Abstract

The ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning. Our method relies on temporal focusing of the illumination pulse. The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration at the focal plane, before stretching again beyond it. This method is applied to obtain depth-resolved two-photon excitation fluorescence (TPEF) images of drosophila egg-chambers with nearly 105 effective pixels using a standard Ti:Sapphire laser oscillator.

© 2005 Optical Society of America

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References

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  • |

  1. M. Minsky, Microscopy apparatus, US patent 3,013,467, Dec. 19 (1961).
  2. T. Wilson, Confocal Microscopy, Academic press, London (1990).
  3. W. Denk, J.H. Strickler, W.W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 248, 73 (1990).
    [CrossRef] [PubMed]
  4. C.J.R. Sheppard, X.Q. Mao, �??Confocal microscopes with slit apertures,�?? J. Mod. Optics 35, 1169 (1988).
    [CrossRef]
  5. G.J. Brakenhoff, J. Squier, T. Norris, A.C. Bliton, M.H. Wade, B. Athey, �??Real-time two-photon confocal microscopy using a femtosecond, amplified, Ti:Sapphire system,�?? J. Microscopy 181, 253 (1995).
    [CrossRef]
  6. M.D. Egger, Petran, �??New reflected-light microscope for viewing unstained brain and ganglion cells,�?? Science 157, 305 (1967).
    [CrossRef] [PubMed]
  7. A.H. Buist, M. Muller, J. Squier, G.J. Brakenhoff, �??Real-time two-photon absorption microscopy using multipoint excitation,�?? J. Microscopy 192, 217 (1998).
    [CrossRef]
  8. J. Bewersdorf, R. Pick, S.W. Hell, �??Multifocal multiphoton microscopy,�?? Opt. Lett. 23, 655 (1998)
    [CrossRef]
  9. S.W. Hell, V. Andersen, �??Space-multiplexed multifocal nonlinear microscopy,�?? J. Microscopy 202, 457 (2001).
    [CrossRef]
  10. D.N. Fittinghoff, P.W. Wiseman, J.A. Squier, �??Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy,�?? Opt. Express 7, 273 (2000)
    [CrossRef] [PubMed]
  11. T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, �??High efficiency beam splitter for multifocal multiphoton microscopy,�?? J. Microscopy 201, 368 (2001).
    [CrossRef]
  12. G.J. Tearney, R.H. Webb, B.E. Bouma, �??Spectrally encoded confocal microscopy,�?? Opt. Lett. 23, 1152 (1998).
    [CrossRef]
  13. A. Egner, S.W. Hell, �??Time multiplexing and parallelization in multifocal multiphoton microscopy�??, J. Opt. Soc. Am. A 17, 1192 (2000)
    [CrossRef]
  14. V. Andersen, A. Egner, S.W. Hell, �??Time-multiplexed multifocal multiphoton microscope,�?? Opt. Lett. 26, 75 (2001).
    [CrossRef]
  15. O.E. Martinez, �??3000 times grating compressor with positive group-velocity dispersion - application to fiber compensation in 1.3-1.6 m region,�?? IEEE J. Quantum Electron. 23, 59 (1987).
    [CrossRef] [PubMed]
  16. A. Hopt, E. Neher, �??Highly nonlinear photodamage in two-photon fluorescence microscopy,�?? Biophys. J. 80, 2029 (2001).
    [CrossRef]
  17. S.H. Cho, B.E. Bouma, E.P. Ippen, J.G. Fujimoto, �??Low-repetition-rate high-peak-power Kerr-lens mode-locked Ti:Al2O3 laser with a multiple-pass cavity,�?? Opt. Lett. 24, 417 (1999).
    [CrossRef] [PubMed]
  18. T.B. Norris, �??Femtosecond pulse amplification at 250KHz with a Ti:Sapphire regenerative amplifier and application to continuum generation,�?? Opt. Lett. 17, 1009 (1992).
    [CrossRef]
  19. M. Straub, S.W. Hell, �??Fluorescnece lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,�?? Appl. Phys. Lett. 73, 1769 (1998).
    [CrossRef]
  20. S. Leveque-Fort, M.P. Fontaine-Aupart, G. Roger, P. Georges, �??Fluorescence-lifetime imaging with a multifocal multiphoton microscope,�?? Opt. Lett. 29, 2884 (2004).
    [CrossRef]
  21. A. M. Weiner, �??Femtosecond pulse shaping using spatial light modulators,�?? Rev. Sci. Inst. 71, 1929 (2000).
    [CrossRef]
  22. G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, M. Linial, �??Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,�?? Bioimaging 4, 215 (1996).
    [CrossRef]
  23. Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, �??Nonlinear scanning laser microscopy by third harmonic generation,�?? Appl. Phys. Lett. 70, 922 (1997).
    [CrossRef] [PubMed]
  24. M. Muller, J. Squier, K.R. Wilson, G.J. Brakenhoff,�??3D-microscopy of transparent objects using third-harmonic generation,�?? J. Microsc 191, 266 (1998).
    [CrossRef] [PubMed]
  25. M.D. Duncan, J. Reintjes, T.J., Manuccia, �??Scanning coherent anti-Stokes Raman microscope,�?? Opt. Lett. 7, 350 (1982).
    [CrossRef]
  26. A. Zumbusch, G.R. Holtom, X.S. Xie, �??Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,�?? Phys. Rev. Lett. 82, 4142 (1999).
    [CrossRef]
  27. M.G.L. Gustaffson, �??Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,�?? J. Microscopy 198, 82 (2000).

Appl. Phys. Lett. (2)

Y. Barad, H. Eisenberg, M. Horowitz, Y. Silberberg, �??Nonlinear scanning laser microscopy by third harmonic generation,�?? Appl. Phys. Lett. 70, 922 (1997).
[CrossRef] [PubMed]

M. Straub, S.W. Hell, �??Fluorescnece lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,�?? Appl. Phys. Lett. 73, 1769 (1998).
[CrossRef]

Bioimaging (1)

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, M. Linial, �??Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,�?? Bioimaging 4, 215 (1996).
[CrossRef]

Biophys. J. (1)

A. Hopt, E. Neher, �??Highly nonlinear photodamage in two-photon fluorescence microscopy,�?? Biophys. J. 80, 2029 (2001).
[CrossRef]

IEEE J. Quantum Electron. (1)

O.E. Martinez, �??3000 times grating compressor with positive group-velocity dispersion - application to fiber compensation in 1.3-1.6 m region,�?? IEEE J. Quantum Electron. 23, 59 (1987).
[CrossRef] [PubMed]

J. Microsc (1)

M. Muller, J. Squier, K.R. Wilson, G.J. Brakenhoff,�??3D-microscopy of transparent objects using third-harmonic generation,�?? J. Microsc 191, 266 (1998).
[CrossRef] [PubMed]

J. Microscopy (5)

G.J. Brakenhoff, J. Squier, T. Norris, A.C. Bliton, M.H. Wade, B. Athey, �??Real-time two-photon confocal microscopy using a femtosecond, amplified, Ti:Sapphire system,�?? J. Microscopy 181, 253 (1995).
[CrossRef]

A.H. Buist, M. Muller, J. Squier, G.J. Brakenhoff, �??Real-time two-photon absorption microscopy using multipoint excitation,�?? J. Microscopy 192, 217 (1998).
[CrossRef]

S.W. Hell, V. Andersen, �??Space-multiplexed multifocal nonlinear microscopy,�?? J. Microscopy 202, 457 (2001).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, �??High efficiency beam splitter for multifocal multiphoton microscopy,�?? J. Microscopy 201, 368 (2001).
[CrossRef]

M.G.L. Gustaffson, �??Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,�?? J. Microscopy 198, 82 (2000).

J. Mod. Optics (1)

C.J.R. Sheppard, X.Q. Mao, �??Confocal microscopes with slit apertures,�?? J. Mod. Optics 35, 1169 (1988).
[CrossRef]

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

Opt. Express (1)

Opt. Lett. (7)

Phys. Rev. Lett. (1)

A. Zumbusch, G.R. Holtom, X.S. Xie, �??Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,�?? Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Rev. Sci. Inst. (1)

A. M. Weiner, �??Femtosecond pulse shaping using spatial light modulators,�?? Rev. Sci. Inst. 71, 1929 (2000).
[CrossRef]

Science (2)

W. Denk, J.H. Strickler, W.W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 248, 73 (1990).
[CrossRef] [PubMed]

M.D. Egger, Petran, �??New reflected-light microscope for viewing unstained brain and ganglion cells,�?? Science 157, 305 (1967).
[CrossRef] [PubMed]

Other (2)

M. Minsky, Microscopy apparatus, US patent 3,013,467, Dec. 19 (1961).

T. Wilson, Confocal Microscopy, Academic press, London (1990).

Supplementary Material (2)

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Figures (5)

Fig. 1.
Fig. 1.

(a) Cartoon of the standard multiphoton microscopy scheme: a high peak intensity at the focus of the objective lens is generated by spatial focusing of the beam, while the temporal profile of the pulse remains unchanged in passing through the sample. (b) Cartoon of the scanningless method. The beam is weakly focused, covering an area which is many orders of magnitude larger than a diffraction limited spot. A high peak intensity at the focus is achieved by modification of the temporal profile of the pulse as it propagates, reaching its peak at the focus of the objective lens.

Fig. 2.
Fig. 2.

(a) Principle of the scanningless TPEF microscope. A short pulse impinges upon a scatterer. At a point P further away, the pulse duration is longer due to the difference in the length of trajectories taken by the rays reaching it from different locations on the scatterer. Only at the image plane of the telescope is the pulse duration restored to its initial value, in accordance with the Fermat principle. (b) The experimental setup: The input beam impinges upon a grating, aligned perpendicular to the optic axis of the microscope. The grating is imaged through a high magnification telescope, comprised of an achromatic lens and the microscope objective, on the sample. Fluorescence is epi-detected and imaged onto a CCD using a dichroic mirror.

Fig. 3.
Fig. 3.

Schematics movies of the temporal focusing mechanism: (a) An ultrashort pulse impinges on a scattering plate. The temporal evolution of scattered rays from 20 different points across the plate is followed (one of these wavefronts is highlighted in blue). The scattered rays denote the ultrashort pulse envelope, typically of a width of several microns. In general, points in space see an extended illumination due to pathlength difference between rays emerging from different locations on the scattering plate. Only at the image plane of the telescope all rays arriving at a point originate from a single point on the scatterer, resulting in an illumination time identical with the original pulse duration (video file of 0.72Mb). (b) Same as in (a) when the incoming pulse impinges on the scattering plate at an angle. This results in larger broadening of the pulse around the focal plane of the telescope due to the effectively increased pathlength difference. Note also the similarity of this scheme to scanning of the illumination beam in one spatial dimension, as the illumination at the focal plane occurs first at the bottom and moves towards the top (video file of 1.21Mb).

Fig. 4.
Fig. 4.

Depth resolution of the scanningless TPEF microscope. The total fluorescence intensity measured from a 0.9µm thick spin-coated fluorescent layer as a function of its distance from the focal plane of the objective. Fluorescence from an area of 104 µm 2 was collected and measured with a photomultiplier tube. A CCD image of the illuminated area was used in the following to normalize for signal intensity due to the Gaussian shape of the excitation pulse.

Fig. 5.
Fig. 5.

Scanningless depth-resolved images of a drosophila egg-chamber stained with DAPI, a fluorescent DNA binding probe. Optical sections of a drosophila egg-chamber containing 15 nurse cells, 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 its top (bottom center image). The area of each image is 140x140µm. Images are separated by 5µm. The integration time for each image was 30 seconds. The intensifier noise was subtracted from each image, and it was corrected for spatial variations in the beam intensity assuming a Gaussian beam profile. On both the bottom and top sections, follicle cells, whose nuclei are approximately 3µm in diameter, are observed. The center images show the nuclei of nurse cells, whose size is of the order of 10µm, as well as the enveloping follicle cells. A smaller egg-chamber is observed on the top right corner of the images. The bottom right image shows, for comparison, a TPEF image where the grating was replaced by a standard mirror, resulting in a non-depth resolved image. This image is to be compared with the one directly above it. While some detail can be seen, the entire egg-chamber shows a strong out-of-focus background.

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