Abstract

This paper proposes a new autofocusing method for observing cells under a transmission illumination. The focusing method uses a quick and simple focus estimation technique termed “depth from diffraction,” which is based on a diffraction pattern in a defocused image of a biological specimen. Since this method can estimate the focal position of the specimen from only a single defocused image, it can easily realize high-speed auto-focusing. To demonstrate the method, it was applied to continuous focus tracking of a swimming paramecium, in combination with two-dimensional position tracking. Three-dimensional tracking of the paramecium for 70 s was successfully demonstrated.

© 2006 Optical Society of America

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References

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  1. J. H. Price and D. A. Gough, "Comparison of Phase-Contrast and Fluorescence Digital Autofocus for Scanning Microscopy," Cytometry 16, 283-297 (1994).
    [CrossRef]
  2. M. Subbarao and J.-K. Tyan, "Selecting the Optimal Focus Measure for Autofocusing and Depth-From-Focus," IEEE Trans. Patternn Anal. Mach. Intell. 20, 864-870 (1998).
    [CrossRef]
  3. J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
    [CrossRef] [PubMed]
  4. M. Born and E. Wolf, Principles of Optics, 7th Edition (Cambridge University Press, Cambridge, 2002).
  5. W. D. Nesse, Introduction to Optical Mineralogy. (Oxford University Press, New York, 1991).
  6. T. Tsuruta. Oyo-kogaku (Applied Optics) I. (Baifukan, Tokyo, 1990). (in Japanese)
  7. H. Toyoda, N. Mukohzaka, K. Nakamura, M. Takumi, S. Mizuno, and M. Ishikawa, "1ms column-parallel vision system coupled with an image intensifier; I-CPV," in Proceedings of Symp. High Speed Photography and Photonics 2001, vol. 5-1, 2001, pp. 89-92 (in Japanese).
  8. Y. Nakabo, M. Ishikawa, H. Toyoda, and S. Mizuno, "1ms column parallel vision system and it’s application of high speed target tracking," in Proceedings of the IEEE International Conference on Robotics & Automation (Institute of Electrical and Electronics Engineers, New York, 2000), pp. 650-655.
  9. H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
    [CrossRef]
  10. J. W. Goodman, Introduction to Fourier Optics. (McGraw-Hill, Inc., Boston, Massachusetts, 1996).

2005 (1)

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

2000 (1)

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

1998 (1)

M. Subbarao and J.-K. Tyan, "Selecting the Optimal Focus Measure for Autofocusing and Depth-From-Focus," IEEE Trans. Patternn Anal. Mach. Intell. 20, 864-870 (1998).
[CrossRef]

1994 (1)

J. H. Price and D. A. Gough, "Comparison of Phase-Contrast and Fluorescence Digital Autofocus for Scanning Microscopy," Cytometry 16, 283-297 (1994).
[CrossRef]

Cornelissen, F.

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

Geusebroek, J.-M.

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

Gough, D. A.

J. H. Price and D. A. Gough, "Comparison of Phase-Contrast and Fluorescence Digital Autofocus for Scanning Microscopy," Cytometry 16, 283-297 (1994).
[CrossRef]

Greets, H.

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

Hashimoto, K.

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

Ishikawa, M.

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

Ogawa, N.

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

Oku, H.

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

Price, J. H.

J. H. Price and D. A. Gough, "Comparison of Phase-Contrast and Fluorescence Digital Autofocus for Scanning Microscopy," Cytometry 16, 283-297 (1994).
[CrossRef]

Smeulders, A. W. M.

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

Subbarao, M.

M. Subbarao and J.-K. Tyan, "Selecting the Optimal Focus Measure for Autofocusing and Depth-From-Focus," IEEE Trans. Patternn Anal. Mach. Intell. 20, 864-870 (1998).
[CrossRef]

Tyan, J.-K.

M. Subbarao and J.-K. Tyan, "Selecting the Optimal Focus Measure for Autofocusing and Depth-From-Focus," IEEE Trans. Patternn Anal. Mach. Intell. 20, 864-870 (1998).
[CrossRef]

Cytometry (2)

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Greets, " Robust Autofocusing in Microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

J. H. Price and D. A. Gough, "Comparison of Phase-Contrast and Fluorescence Digital Autofocus for Scanning Microscopy," Cytometry 16, 283-297 (1994).
[CrossRef]

IEEE Trans. Patternn Anal. Mach. Intell. (1)

M. Subbarao and J.-K. Tyan, "Selecting the Optimal Focus Measure for Autofocusing and Depth-From-Focus," IEEE Trans. Patternn Anal. Mach. Intell. 20, 864-870 (1998).
[CrossRef]

Rev. Sci. Instrum. (1)

H. Oku, N. Ogawa, K. Hashimoto, and M. Ishikawa, " Two-dimensional tracking of a motile microorganism allowing high-resolution observation with various imaging techniques," Rev. Sci. Instrum. 76, 034301 (2005).
[CrossRef]

Other (6)

J. W. Goodman, Introduction to Fourier Optics. (McGraw-Hill, Inc., Boston, Massachusetts, 1996).

M. Born and E. Wolf, Principles of Optics, 7th Edition (Cambridge University Press, Cambridge, 2002).

W. D. Nesse, Introduction to Optical Mineralogy. (Oxford University Press, New York, 1991).

T. Tsuruta. Oyo-kogaku (Applied Optics) I. (Baifukan, Tokyo, 1990). (in Japanese)

H. Toyoda, N. Mukohzaka, K. Nakamura, M. Takumi, S. Mizuno, and M. Ishikawa, "1ms column-parallel vision system coupled with an image intensifier; I-CPV," in Proceedings of Symp. High Speed Photography and Photonics 2001, vol. 5-1, 2001, pp. 89-92 (in Japanese).

Y. Nakabo, M. Ishikawa, H. Toyoda, and S. Mizuno, "1ms column parallel vision system and it’s application of high speed target tracking," in Proceedings of the IEEE International Conference on Robotics & Automation (Institute of Electrical and Electronics Engineers, New York, 2000), pp. 650-655.

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Figures (8)

Fig. 1.
Fig. 1.

Diffraction images of yeast and paramecium cells at three focal positions. The yeast cell has a spherical body about 5 μm in diameter. Paramecium is a motile cell with an ellipsoidal body whose longitudinal length is from 100 to 200 μm. The paramecium cell was held in a micro-capillary to keep it in the field of view. In the case of the yeast cell, an intensity variance was observed in the interior of the cell. No clear inner fringe was observed.

Fig. 2.
Fig. 2.

Block diagram of the experimental set-up.

Fig. 3.
Fig. 3.

Block diagram of image processing to extract features of bright fringe in Becke pattern. All thresholds were determined by humans using three images (one focused and two defocused in different direction) of the specimen captured under the environment for the experiments.

Fig. 4.
Fig. 4.

(a) Profile of bright fringe and (b) profile of estimated Z position of the object.

Fig. 5.
Fig. 5.

(a) Sequence of photographs acquired by the CCD camera. The maximum speed of the focus position movment during this sequence was 346 μm/s. (b) Computer-generated movie based on the acquired trajectory. Two small movies are superimposed: one is a movie of the target paramecium captured by a CCD camera placed at the top left in the movie, and the other is a computer-generated animation from the same viewpoint of a CCD camera placed at the bottom-right in the movie. [Media 1]

Fig. 6.
Fig. 6.

Relationship between focal plane position and features of the acquired image. Two schematics are shown to explain the diffraction pattern generation qualitatively: (a) the object is in front of the focal plane and a bright fringe appears inside of the cell or (b) the object is behind the focal plane and a bright fringe appears outside of the cell.

Fig. 7.
Fig. 7.

Arrangement used in calculating theoretical intensity distribution.

Fig. 8.
Fig. 8.

Theoretical intensity profiles of the amplitude knife-edge and phase knife-edge [6] : (a) amplitude knife-edge (z > 0), (b) amplitude knife-edge (z < 0), (c) phase knife-edge (z > 0), and (d) phase knife-edge (z < 0).

Tables (1)

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Table 1. Specifications of the XYZ automated stage

Equations (5)

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z ̂ = { a 1 i outer + a 0 ( z 0 ) b 1 i inner + b 0 ( z < 0 )
z d = z k ( i outer i inner )
I ( x , y ) = U ( x , y , 0 ) 2 = I 0 λ 2 z 2 g ( ξ , η ) · exp [ j π λz { ( x ξ ) 2 + ( y η ) 2 } ] dξdη 2
g = { 1 ( ξ 0 ) Amplitude knife edge 0 ( ξ < 0 )
g = { 1 ( ξ 0 ) Phase knife edge e ( ξ < 0 )

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