Abstract

In an accompanying paper [G. Indebetouw and P. Klysubun, J. Opt. Soc. Am. A 18, 319 (2001)], the theoretical background of a spatiotemporal digital microholographic method was described, and some experimental results were presented. Here the usefulness of the method for microholographic imaging of biological specimens such as cells is demonstrated. The vast possibility of a posteriori processing of the microholograms is discussed. Dark-field, phase-contrast, and interference-contrast images, as well as quantitative phase maps, all obtained a posteriori from the same microhologram, are illustrated as examples.

© 2001 Optical Society of America

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References

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  1. G. Indebetouw, P. Klysubun, “Spatiotemporal digital microholography,” J. Opt. Soc. Am. A 18, 319–325 (2000).
    [CrossRef]
  2. G. Indebetouw, P. Klysubun, “Space–time digital holography: a three-dimensional microscopic imaging scheme with an arbitrary degree of spatial coherence,” Appl. Phys. Lett. 75, 2017–2019 (1999).
    [CrossRef]
  3. G. W. Ellis, “Holomicrography: transformation of images during reconstruction a posteriori,” Science 154, 1195–1196 (1966).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]

2000 (1)

1999 (5)

1997 (2)

1987 (1)

L. Onural, P. D. Scott, “Digital decoding of in-line holograms,” Opt. Eng. 26, 1124–1132 (1987).
[CrossRef]

1966 (1)

G. W. Ellis, “Holomicrography: transformation of images during reconstruction a posteriori,” Science 154, 1195–1196 (1966).
[CrossRef] [PubMed]

Bevilacqua, F.

Boccara, A. C.

Cuche, E.

Depeursinge, C.

Dubois, A.

Ellis, G. W.

G. W. Ellis, “Holomicrography: transformation of images during reconstruction a posteriori,” Science 154, 1195–1196 (1966).
[CrossRef] [PubMed]

Indebetouw, G.

G. Indebetouw, P. Klysubun, “Spatiotemporal digital microholography,” J. Opt. Soc. Am. A 18, 319–325 (2000).
[CrossRef]

G. Indebetouw, P. Klysubun, “Space–time digital holography: a three-dimensional microscopic imaging scheme with an arbitrary degree of spatial coherence,” Appl. Phys. Lett. 75, 2017–2019 (1999).
[CrossRef]

Kawai, H.

Klysubun, P.

G. Indebetouw, P. Klysubun, “Spatiotemporal digital microholography,” J. Opt. Soc. Am. A 18, 319–325 (2000).
[CrossRef]

G. Indebetouw, P. Klysubun, “Space–time digital holography: a three-dimensional microscopic imaging scheme with an arbitrary degree of spatial coherence,” Appl. Phys. Lett. 75, 2017–2019 (1999).
[CrossRef]

Lebec, M.

Ohzu, H.

Onural, L.

L. Onural, P. D. Scott, “Digital decoding of in-line holograms,” Opt. Eng. 26, 1124–1132 (1987).
[CrossRef]

Scott, P. D.

L. Onural, P. D. Scott, “Digital decoding of in-line holograms,” Opt. Eng. 26, 1124–1132 (1987).
[CrossRef]

Surrel, Y.

Takaki, Y.

Yamaguchi, I.

Zhang, T.

Appl. Opt. (3)

Appl. Phys. Lett. (1)

G. Indebetouw, P. Klysubun, “Space–time digital holography: a three-dimensional microscopic imaging scheme with an arbitrary degree of spatial coherence,” Appl. Phys. Lett. 75, 2017–2019 (1999).
[CrossRef]

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

Opt. Eng. (1)

L. Onural, P. D. Scott, “Digital decoding of in-line holograms,” Opt. Eng. 26, 1124–1132 (1987).
[CrossRef]

Opt. Lett. (3)

Science (1)

G. W. Ellis, “Holomicrography: transformation of images during reconstruction a posteriori,” Science 154, 1195–1196 (1966).
[CrossRef] [PubMed]

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Figures (11)

Fig. 1
Fig. 1

Sketch of the spatiotemporal holographic microscope used for the experiments. M1,2,3, mirrors; BS1,2, beam splitters; P1, P2, imaging pupil and pinhole pupil, respectively; Δ, defocus distance; L1,2, afocal relay optics.

Fig. 2
Fig. 2

Real part of the on-line, single-sideband hologram of an unstained oral epithelial cell obtained with a 40× objective (effective numerical aperture ∼ 0.45), bright-field imaging, and spatially partially coherent illumination.

Fig. 3
Fig. 3

(a) Magnitude of the complex field reconstructed from the hologram of Fig. 2, (b) intensity of the reconstructed image, (c) phase distribution of the reconstructed complex field (wrapped).

Fig. 4
Fig. 4

(a) Real part and (b) imaginary part of the complex field reconstructed from the hologram of Fig. 2.

Fig. 5
Fig. 5

3-D quantitative phase map of the reconstructed unstained epithelial cell.

Fig. 6
Fig. 6

Examples of a posteriori processing of the holographic data. (All are from the same hologram recorded with bright-field illumination. See the text for details.) (a) Dark-field reconstruction, (b) mixed gray field and phase contrast, (c) phase-contrast reconstruction (Zernike style).

Fig. 7
Fig. 7

Differential phase contrast obtained by subtracting two images of the reconstructed phase distribution with a shift of 10 pixels along the horizontal direction.

Fig. 8
Fig. 8

(a) Amplitude, (b) phase, and (c) quantitative phase map of the reconstruction of an unstained blood smear (40× objective, effective numerical aperture ∼0.45).

Fig. 9
Fig. 9

Transverse differential contrast obtained by reconstructing the hologram of the blood smear with a transverse differential reconstruction function [Eq. (3)] with a shear of 4 pixels along x and 4 pixels along y. The real part is displayed.

Fig. 10
Fig. 10

Transverse differential contrast obtained with a differential pupil filter [relation (4)] applied to the bright-field complex amplitude of the reconstructed field (real part displayed).

Fig. 11
Fig. 11

Axial differential contrast obtained by reconstructing the holographic data with an axial differential reconstruction function [Eq. (5)].  

Equations (5)

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H(r)Γg(r1-r2)Tg(r1, z)Tg*(r2, z)×p1(r-r1, z)p2*(r-r2, z)d2r1d2r2dz,
F(ρ)[1-(1-ia)circ(ρ/ρ0)]circ(ρ/ρC)
pR=p1(r-12ξ; zR)-p1(r+12ξ; zR),
F(ρ)sin(πρξ/ρC)circ(ρ/ρC),
pR=p1(r1; zR-12η)-p1(r; zR+12η),

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