Abstract

We present a technique for imaging fluorescent particles based on the axial modulation of the objective’s focal plane position. This technique provides full-field optical sectioning and can be used to localize the fluorophores in three dimensions. We describe the technique and apply it to image 200nm diameter fluorescent beads immobilized in a gel. We show that full-field optical sectioning is obtained and that the beads are localized with a precision of 10nm in the transverse plane and 14nm in the axial direction.

© 2006 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
    [CrossRef] [PubMed]
  2. L. Kastrup and S. W. Hell, Angew. Chem., Int. Ed. Engl. 43, 6646 (2004).
    [CrossRef]
  3. M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
    [CrossRef]
  4. F. Kulzer, M. Orrit, Annu. Rev. Phys. Chem. 55, 585 (2004).
    [CrossRef] [PubMed]
  5. R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
    [CrossRef] [PubMed]
  6. R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
    [CrossRef] [PubMed]
  7. M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
    [CrossRef]
  8. M. Speidel, A. Jonás, and E. L. Florin, Opt. Lett. 28, 69 (2003).
    [CrossRef] [PubMed]
  9. H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
    [CrossRef] [PubMed]
  10. T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).
  11. A. V. Oppenheim and R. W. Schafer, Digital Signal Processing (Prentice-Hall, 1975), Chap. 3.
  12. S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
    [CrossRef]

2004 (2)

L. Kastrup and S. W. Hell, Angew. Chem., Int. Ed. Engl. 43, 6646 (2004).
[CrossRef]

F. Kulzer, M. Orrit, Annu. Rev. Phys. Chem. 55, 585 (2004).
[CrossRef] [PubMed]

2003 (2)

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

M. Speidel, A. Jonás, and E. L. Florin, Opt. Lett. 28, 69 (2003).
[CrossRef] [PubMed]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
[CrossRef] [PubMed]

2000 (1)

M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

1999 (1)

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

1996 (1)

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

1994 (1)

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

1993 (1)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

1984 (1)

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

1975 (1)

A. V. Oppenheim and R. W. Schafer, Digital Signal Processing (Prentice-Hall, 1975), Chap. 3.

Cremer, C.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Dahan, M.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Dickson, R. M.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

Florin, E. L.

Hell, S.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Hell, S. W.

L. Kastrup and S. W. Hell, Angew. Chem., Int. Ed. Engl. 43, 6646 (2004).
[CrossRef]

M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Jonás, A.

Kao, H. P.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Kastrup, L.

L. Kastrup and S. W. Hell, Angew. Chem., Int. Ed. Engl. 43, 6646 (2004).
[CrossRef]

Kulzer, F.

F. Kulzer, M. Orrit, Annu. Rev. Phys. Chem. 55, 585 (2004).
[CrossRef] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
[CrossRef] [PubMed]

Levi, S.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Luccardini, C.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Moerner, W. E.

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

Nagorni, M.

M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Norris, D. J.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

Oppenheim, A. V.

A. V. Oppenheim and R. W. Schafer, Digital Signal Processing (Prentice-Hall, 1975), Chap. 3.

Orrit, M.

F. Kulzer, M. Orrit, Annu. Rev. Phys. Chem. 55, 585 (2004).
[CrossRef] [PubMed]

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

Reiner, G.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Riveau, B.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Rostaing, P.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Schafer, R. W.

A. V. Oppenheim and R. W. Schafer, Digital Signal Processing (Prentice-Hall, 1975), Chap. 3.

Schimdt, M.

M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Sheppard, C.

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

Speidel, M.

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
[CrossRef] [PubMed]

Triller, A.

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Tzeng, Y.-L.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

Verkman, A. S.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

Angew. Chem., Int. Ed. Engl. (1)

L. Kastrup and S. W. Hell, Angew. Chem., Int. Ed. Engl. 43, 6646 (2004).
[CrossRef]

Annu. Rev. Phys. Chem. (1)

F. Kulzer, M. Orrit, Annu. Rev. Phys. Chem. 55, 585 (2004).
[CrossRef] [PubMed]

Biophys. J. (2)

R. E. Thompson, D. R. Larson, and W. W. Webb, Biophys. J. 82, 2775 (2002).
[CrossRef] [PubMed]

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

J. Microsc. (1)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Opt. Lett. (1)

Rev. Sci. Instrum. (1)

M. Schimdt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Science (3)

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, and W. E. Moerner, Science 274, 966 (1996).
[CrossRef] [PubMed]

M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, Science 300, 442 (2003).
[CrossRef]

Other (2)

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

A. V. Oppenheim and R. W. Schafer, Digital Signal Processing (Prentice-Hall, 1975), Chap. 3.

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 (4)

Fig. 1
Fig. 1

Axial modulation of the focal plane at the frequency ω induces an intensity modulation of the fluorescence intensity both at ω and 2 ω . Inset, scheme of image acquisition; the phase between the modulation (solid curve) and the acquisition must be correctly adjusted to extract the signal at 2 ω (dashed curve).

Fig. 2
Fig. 2

Left, results of simulations. (a) Intensity along the axis evaluated with Eq. (1) for an oil immersion objective with NA = 1.35 . (b) I ω calculated by using Eq. (5) and d = 300 nm . (c) I 2 ω calculated by using Eq. (6) and d = 300 nm . (d) ϕ = arctan ( I ω I 2 ω ) . A linear fit gives ϕ = ( 5.18 ± 0.02 ) z . Right, data from images of fluorescent beads in a gel. d is 600 nm . (a) Intensity along the axis; this is the experimental PSF. (b) I ω calculated by using Eq. (3) (c) I 2 ω calculated by using Eq. (4) (d) ϕ. For all of these graphics, signals were measured and calculated each 50 nm (crosses), and we plotted the best curve (solid curves).

Fig. 3
Fig. 3

(a) Standard epifluorescence image ( 7.9 μ m × 7.7 μ m ) . (b) 2 ω image without negative values ( 7.9 μ m × 7.7 μ m ) . (c) 3D representation of the samples. Coordinates ( x , y , z ) of each beads in nanometers are bead 1, ( 3079 , 1018 , 549 ) ; bead 2, ( 880 , 1386 , 226 ) ; bead 3, ( 5487 , 5418 , 65 ) ; bead 4, (1627, 5494, 210), with the origin in the upper left-hand corner. The precision on x and y is 10 nm and 14 nm for z.

Fig. 4
Fig. 4

Left, stack of images axially separated by 50 nm . By combining three images we can obtain an optical section and the precise localization of objects. Right, calculation of I ω , I 2 ω , and ϕ from a stack. K = 302.7 nm rad 1 .

Equations (7)

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

I ( u ) = I 0 [ sin ( u 4 ) u 4 ] 2
u = ( 8 π λ ) n z sin 2 ( α 2 ) .
I ω ( x , y , z ) = ( 1 4 ) [ ( p 0 p 2 ) + i ( p 1 p 3 ) ] ,
I 2 ω ( x , y , z ) = ( 1 4 ) [ ( p 1 + p 3 ) ( p 0 + p 2 ) ] .
I ω ( x , y , z ) = ( 1 4 ) [ p ( z + d ) p ( z d ) ] ,
I 2 ω ( x , y , z ) = ( 1 4 ) [ 2 p ( z ) p ( z + d ) p ( z d ) ] ,
z = K × ϕ ,

Metrics