Abstract

A modification of the phase contrast method in microscopy is presented, which reduces inherent artifacts and improves the spatial resolution. In standard Zernike phase contrast microscopy the illumination is achieved through an annular ring aperture, and the phase filtering operation is performed by a corresponding phase ring in the back focal plane of the objective. The Zernike method increases the spatial resolution as compared to plane wave illumination, but it also produces artifacts, such as the halo- and the shade-off effect. Our modification consists in replacing the illumination ring by a set of point apertures which are randomly distributed over the whole aperture of the condenser, and in replacing the Zernike phase ring by a matched set of point-like phase shifters in the back focal plane of the objective. Experimentally this is done by illuminating the sample with light diffracted from a phase hologram displayed at a spatial light modulator (SLM). The subsequent filtering operation is then done with a second matched phase hologram displayed at another SLM in a Fourier plane of the imaging pathway. This method significantly reduces the halo- and shade-off artifacts whilst providing the full spatial resolution of the microscope.

© 2008 Optical Society of America

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  1. F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung," Z. Techn. Physik. 16, 454-457 (1935).
  2. R. Barer, "Some Applications of Phase-contrast Microscopy," Quarterly Journal of Microscopic Sciences 88, 491-499 (1947).
  3. P. C. Mogensen and J. Gluckstad, "Dynamic array generation and pattern formation for optical tweezers," Opt. Commun. 175, 7581 (2000).
    [CrossRef]
  4. J. Gluckstad, D. Palima, P. J. Rodrigo, and C. A. Alonzo, "Laser projection using generalized phase contrast," Opt. Lett. 32, 3281-3283 (2007).
    [CrossRef] [PubMed]
  5. A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
    [CrossRef]
  6. H. Siedentopf, "Uber das Auflosungsvermogen der Mikroskope bei Hellfeld- und Dunkelfeldbeleuchtung," Z. Wiss. Mikroskopie 32, 1-42 (1915).
  7. H. H. Hopkins and P. M. Barham, "The Influence of the Condenser onMicroscopic Resolution," Proc. Phys. Soc. London Sect. B 63, 737-744 (1950).
    [CrossRef]
  8. M. Born and H. Wolf, Principles of Optics (Pergamon, London, 1959).
  9. W. Singer, M. Totzeck, and H. Gross, Handbook of Optics - Physical Image Formation ed. H. Gross, (Wiley-vch, Weinheim, 2005).
  10. E. C. Kintner, "Method for the calculation of partially coherent imagery," Appl. Opt. 17, 2747-2753 (1978).
    [CrossRef] [PubMed]
  11. R. Liang, J. K. Erwin, and M. Mansuripur, "Variation on Zernike’s phase contrast microscope," Appl. Opt. 39,2152-2158 (2000).
    [CrossRef]
  12. G. Indebetouw and C. Varamit, "Spatial filtering with complementary source-pupil masks," J. Opt. Soc. Am. A 2, 794-798 (1985).
    [CrossRef]
  13. T. Otaki, "Artifact Halo reduction in Phase Contrast microscopy using Apodization," Opt. Rev. 7, 119-122 (2000).
    [CrossRef]
  14. S. Furhapter, A. Jesacher, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Spiral phase microscopy," Adv. Imag. Electron Physics 146,1-56, (2007).
    [CrossRef]
  15. S. Bernet, A. Jesacher, S. Furhapter, C. Maurer, and M. Ritsch-Marte, "Quantitative imaging of complex samples by spiral phase contrast microscopy," Opt. Express 14, 3792-3805 (2006).
    [CrossRef] [PubMed]
  16. S. Furhapter, A. Jesacher, S. Bernet, and M. Ritsch-Marte, "Spiral interferometry," Opt. Lett. 30, 1953-1955 (2005).
    [CrossRef] [PubMed]
  17. E. R. Dufresne and D. G. Grier, "Optical tweezer arrays and optical substrates created with diffractive optical elements," Rev. Sci. Instrum. 69, 1974-1977 (1998).
    [CrossRef]
  18. V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
    [CrossRef]
  19. H. Melville, G. Milne, G. Spalding, W. Sibbett, K. Dholakia, and D. McGloin, "Optical trapping of threedimensional structures using dynamic holograms," Opt. Express 11, 3562-3567 (2003).
    [CrossRef] [PubMed]
  20. A. Jesacher, S. Furhapter, S. Bernet, and M. Ritsch-Marte, "Diffractive optical tweezers in the Fresnel regime," Opt. Express 12, 2243-2250 (2004).
    [CrossRef] [PubMed]
  21. R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of phase from image and diffraction plane pictures," Optik 35, 237-246 (1972).
  22. A description of the Gerchberg-Saxton and other algorithms for hologram calculation is given in B. Kress and P. Meyrueis, "Digital Diffractive Optics," pp. 90-92, John Wiley & Sons Ltd., Chichester, 2000: Briefly, the algorithm starts with a complex image having the desired intensity distribution as its (squared) absolute value, whereas the phase of each pixel is randomized. Using the fast two-dimensional Fourier algorithm the image is transformed into its Fourier (=hologram) plane, resulting again in a complex image with both amplitude and phase modulations. Since the SLM can only display phase values, the image amplitude of each pixel is set to unity, whereas the phase values are maintained, and the resulting pixel array is Fourier back-transformed into the image plane. There the intensity distribution (which is already an approximation of the desired one) is now substituted by the desired image, whereas the phase is maintained, and the whole procedure starts again by Fourier transforming into the hologram plane. After typically less than 10 iterations, the output of this algorithm will be a pure phase hologram, which accurately reconstructs the desired image intensity distribution.
  23. N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
    [CrossRef]
  24. G. A. Swartzlander, Jr., "Peering into darkness with a vortex spatial filter," Opt. Lett. 26, 497-499 (2001).
    [CrossRef]

2007 (2)

S. Furhapter, A. Jesacher, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Spiral phase microscopy," Adv. Imag. Electron Physics 146,1-56, (2007).
[CrossRef]

J. Gluckstad, D. Palima, P. J. Rodrigo, and C. A. Alonzo, "Laser projection using generalized phase contrast," Opt. Lett. 32, 3281-3283 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (1)

2004 (1)

2003 (3)

A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
[CrossRef]

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

H. Melville, G. Milne, G. Spalding, W. Sibbett, K. Dholakia, and D. McGloin, "Optical trapping of threedimensional structures using dynamic holograms," Opt. Express 11, 3562-3567 (2003).
[CrossRef] [PubMed]

2001 (1)

2000 (3)

T. Otaki, "Artifact Halo reduction in Phase Contrast microscopy using Apodization," Opt. Rev. 7, 119-122 (2000).
[CrossRef]

R. Liang, J. K. Erwin, and M. Mansuripur, "Variation on Zernike’s phase contrast microscope," Appl. Opt. 39,2152-2158 (2000).
[CrossRef]

P. C. Mogensen and J. Gluckstad, "Dynamic array generation and pattern formation for optical tweezers," Opt. Commun. 175, 7581 (2000).
[CrossRef]

1998 (1)

E. R. Dufresne and D. G. Grier, "Optical tweezer arrays and optical substrates created with diffractive optical elements," Rev. Sci. Instrum. 69, 1974-1977 (1998).
[CrossRef]

1992 (1)

N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
[CrossRef]

1985 (1)

1978 (1)

1972 (1)

R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of phase from image and diffraction plane pictures," Optik 35, 237-246 (1972).

1950 (1)

H. H. Hopkins and P. M. Barham, "The Influence of the Condenser onMicroscopic Resolution," Proc. Phys. Soc. London Sect. B 63, 737-744 (1950).
[CrossRef]

1947 (1)

R. Barer, "Some Applications of Phase-contrast Microscopy," Quarterly Journal of Microscopic Sciences 88, 491-499 (1947).

1935 (1)

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung," Z. Techn. Physik. 16, 454-457 (1935).

1915 (1)

H. Siedentopf, "Uber das Auflosungsvermogen der Mikroskope bei Hellfeld- und Dunkelfeldbeleuchtung," Z. Wiss. Mikroskopie 32, 1-42 (1915).

Alonzo, C. A.

Barer, R.

R. Barer, "Some Applications of Phase-contrast Microscopy," Quarterly Journal of Microscopic Sciences 88, 491-499 (1947).

Barham, P. M.

H. H. Hopkins and P. M. Barham, "The Influence of the Condenser onMicroscopic Resolution," Proc. Phys. Soc. London Sect. B 63, 737-744 (1950).
[CrossRef]

Bernet, S.

Bingelyte, V.

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

Courtial, J.

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

Dholakia, K.

Dufresne, E. R.

E. R. Dufresne and D. G. Grier, "Optical tweezer arrays and optical substrates created with diffractive optical elements," Rev. Sci. Instrum. 69, 1974-1977 (1998).
[CrossRef]

Erwin, J. K.

F¨urhapter, S.

S. Furhapter, A. Jesacher, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Spiral phase microscopy," Adv. Imag. Electron Physics 146,1-56, (2007).
[CrossRef]

Furhapter, S.

Gerchberg, R. W.

R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of phase from image and diffraction plane pictures," Optik 35, 237-246 (1972).

Gluckstad, J.

J. Gluckstad, D. Palima, P. J. Rodrigo, and C. A. Alonzo, "Laser projection using generalized phase contrast," Opt. Lett. 32, 3281-3283 (2007).
[CrossRef] [PubMed]

P. C. Mogensen and J. Gluckstad, "Dynamic array generation and pattern formation for optical tweezers," Opt. Commun. 175, 7581 (2000).
[CrossRef]

Grier, D. G.

E. R. Dufresne and D. G. Grier, "Optical tweezer arrays and optical substrates created with diffractive optical elements," Rev. Sci. Instrum. 69, 1974-1977 (1998).
[CrossRef]

Heckenberg, N. R.

N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
[CrossRef]

Hopkins, H. H.

H. H. Hopkins and P. M. Barham, "The Influence of the Condenser onMicroscopic Resolution," Proc. Phys. Soc. London Sect. B 63, 737-744 (1950).
[CrossRef]

Indebetouw, G.

Jesacher, A.

Kintner, E. C.

Leach, J.

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

Liang, R.

Mansuripur, M.

Maurer, C.

S. Furhapter, A. Jesacher, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Spiral phase microscopy," Adv. Imag. Electron Physics 146,1-56, (2007).
[CrossRef]

S. Bernet, A. Jesacher, S. Furhapter, C. Maurer, and M. Ritsch-Marte, "Quantitative imaging of complex samples by spiral phase contrast microscopy," Opt. Express 14, 3792-3805 (2006).
[CrossRef] [PubMed]

McDuff, R.

N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
[CrossRef]

McGloin, D.

Melville, H.

Milne, G.

Mogensen, P. C.

P. C. Mogensen and J. Gluckstad, "Dynamic array generation and pattern formation for optical tweezers," Opt. Commun. 175, 7581 (2000).
[CrossRef]

Ng, A. Y. M.

A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
[CrossRef]

Otaki, T.

T. Otaki, "Artifact Halo reduction in Phase Contrast microscopy using Apodization," Opt. Rev. 7, 119-122 (2000).
[CrossRef]

Padgett, M. J.

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

Palima, D.

Ritsch-Marte, M.

Rodrigo, P. J.

Saxton, W. O.

R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of phase from image and diffraction plane pictures," Optik 35, 237-246 (1972).

See, C. W.

A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
[CrossRef]

Sibbett, W.

Siedentopf, H.

H. Siedentopf, "Uber das Auflosungsvermogen der Mikroskope bei Hellfeld- und Dunkelfeldbeleuchtung," Z. Wiss. Mikroskopie 32, 1-42 (1915).

Smith, C. P.

N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
[CrossRef]

Somekh, M. G.

A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
[CrossRef]

Spalding, G.

Swartzlander, G. A.

Varamit, C.

White, A. G.

N. R. Heckenberg, R. McDuff, C. P. Smith, and A. G. White, "Generation of optical phase singularities by computer-generated holograms," Opt. Lett. 17, 221223 (1992).
[CrossRef]

Zernike, F.

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung," Z. Techn. Physik. 16, 454-457 (1935).

Adv. Imag. Electron Physics (1)

S. Furhapter, A. Jesacher, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Spiral phase microscopy," Adv. Imag. Electron Physics 146,1-56, (2007).
[CrossRef]

Appl. Opt. (2)

Appl. Phys. Lett. (1)

V. Bingelyte, J. Leach, J. Courtial, and M. J. Padgett, "Optically controlled three-dimensional rotation of microscopic objects," Appl. Phys. Lett. 82, 829-831 (2003).
[CrossRef]

J. Microsc. (1)

A. Y. M. Ng, C. W. See, and M. G. Somekh, "Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator," J. Microsc. 214, 334-304 (2003).
[CrossRef]

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

Opt. Commun. (1)

P. C. Mogensen and J. Gluckstad, "Dynamic array generation and pattern formation for optical tweezers," Opt. Commun. 175, 7581 (2000).
[CrossRef]

Opt. Express (3)

Opt. Lett. (4)

Opt. Rev. (1)

T. Otaki, "Artifact Halo reduction in Phase Contrast microscopy using Apodization," Opt. Rev. 7, 119-122 (2000).
[CrossRef]

Optik (1)

R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of phase from image and diffraction plane pictures," Optik 35, 237-246 (1972).

Proc. Phys. Soc. London Sect. B (1)

H. H. Hopkins and P. M. Barham, "The Influence of the Condenser onMicroscopic Resolution," Proc. Phys. Soc. London Sect. B 63, 737-744 (1950).
[CrossRef]

Quarterly Journal of Microscopic Sciences (1)

R. Barer, "Some Applications of Phase-contrast Microscopy," Quarterly Journal of Microscopic Sciences 88, 491-499 (1947).

Rev. Sci. Instrum. (1)

E. R. Dufresne and D. G. Grier, "Optical tweezer arrays and optical substrates created with diffractive optical elements," Rev. Sci. Instrum. 69, 1974-1977 (1998).
[CrossRef]

Z. Techn. Physik. (1)

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung," Z. Techn. Physik. 16, 454-457 (1935).

Z. Wiss. Mikroskopie (1)

H. Siedentopf, "Uber das Auflosungsvermogen der Mikroskope bei Hellfeld- und Dunkelfeldbeleuchtung," Z. Wiss. Mikroskopie 32, 1-42 (1915).

Other (3)

M. Born and H. Wolf, Principles of Optics (Pergamon, London, 1959).

W. Singer, M. Totzeck, and H. Gross, Handbook of Optics - Physical Image Formation ed. H. Gross, (Wiley-vch, Weinheim, 2005).

A description of the Gerchberg-Saxton and other algorithms for hologram calculation is given in B. Kress and P. Meyrueis, "Digital Diffractive Optics," pp. 90-92, John Wiley & Sons Ltd., Chichester, 2000: Briefly, the algorithm starts with a complex image having the desired intensity distribution as its (squared) absolute value, whereas the phase of each pixel is randomized. Using the fast two-dimensional Fourier algorithm the image is transformed into its Fourier (=hologram) plane, resulting again in a complex image with both amplitude and phase modulations. Since the SLM can only display phase values, the image amplitude of each pixel is set to unity, whereas the phase values are maintained, and the resulting pixel array is Fourier back-transformed into the image plane. There the intensity distribution (which is already an approximation of the desired one) is now substituted by the desired image, whereas the phase is maintained, and the whole procedure starts again by Fourier transforming into the hologram plane. After typically less than 10 iterations, the output of this algorithm will be a pure phase hologram, which accurately reconstructs the desired image intensity distribution.

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Figures (3)

Fig. 1.
Fig. 1.

Explanation of the artifacts in Zernike phase contrast microscopy, and by random light dot illumination: (A) sketches the phase-only Zernike filter in the back focal objective plane, whereas (B) sketches a filter used for random light dot illumination (not to scale): (A) shows the Zernike type phase ring (brighter) which coincides with the ring shaped image of the illumination aperture. In the ideal case only the zero-order wave should pass through the phase ring. In the figure only a small portion (small dot) of the illumination light ring is indicated. This dot corresponds to a part of the ring-shaped zero-order wave, with its diffracted components spread-out around it (indicated as a dimmer disk around the central dot). As shown in the figure, also a part of this diffracted light passes through the adjacent areas of the phase ring, and is thus erroneously shifted in its phase, giving rise to image artifacts. In (B) the situation is sketched for “random dot” illumination, i.e. the sample is illuminated with a variety of plane waves which are incident from randomly chosen directions. In the sketched filter plane, these illumination directions focus at randomly distributed spots, but at known positions. The corresponding phase filter is designed such that it exactly matches with the focussed points, shifting their phases by π/2 with respect to the surrounding diffracted light components. Compared to the situation (A) there is now much less intensity of the scattered light which erroneously passes through phase-shifting areas of the filter.

Fig. 2.
Fig. 2.

Sketch of the experimental setup: A collimated laser beam illuminates a Fourier hologram displayed at a first phase-only SLM. With a Fourier transforming lens, the holographically programmed illumination pattern is reconstructed in the plane of a rotating diffuser, acting as the effective incoherent illumination source. A further Fourier transforming (condenser) lens leads to a uniformly illuminated sample, which is then imaged with a microscope objective. In its back focal plane the programmed illumination pattern (which was displayed at the rotating diffuser screen) is sharply imaged in the plane of a second SLM which acts as a programmable phase-only Fourier filter, displaying for example the phase masks of Fig. 1. A final Fourier transforming imaging lens then produces a sharp, processed image of the sample at a camera.

Fig. 3.
Fig. 3.

Phase contrast images of polystyrene beads with a diameter of 10 µm surrounded by immersion oil (left) and oil smears sandwiched between two cover slips (right), imaged with Zernike or random dot phase contrast (indicated in the images), respectively. The profile plots under the images illustrate the intensity variations along the indicated horizontal lines.

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