.

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

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Lewis, A.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Lindek, S.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Linial, M.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Loew, L.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Manuccia, T. J.

Martinez, O. E.

O. E. Martinez, IEEE J. Quantum Electron. 23, 59 (1987).
[CrossRef]

Muller, M.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

Neher, E.

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

Nielsen, T.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

Norris, T.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Oron, D.

Parnas, D.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Peleg, G.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Pick, R.

J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
[CrossRef]

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Reintjes, J.

Ritter, G.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Salmon, N.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Silberberg, Y.

Squier, J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Stelzer, E.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Storz, C.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Stricker, R.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Tal, E.

Wade, M. H.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wilson, K. R.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

Xie, X. S.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Zumbusch, A.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Appl. Phys. Lett.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Bioimaging

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Biophys. J.

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

IEEE J. Quantum Electron.

O. E. Martinez, IEEE J. Quantum Electron. 23, 59 (1987).
[CrossRef]

J. Microsc.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, J. Microsc. (Oxford) 181, 253 (1995).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, J. Microsc. (Oxford)  192, 217 (1998).
[CrossRef]

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. (Oxford)  191, 266 (1998).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, J. Microsc. (Oxford)  201, 368 (2001).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

Opt. Commun.

E. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, Opt. Commun. 104, 223 (1994).
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Science

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

Experimental setup: After passing through a grating compressor to compensate for dispersion and through a cylindrical (cyl.) 2 × magnifying telescope, the input beam is scanned and focused in one dimension (1D, not shown) on a grating, aligned perpendicular to the optic axis of the microscope. The grating is imaged onto the sample through a high-magnification telescope that comprises an achromatic lens and the microscope objective. Fluorescence is epidetected and imaged onto a CCD by a dichroic mirror.

Fig. 2
Fig. 2

Depth resolution of the TPEF microscope. The total fluorescence intensity is measured from a 0.8 μ m thick spin-coated fluorescent layer as a function of its distance from the focal plane of the objective. Fluorescence from a line with dimensions of 80 μ m × 0.5 μ m was collected and measured with a photomultiplier tube.

Fig. 3
Fig. 3

Depth-resolved images of a Drosophila egg chamber stained with DAPI. Optical sections of a Drosophila egg chamber containing 15 nurse cells and a single oocyte and wrapped by a layer of follicle cells are presented. The images go from the bottom of the egg chamber (top left image) to the center of the egg chamber (bottom right image). The approximate area of each image is 100 μ m × 80 μ m . The scale bar is 10 μ m . Images are slices separated by 2 μ m . The integration time for each image was 100 ms . The intensifier noise was subtracted from each image, which was corrected for spatial variations in the beam intensity, assuming a Gaussian beam profile. In the top row, follicle cells, whose nuclei are approximately 3 μ m in diameter, are shown. The images in the bottom row show the nuclei of nurse cells, whose size is of the order of 10 μ m , as well as the enveloping follicle cells.

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