Abstract

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.

© 2006 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
    [CrossRef] [PubMed]
  2. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).
  3. A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
    [CrossRef] [PubMed]
  4. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-677 (1998).
    [CrossRef]
  5. A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
    [CrossRef]
  6. D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, "Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy," Opt. Express 7, 273-279 (2000).
    [CrossRef] [PubMed]
  7. S. C. Tucker, W. T. Cathey, and E. R. Dowski, "Extended depth of field and aberration control for inexpensive digital microscope systems," Opt. Express 4, 467-474 (1999).
    [CrossRef] [PubMed]
  8. P. Potuluri, M. R. Fetterman, and D. J. Brady, "High depth of field microscopic imaging using an interferometric camera," Opt. Express 8, 624-630 (2001).
    [CrossRef] [PubMed]
  9. S. Fujiwara, "Optical properties of conic surfaces. I. Reflecting cone," J. Opt. Soc. Am. 52, 287-292 (1962).
    [CrossRef]
  10. G. Roy and R. Tremblay, "Influence of the divergence of a laser beam on the axial intensity distribution of an axicon," Opt. Commun. 34, 1-3 (1980).
    [CrossRef]
  11. J. H. McLeod, "The axicon: a new type of optical element," J. Opt. Soc. Am. 44, 592-597 (1953).
    [CrossRef]
  12. M. Rioux, R. Tremblay, and P. A. Bélanger, "Linear, annular, and radial focusing with axicons and applications to laser machining," Appl. Opt. 17, 1532-1536 (1978).
    [CrossRef] [PubMed]
  13. R. M. Herman and T. A. Wiggins, "High-efficiency diffractionless beams of constant size and intensity," Appl. Opt. 33, 7297-7306 (1994).
    [CrossRef] [PubMed]
  14. J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
    [CrossRef]
  15. A. Vasara, J. Turunen, and A. T. Friberg, "Realization of general nondiffracting beams with computer-generated holograms," J. Opt. Soc. Am. A 6, 1748-1754 (1989).
    [CrossRef] [PubMed]
  16. Z. Ding, H. Ren, Y. Zhao, J. S. Nelson, and Z. Chen, "High-resolution optical coherence tomography over a large depth range with an axicon lens," Opt. Lett. 27, 243-245 (2002).
    [CrossRef]
  17. D. McGloin and K. Dholakia, "Bessel beams: diffraction in a new light," Contemp. Phys. 46, 15-28 (2005).
    [CrossRef]
  18. H. Little, C. T. A. Brown, V. Garcés-Chávez, W. Sibbett, and K. Dholakia, "Optical guiding of microscopic particles in femtosecond and continuous wave Bessel light beams," Opt. Express 12, 2560-2563 (2004).
    [CrossRef] [PubMed]
  19. V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
    [CrossRef] [PubMed]
  20. F. W. J. Olver, Asymptotics and Special Functions (Academic, 1974), p. 563.
  21. R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
    [CrossRef]
  22. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
    [CrossRef]

2005 (2)

D. McGloin and K. Dholakia, "Bessel beams: diffraction in a new light," Contemp. Phys. 46, 15-28 (2005).
[CrossRef]

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef]

2004 (1)

2003 (2)

R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
[CrossRef] [PubMed]

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

2002 (2)

Z. Ding, H. Ren, Y. Zhao, J. S. Nelson, and Z. Chen, "High-resolution optical coherence tomography over a large depth range with an axicon lens," Opt. Lett. 27, 243-245 (2002).
[CrossRef]

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

2001 (2)

2000 (1)

1999 (1)

1998 (2)

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-677 (1998).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

1994 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).

1989 (1)

1987 (1)

J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
[CrossRef]

1980 (1)

G. Roy and R. Tremblay, "Influence of the divergence of a laser beam on the axial intensity distribution of an axicon," Opt. Commun. 34, 1-3 (1980).
[CrossRef]

1978 (1)

1962 (1)

1953 (1)

Aronov, D.

R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
[CrossRef] [PubMed]

Bélanger, P. A.

Bewersdorf, J.

Brackenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Brady, D. J.

Brown, C. T. A.

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Cathey, W. T.

Chen, Z.

Cossart, R.

R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).

Dholakia, K.

D. McGloin and K. Dholakia, "Bessel beams: diffraction in a new light," Contemp. Phys. 46, 15-28 (2005).
[CrossRef]

H. Little, C. T. A. Brown, V. Garcés-Chávez, W. Sibbett, and K. Dholakia, "Optical guiding of microscopic particles in femtosecond and continuous wave Bessel light beams," Opt. Express 12, 2560-2563 (2004).
[CrossRef] [PubMed]

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

Ding, Z.

Dowski, E. R.

Dumin, J.

J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
[CrossRef]

Eberly, J. H.

J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
[CrossRef]

Fetterman, M. R.

Fittinghoff, D. N.

Fortin, M.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Friberg, A. T.

Fujiwara, S.

Garcés-Chavez, V.

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

Garcés-Chávez, V.

Griebner, U.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Grunwald, R.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Hell, S. W.

Helmchen, F.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef]

Herman, R. M.

Hopt, A.

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

Kebbel, V.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Kummrow, A.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Little, H.

McGloin, D.

D. McGloin and K. Dholakia, "Bessel beams: diffraction in a new light," Contemp. Phys. 46, 15-28 (2005).
[CrossRef]

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

McLeod, J. H.

Melville, H.

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

Miceli, J. J.

J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
[CrossRef]

Muller, M.

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Neher, E.

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

Nelson, J. S.

Neumann, U.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Nibbering, E. T. J.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Olver, F. W. J.

F. W. J. Olver, Asymptotics and Special Functions (Academic, 1974), p. 563.

Piché, M.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Pick, R.

Potuluri, P.

Ren, H.

Rini, M.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Rioux, M.

Rousseau, G.

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Roy, G.

G. Roy and R. Tremblay, "Influence of the divergence of a laser beam on the axial intensity distribution of an axicon," Opt. Commun. 34, 1-3 (1980).
[CrossRef]

Sibbett, W.

H. Little, C. T. A. Brown, V. Garcés-Chávez, W. Sibbett, and K. Dholakia, "Optical guiding of microscopic particles in femtosecond and continuous wave Bessel light beams," Opt. Express 12, 2560-2563 (2004).
[CrossRef] [PubMed]

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

Squier, J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Squier, J. A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).

Tremblay, R.

G. Roy and R. Tremblay, "Influence of the divergence of a laser beam on the axial intensity distribution of an axicon," Opt. Commun. 34, 1-3 (1980).
[CrossRef]

M. Rioux, R. Tremblay, and P. A. Bélanger, "Linear, annular, and radial focusing with axicons and applications to laser machining," Appl. Opt. 17, 1532-1536 (1978).
[CrossRef] [PubMed]

Tucker, S. C.

Turunen, J.

Vasara, A.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).

Wiggins, T. A.

Wiseman, P. W.

Yuste, R.

R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
[CrossRef] [PubMed]

Zhao, Y.

Appl. Opt. (2)

Biophys. J. (1)

A. Hopt and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

Contemp. Phys. (1)

D. McGloin and K. Dholakia, "Bessel beams: diffraction in a new light," Contemp. Phys. 46, 15-28 (2005).
[CrossRef]

J. Microsc. (1)

A. H. Buist, M. Muller, J. Squier, and G. J. Brackenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

J. Opt. Soc. Am. (2)

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

Nat. Methods (1)

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef]

Nature (3)

V. Garcés-Chavez, D. McGloin, H. Melville, W. Sibbett, and K. Dholakia, "Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam," Nature 419, 145-147 (2002).
[CrossRef] [PubMed]

R. Cossart, D. Aronov, and R. Yuste, "Attractor dynamics of network UP states in the neocortex," Nature 423, 283-288 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Nature 248, 73-76 (1990).

Opt. Commun. (1)

G. Roy and R. Tremblay, "Influence of the divergence of a laser beam on the axial intensity distribution of an axicon," Opt. Commun. 34, 1-3 (1980).
[CrossRef]

Opt. Express (4)

Opt. Lett. (2)

Phys. Rev. A (1)

R. Grunwald, V. Kebbel, U. Griebner, U. Neumann, A. Kummrow, M. Rini, E. T. J. Nibbering, M. Piché, G. Rousseau, and M. Fortin, "Generation and characterization of spatially and temporally localized few-cycle optical wavepackets," Phys. Rev. A 67, 063820 (2003).
[CrossRef]

Phys. Rev. Lett. (1)

J. Dumin, J. J. Miceli, Jr., and J. H. Eberly, "Diffraction-free beams," Phys. Rev. Lett. 58, 1499-1501 (1987).
[CrossRef]

Other (1)

F. W. J. Olver, Asymptotics and Special Functions (Academic, 1974), p. 563.

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 (7)

Fig. 1
Fig. 1

Illustration of the excitation volume achieved with an objective (top) and with an axicon (bottom).

Fig. 2
Fig. 2

Experimental setup of the two-photon excitation fluorescence microscopy system using an axicon. Thickness of the beam splitter was 1   mm ; thickness of the axicon at the center was 6.8 mm.

Fig. 3
Fig. 3

(a) Comparison in the lateral plane of the square of the intensity of a Bessel beam produced with a BK7 axicon ( α = 30 ° ) and the focal spot obtained with a conventional objective ( NA = 0.5 ) ; both have the same radial resolution ( ρ 0 = 1.0   μm ) . A picture of the Bessel beam is shown in the inset. (b) Squared intensity along the z axis for the same incident beam passing through the axicon and the objective. In both figures, the continuous curve is used for the beam generated with the axicon, and the dashed curve is used for the beam generated with the objective. (c) Squared intensity along the z axis: experimental results (broken curve) and theoretical results (continuous curve) for an axicon of 25°.

Fig. 4
Fig. 4

Image of two 15   μm fluorescent microspheres taken with the two-photon microscope with an axicon have an angle α = 25 ° .

Fig. 5
Fig. 5

Twelve images of fluorescence obtained by scanning the objective in different planes. These images are 100 × 100   pixels with a pixel size of 3   μm × 3   μm and a pixel time of 1   ms (total acquisition time of 10 s). The distance between consecutive planes is 50   μs . The laser power on the sample was 0 .5   mW .

Fig. 6
Fig. 6

Comparison of projection images of the same sample, resulting from (a) the sum of 12 scans of the objective and (b) a single scan of the axicon ( 120 × 120 pixels with a pixel size of 2 .5   μm × 2 .5   μm and a pixel time of 1 ms). The laser power on the sample was 30   mW .

Fig. 7
Fig. 7

Lateral profile of two-photon fluorescence with (a) the NA = 0.5 objective and (b) the 30° axicon. The 2D image is shown in the insets for both graphs.

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

I ( ρ , z ) = I 0 z exp [ 2 ( ( n 1 ) z tan α w 0 ) 2 ] × J 0 2 [ 2 π λ 0 ( n 1 ) ρ tan α ] ,
I 0 = A 0 2 π 2 ( n 1 ) 2 tan 2 α λ ,
ρ 0 = 2.4048 λ 0 2 π ( n 1 ) tan α ,
L = w 0 tan [ arcsin ( n sin α ) α ] ,

Metrics