Abstract

A method to obtain the axial locations of objects reconstructed by scanning holographic microscopy with submicrometer accuracy is presented and demonstrated. The method combines the holographic advantage of capturing three-dimensional (3D) information in a single two-dimensional scan, with the possibility of optically sectioning the 3D holographic reconstruction a posteriori. The method is demonstrated experimentally for pointlike features (fluorescent beads), and the limitation of the method for features of arbitrary size and shape is discussed.

© 2006 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. T. Wilson, Confocal Microscopy (Academic, 1990).
  2. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  3. E. R. Dowski and W. T. Cathey, "Extended depth of field through wavefront coding," Appl. Opt. 34, 1859-1866 (1995).
    [CrossRef] [PubMed]
  4. S. Bradburn, W. T. Cathey, and E. R. Dowski, "Realization of focus invariance in optical-digital systems with wavefront coding," Appl. Opt. 36, 9157-9166 (1997).
    [CrossRef]
  5. G. Indebetouw, A. El Maghnouji, and R. Foster, "Scanning holographic microscopy with transverse resolution exceeding the Rayleigh limit, and extended depth of focus," J. Opt. Soc. Am. A 22, 892-898 (2005).
    [CrossRef]
  6. M. A. A. Neil, R. Juskaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905-1907 (1997).
    [CrossRef]
  7. C. Vantalon and J. Mertz, "Quasi-confocal fluorescence sectioning with dynamic speckle illumination," Opt. Lett. 30, 3350-3352 (2005).
    [CrossRef]
  8. G. Indebetouw, P. Klysubun, T. Kim, and T.-C. Poon,"Imaging properties of scanning holographic microscopy," J. Opt. Soc. Am. A 17, 380-390 (2000).
    [CrossRef]
  9. G. Indebetouw and W. Zhong, "Scanning holographic microscopy of three-dimensional fluorescent specimens," J. Opt. Soc. Am. A 23, 1699-1707 (2006).
    [CrossRef]
  10. J. Rosen, G. Indebetouw, and G. Brooker, "Homodyne scanning holography," Opt. Express 14, 4280-4285 (2006).
    [CrossRef]
  11. T. Kim, "Optical sectioning by optical scanning holography and a Wiener filter," Appl. Opt. 45, 872-879 (2006).
    [CrossRef] [PubMed]

2006 (3)

2005 (2)

2000 (1)

1997 (2)

1995 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Bradburn, S.

Brooker, G.

Cathey, W. T.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Dowski, E. R.

El Maghnouji, A.

Foster, R.

Indebetouw, G.

Juskaitis, R.

Kim, T.

Klysubun, P.

Mertz, J.

Neil, M. A. A.

Poon, T.-C.

Rosen, J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Vantalon, C.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Wilson, T.

Zhong, W.

Appl. Opt. (3)

J. Opt. Soc. Am. A (3)

Opt. Express (1)

Opt. Lett. (2)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Other (1)

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

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

Section measuring 40 μ m × 40 μ m of a 160 μ m × 160 μ m 3D field of 1 μ m fluorescent beads reconstructed from a scanning holographic hologram at a depth of 3 μ m from the focal plane of the objective (excitation 532 nm , emission 600 nm , objective 20 × , NA 0.42, effective FZP NA 0.32 ). (a) Amplitude of the reconstruction (the scale bar is 5 μ m ), (b) phase map of the reconstruction from which the axial location of the beads are calculated using a sensitivity s = 0.306 rad μ m .

Fig. 2
Fig. 2

Same as Fig. 1 for a reconstruction at a depth of 15 μ m from the focal plane of the objective: (a) amplitude, (b) phase map.

Fig. 3
Fig. 3

Phase of six selected beads as functions of the axial depth of a series of reconstructions focused in planes 0.5 μ m apart. The average measured sensitivity is s = d ϕ d z = 0.306 ± 0.014 rad μ m matching the theoretical value s = π 2 ε Z 0.303 rad μ m .

Fig. 4
Fig. 4

Optical sections at depths of (a) 3 μ m and (b) 15 μ m from the focal plane of the objective, obtained a posteriori by selecting the pixels of the reconstructions having a phase in the range of 0.24 rad , corresponding to an axial section thickness of 1.6 μ m . The spurious rings around the out-of-focus beads are the regions where the phase of the out-of focus reconstructions cross zero. The scale bars are 5 μ m .

Equations (6)

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

P ̃ 1 ( ρ ) = exp ( i π λ z 0 ρ 2 ) circ ( ρ ρ MAX ) ,
P ̃ 2 ( ρ ) = δ ( ρ ) ,
R ̃ ( ρ ; z R ) = d z I ̃ ( ρ ; z ) exp [ i π λ ( z z R ) ρ 2 ] circ ( ρ ρ MAX ) ,
R ( μ , ξ ) δ ( μ μ P ) = R ̃ ( ρ ; ξ ) d 2 ρ = π ( ρ MAX ) 2 exp [ i π ξ 2 ] sin ( π ξ 2 ) ( π ξ 2 ) .
s = d ϕ d z = π 2 ε Z .
Δ z F = 2 ε Z .

Metrics