Abstract

We demonstrate experimentally the three-dimensional reconstructions of fluorescent biological specimens using scanning holographic microscopy. Three-dimensional reconstructions with transverse resolution below about 1μm of transmission and fluorescence emission images are presented and analyzed. The limitations of the method are discussed.

© 2006 Optical Society of America

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  1. J. W. Goodman and R. W. Lawrence, 'Digital image information from electronically detected holograms,' Appl. Phys. Lett. 11, 77-79 (1967).
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  12. B. Schilling, T.-C. Poon, G. Indebetouw, B. Storie, K. Shinoda, and M. Wu, 'Three-dimensional holographic fluorescence microscopy,' Opt. Lett. 22, 1506-1508 (1997).
    [CrossRef]
  13. G. Indebetouw, 'Properties of a scanning holographic microscope: improved resolution, extended depth of focus, and/or optical sectioning,' J. Mod. Opt. 49, 1479-1500 (2002).
    [CrossRef]
  14. G. Indebetouw, W. Zhong, and D. Chamberlin-Long, 'Point-spread function synthesis in scanning holographic microscopy,' J. Opt. Soc. Am. A 23, 1708-1717 (2006).
    [CrossRef]
  15. 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, 829-898 (2005).
    [CrossRef]
  16. A.Diaspro, ed., Confocal and Two-Photon Microscopy: Foundation, Applications, and Advances (Wiley, 2002).
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  18. P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).
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    [CrossRef]
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    [CrossRef]

2006 (1)

2005 (1)

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, 829-898 (2005).
[CrossRef]

2002 (3)

U. Schnars and W. P. O. Juptner, 'Digital recording and numerical reconstruction of holograms,' Meas. Sci. Technol. 13, R85-R101 (2002).
[CrossRef]

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

G. Indebetouw, 'Properties of a scanning holographic microscope: improved resolution, extended depth of focus, and/or optical sectioning,' J. Mod. Opt. 49, 1479-1500 (2002).
[CrossRef]

2000 (1)

1999 (2)

E. Cuche, F. Bevilacqua, and C. Depeursinge, 'Digital holography for quantitative phase-contrast imaging,' Opt. Lett. 24, 291-293 (1999).
[CrossRef]

P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).

1997 (1)

1985 (1)

1979 (1)

1978 (1)

1967 (1)

J. W. Goodman and R. W. Lawrence, 'Digital image information from electronically detected holograms,' Appl. Phys. Lett. 11, 77-79 (1967).
[CrossRef]

1964 (1)

1962 (1)

1948 (1)

D. Gabor, 'A new microscopic principle,' Nature 161, 777-778 (1948).
[CrossRef]

Bevilacqua, F.

Chamberlin-Long, D.

Cuche, E.

E. Cuche, F. Bevilacqua, and C. Depeursinge, 'Digital holography for quantitative phase-contrast imaging,' Opt. Lett. 24, 291-293 (1999).
[CrossRef]

E. Cuche, P. Poscio, and C. Depeursinge, 'Optical tomography at the microscopic scale by means of a numerical low-coherence holographic technique,' in Optical and Imaging Techniques for Biomonitoring II, H. J. Foth, R. Marchesini, and H. Podbielska, eds., Proc. SPIE 2927, 61-66 (1996).

Depeursinge, C.

E. Cuche, F. Bevilacqua, and C. Depeursinge, 'Digital holography for quantitative phase-contrast imaging,' Opt. Lett. 24, 291-293 (1999).
[CrossRef]

E. Cuche, P. Poscio, and C. Depeursinge, 'Optical tomography at the microscopic scale by means of a numerical low-coherence holographic technique,' in Optical and Imaging Techniques for Biomonitoring II, H. J. Foth, R. Marchesini, and H. Podbielska, eds., Proc. SPIE 2927, 61-66 (1996).

El Maghnouji, A.

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, 829-898 (2005).
[CrossRef]

Foster, R.

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, 829-898 (2005).
[CrossRef]

Gabor, D.

D. Gabor, 'A new microscopic principle,' Nature 161, 777-778 (1948).
[CrossRef]

Gemkow, M. J.

P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).

Goodman, J. W.

J. W. Goodman and R. W. Lawrence, 'Digital image information from electronically detected holograms,' Appl. Phys. Lett. 11, 77-79 (1967).
[CrossRef]

Indebetouw, G.

G. Indebetouw, W. Zhong, and D. Chamberlin-Long, 'Point-spread function synthesis in scanning holographic microscopy,' J. Opt. Soc. Am. A 23, 1708-1717 (2006).
[CrossRef]

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, 829-898 (2005).
[CrossRef]

G. Indebetouw, 'Properties of a scanning holographic microscope: improved resolution, extended depth of focus, and/or optical sectioning,' J. Mod. Opt. 49, 1479-1500 (2002).
[CrossRef]

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]

B. Schilling, T.-C. Poon, G. Indebetouw, B. Storie, K. Shinoda, and M. Wu, 'Three-dimensional holographic fluorescence microscopy,' Opt. Lett. 22, 1506-1508 (1997).
[CrossRef]

Jerico, M. H.

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

Jovin, T. M.

P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).

Juptner, W. P. O.

U. Schnars and W. P. O. Juptner, 'Digital recording and numerical reconstruction of holograms,' Meas. Sci. Technol. 13, R85-R101 (2002).
[CrossRef]

Kim, T.

Klysubun, P.

Korpel, A.

Kreuzer, H. J.

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

Lawrence, R. W.

J. W. Goodman and R. W. Lawrence, 'Digital image information from electronically detected holograms,' Appl. Phys. Lett. 11, 77-79 (1967).
[CrossRef]

Leith, E. N.

Lohmann, A. W.

McCutchen, C. W.

Meinertzhagen, I. A.

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

Poon, T.-C.

Poscio, P.

E. Cuche, P. Poscio, and C. Depeursinge, 'Optical tomography at the microscopic scale by means of a numerical low-coherence holographic technique,' in Optical and Imaging Techniques for Biomonitoring II, H. J. Foth, R. Marchesini, and H. Podbielska, eds., Proc. SPIE 2927, 61-66 (1996).

Rhodes, W. T.

Saleh, B. E. A.

B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley, 1991).
[CrossRef]

Schilling, B.

Schnars, U.

U. Schnars and W. P. O. Juptner, 'Digital recording and numerical reconstruction of holograms,' Meas. Sci. Technol. 13, R85-R101 (2002).
[CrossRef]

Shaw, P.

P. Shaw, 'Comparison of wide-field deconvolution and confocal microscopy for 3D imaging,' in Handbook of Biological Confocal Microscopy, 2nd ed., J.B.Pawley, ed. (Plenum, 1995), pp. 373-387.

Shinoda, K.

Storie, B.

Teich, M. C.

B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley, 1991).
[CrossRef]

Upatnieks, J.

Verveer, P. J.

P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).

Wu, M.

Xu, W.

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

Zhong, W.

Appl. Opt. (1)

Appl. Phys. Lett. (1)

J. W. Goodman and R. W. Lawrence, 'Digital image information from electronically detected holograms,' Appl. Phys. Lett. 11, 77-79 (1967).
[CrossRef]

J. Microsc. (1)

P. J. Verveer, M. J. Gemkow, and T. M. Jovin, 'A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy,' J. Microsc. 183, 50-61 (1999).

J. Mod. Opt. (1)

G. Indebetouw, 'Properties of a scanning holographic microscope: improved resolution, extended depth of focus, and/or optical sectioning,' J. Mod. Opt. 49, 1479-1500 (2002).
[CrossRef]

J. Opt. Soc. Am. (2)

J. Opt. Soc. Am. A (4)

Meas. Sci. Technol. (1)

U. Schnars and W. P. O. Juptner, 'Digital recording and numerical reconstruction of holograms,' Meas. Sci. Technol. 13, R85-R101 (2002).
[CrossRef]

Nature (1)

D. Gabor, 'A new microscopic principle,' Nature 161, 777-778 (1948).
[CrossRef]

Opt. Lett. (3)

Proc. Natl. Acad. Sci. USA (1)

W. Xu, M. H. Jerico, I. A. Meinertzhagen, and H. J. Kreuzer, 'Digital in-line holography for biological applications,' Proc. Natl. Acad. Sci. USA 98, 11301-11305 (2002).
[CrossRef]

Other (4)

B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley, 1991).
[CrossRef]

E. Cuche, P. Poscio, and C. Depeursinge, 'Optical tomography at the microscopic scale by means of a numerical low-coherence holographic technique,' in Optical and Imaging Techniques for Biomonitoring II, H. J. Foth, R. Marchesini, and H. Podbielska, eds., Proc. SPIE 2927, 61-66 (1996).

A.Diaspro, ed., Confocal and Two-Photon Microscopy: Foundation, Applications, and Advances (Wiley, 2002).

P. Shaw, 'Comparison of wide-field deconvolution and confocal microscopy for 3D imaging,' in Handbook of Biological Confocal Microscopy, 2nd ed., J.B.Pawley, ed. (Plenum, 1995), pp. 373-387.

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Figures (11)

Fig. 1
Fig. 1

Generic sketch of a two-pupil synthesis set up in scanning mode. SLM, spatial light modulator; EO, electro-optic.

Fig. 2
Fig. 2

Sketch of a scanning holographic microscope based on two-pupil synthesis. M, mirror; BS, beam splitter; DBS, dichroic beam splitter; EOM, electro-optic phase modulator; microscope objective, Mitutoyo Plan Apo 20 × , NA 0.42 .

Fig. 3
Fig. 3

(a) Phase distribution modulo 2 π of the scanning distribution obtained from the hologram of a 1 μ m pinhole. Diameter 70 μ m . Fresnel number 15 . Effective NA 0.23 . (b) Amplitude of the reconstruction of the pinhole using Fresnel–Kirchhoff propagation, representing the in-focus point-spread function with obvious spherical aberration and astigmatism. (c) Amplitude of the reconstruction of the pinhole by correlation with the experimental scanning distribution, showing aberration compensation.

Fig. 4
Fig. 4

Reconstruction of the intensity transmission of Oscillatoria leaves (small algae) at three axial positions from the center of curvature of the scanning distribution: (a) z = 130 μ m , (b) z = 155 μ m , (c) z = 180 μ m . The reconstructions were obtained using digital Fresnel–Kirchhoff backpropagation to the desired axial planes.

Fig. 5
Fig. 5

Absolute value of the complex single-sideband hologram from which the images of Fig. 4 are reconstructed.

Fig. 6
Fig. 6

Intensity of the reconstruction of the same hologram shown in Fig. 5 but reconstructed by correlation with the phase of the experimental scanning distribution and equalized amplitude.

Fig. 7
Fig. 7

Amplitude of the reconstructions of the hologram of fluorescent Oscillatoria dyed with phloxine. The amplitude is proportional to the dye concentration. Excitation: 532 nm . Fluorescence emission: 600 nm . Hologram recorded with fluorescence reflection. (a) z = 130 μ m , (b) z = 180 μ m .

Fig. 8
Fig. 8

Phase (modulo 2 π ) of the reconstruction of the two sections shown in Fig. 7. The arrows point to two disturbances due to the nonfluorescent strands located between the two fluorescent ones.

Fig. 9
Fig. 9

Same as Fig. 7 but for a hologram recorded with fluorescence transmission.

Fig. 10
Fig. 10

Phase (modulo 2 π ) of the reconstructions of the two sections shown in Fig. 9. The arrows point to two disturbances due to the nonfluorescent strands located between the two fluorescent ones.

Fig. 11
Fig. 11

(a) Amplitude and (b) phase of the reconstruction of a hologram of fluorescent pollen grains stained with phloxine. Excitation: 532 nm . Emission: 600 nm . Hologram recorded with fluorescence reflection and reconstructed using the experimental scanning distribution for aberration compensation.

Equations (12)

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

P ̃ 1 ( u , v ) δ ( u , v ) ,
P ̃ 2 ( u , v ) = exp { i k z 0 [ 1 1 λ 2 ( u 2 + v 2 ) ] } P ̃ 0 ( u , v ) .
P 2 ( x , y ) ( i λ z 0 ) 1 exp [ i π ( x 2 + y 2 ) λ z 0 ] circ [ x 2 + y 2 z 0 sin ( α ) ] ,
S ( x , y , z ) = P 1 * ( x , y , z ) P 2 ( x , y , z ) .
P j ( x , y , z ) = d u d v d w P ̃ j ( u , v ) exp { i 2 π ( u x + v y + w z ) } δ ( λ 1 u 2 + v 2 + w 2 ) .
S ( x , y , z ) = d u d v exp [ i 2 π ( u x + v y ) ] exp { i k ( z 0 + z ) [ 1 1 λ 2 ( u 2 + v 2 ) ] } P ̃ 0 ( u , v ) .
H ( x , y ) = d z I ( x , y , z ) S ( x , y , z ) ,
R ( x , y , z R ) = H ( x , y ) S ( x , y , z R ) = H ( x , y ) S * ( x , y , z R ) ,
R ̃ ( u , v ; z R ) = d z I ̃ ( u , v ; z ) S ̃ ( u , v ; z ) S ̃ * ( u , v ; z R ) .
T ̃ ( u , v ; z z R ) = S ̃ ( u , v ; z ) S ̃ * ( u , v ; z R ) = exp { i k ( z z R ) [ 1 1 λ 2 ( u 2 + v 2 ) ] } P ̃ 0 ( u , v ) 2 .
R ( 0 , z z R ) = π ( ρ MAX ) 2 exp ( i Δ ) sin ( Δ ) Δ ,
Δ = ( π 2 ) λ ( z z R ) ( ρ MAX ) 2 .

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