Abstract

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.

© 2012 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. A. Diaspro, ed., Confocal and Two-Photon Microscopy(Wiley-Liss, 2001).
  2. J. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006).
  3. S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
    [CrossRef]
  4. S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
    [CrossRef]
  5. J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
    [CrossRef]
  6. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
    [CrossRef]
  7. H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
    [CrossRef]
  8. E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
    [CrossRef]
  9. E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
    [CrossRef]
  10. P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
    [CrossRef]
  11. H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
    [CrossRef]
  12. G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, Opt. Express 18, 10154 (2010).
    [CrossRef]

2011 (1)

2010 (2)

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, Opt. Express 18, 10154 (2010).
[CrossRef]

2006 (1)

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef]

2004 (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

1999 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef]

1998 (1)

P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
[CrossRef]

1992 (2)

S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

1989 (1)

H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
[CrossRef]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef]

Bewersdorf, J.

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

Chaumet, P. C.

E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
[CrossRef]

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Diaspro, A.

A. Diaspro, ed., Confocal and Two-Photon Microscopy(Wiley-Liss, 2001).

Egner, A.

Engelhardt, J.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

Ferrand, P.

E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
[CrossRef]

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Gugel, H.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

Gustafsson, M. G. L.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef]

Hamilton, D. K.

H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
[CrossRef]

Hell, S. W.

G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, Opt. Express 18, 10154 (2010).
[CrossRef]

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Higdon, P. D.

P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
[CrossRef]

Jakobs, S.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

Le Moal, E.

E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
[CrossRef]

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Matthews, H. J.

H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
[CrossRef]

Mudry, E.

E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
[CrossRef]

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Pawley, J.

J. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006).

Schmidt, R.

Schönle, A.

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef]

Sentenac, A.

E. Le Moal, E. Mudry, P. C. Chaumet, P. Ferrand, and A. Sentenac, J. Opt. Soc. Am. A 28, 1586 (2011).
[CrossRef]

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Sheppard, C. J. R.

H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
[CrossRef]

Stelzer, E. H. K.

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

Storz, R.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

Török, P.

P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
[CrossRef]

Vicidomini, G.

Wilson, T.

P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
[CrossRef]

Biophys. J. (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef]

J. Microsc. (2)

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef]

J. Mod. Opt. (1)

H. J. Matthews, D. K. Hamilton, and C. J. R. Sheppard, J. Mod. Opt. 36, 233 (1989).
[CrossRef]

J. Opt. Soc. Am. A (2)

Opt. Commun. (2)

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

P. Török, P. D. Higdon, and T. Wilson, Opt. Commun. 148, 300 (1998).
[CrossRef]

Opt. Express (1)

Phys. Rev. Lett. (1)

E. Mudry, E. Le Moal, P. Ferrand, P. C. Chaumet, and A. Sentenac, Phys. Rev. Lett. 105, 203903 (2010).
[CrossRef]

Other (2)

A. Diaspro, ed., Confocal and Two-Photon Microscopy(Wiley-Liss, 2001).

J. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1.
Fig. 1.

Schematic view of our experimental two-photon fluorescence isotropic-single-objective microscopy setup. (Inset) Typical phase mask for isotropic focusing at 6 µm above the reflective substrate. HWP, half-wave plate; GP, Glan prism; L, achromatic doublet; M, mirror; obj., objective; DC, dichroic beam splitter; BP, bandpass filter; TL, microscope tube lens; APD, avalanche-photodiode photon counter. See text for details.

Fig. 2.
Fig. 2.

Axial (zx) and transverse (xy) views of (a) the theoretical PSF in ISO-2PFM and standard 2PFM and (b) 3D fluorescence images of 200 nm beads experimentally measured by ISO-2PFM and standard 2PFM. Both microscopy techniques are based on nonconfocal detection. Images are displayed with a linear gray-level scale.

Fig. 3.
Fig. 3.

Intensity profile measured along the z axis on 3D fluorescence images of 200 nm beads [see Fig. 2b] by ISO-2PFM (solid line) and standard 2PFM (dashed line). The origin of the bottom axis corresponds for ISO-2PFM to the position of the mirror surface.

Tables (1)

Tables Icon

Table 1. Specifications of PSFs

Metrics