Abstract

We describe the design, construction, and testing of a variant of Zernike’s phase-contrast microscope. The sample is illuminated with a white-light source through an annular aperture, which is projected onto the entrance pupil of the objective lens. In the return path the light diffracted by the sample and appearing in the interior of the objective’s aperture (i.e., the test beam) is separated from the light returning in the annular region near the rim of the objective (i.e., the reference beam). The separated beams are relatively phase shifted and then combined to create an interferogram of the sample’s surface on a CCD camera. It is fairly straightforward to use this system as a conventional bright-field or dark-field microscope, but its most interesting application is as a Zernike phase-contrast microscope with adjustable amplitude ratio and phase shift between test and reference beams. The ability to continuously adjust the phase of the reference beam also enables quantitative measurement of the phase imparted by the sample to the incident beam.

© 2000 Optical Society of America

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References

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  1. F. Zernike, “How I discovered phase contrast,” Science 121, 345–349 (1995).
    [CrossRef]
  2. X. Kong, T. Feng, G. Jin, “Reflection Zernike phase contrast microscope,” Appl. Opt. 29, 1408–1409 (1990).
    [CrossRef] [PubMed]
  3. A. H. Bennet, Phase Microscope: Principles and Applications (Wiley, New York, 1951).
  4. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, UK, 1980).
  5. M. Mansuripur, “Zernike’s method of phase contrast,” Opt. Photon. News 8(11), 40–43 (1997).
  6. S. S. C. Chim, G. S. Kino, “Phase measurements using the Mirau correlation microscope,” Appl. Opt. 30, 2197–2201 (1991).
    [CrossRef] [PubMed]
  7. B. S. Lee, T. C. Strand, “Profilometry with a coherence scanning microscope,” Appl. Opt. 29, 3784–3788 (1990).
    [CrossRef] [PubMed]
  8. P. Sandoz, “Wavelet transform as a processing tool in white-light interferometry,” Opt. Lett. 22, 1065–1067 (1997).
    [CrossRef] [PubMed]

1997 (2)

M. Mansuripur, “Zernike’s method of phase contrast,” Opt. Photon. News 8(11), 40–43 (1997).

P. Sandoz, “Wavelet transform as a processing tool in white-light interferometry,” Opt. Lett. 22, 1065–1067 (1997).
[CrossRef] [PubMed]

1995 (1)

F. Zernike, “How I discovered phase contrast,” Science 121, 345–349 (1995).
[CrossRef]

1991 (1)

1990 (2)

Bennet, A. H.

A. H. Bennet, Phase Microscope: Principles and Applications (Wiley, New York, 1951).

Born, M.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, UK, 1980).

Chim, S. S. C.

Feng, T.

Jin, G.

Kino, G. S.

Kong, X.

Lee, B. S.

Mansuripur, M.

M. Mansuripur, “Zernike’s method of phase contrast,” Opt. Photon. News 8(11), 40–43 (1997).

Sandoz, P.

Strand, T. C.

Wolf, E.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, UK, 1980).

Zernike, F.

F. Zernike, “How I discovered phase contrast,” Science 121, 345–349 (1995).
[CrossRef]

Appl. Opt. (3)

Opt. Lett. (1)

Opt. Photon. News (1)

M. Mansuripur, “Zernike’s method of phase contrast,” Opt. Photon. News 8(11), 40–43 (1997).

Science (1)

F. Zernike, “How I discovered phase contrast,” Science 121, 345–349 (1995).
[CrossRef]

Other (2)

A. H. Bennet, Phase Microscope: Principles and Applications (Wiley, New York, 1951).

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, UK, 1980).

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Figures (10)

Fig. 1
Fig. 1

Diagram of the adjustable phase-contrast microscope.

Fig. 2
Fig. 2

(a) Simulated annular light source consisting of 36 independent, quasi-monochromatic point sources. (b) Intensity image of the 400λ-wide annular ring at the entrance pupil of the objective.

Fig. 3
Fig. 3

(a) Pattern of simulated phase objects (marks) on a uniform background. The reflectivity is uniform over the entire area of this object, and the marks impart to the incident beam a 36° phase shift in double path on reflection. (b) Distribution of the logarithm of intensity at the exit pupil of the 0.6-N.A. objective lens. The annular phase mask projected onto this pupil has a width of 400λ and an inner radius of 5600λ. (c) Distribution of the logarithm of intensity in the interior region of the exit pupil, with a radius of 5600λ. (d) Distribution of the directly reflected intensity in the region of the exit pupil corresponding to the area covered by the image of the annular mask.

Fig. 4
Fig. 4

Computed distributions of the logarithm of intensity at the image plane. (a) Phase-contrast image obtained when the reference is phase shifted by 90° and its amplitude is attenuated by 70%. (b) When no phase shift or attenuation is applied to the reflected beam, a conventional bright-field image of the sample is obtained. (c) When the reflected light within the annular ring (i.e., the reference beam) is blocked, one obtains the dark-field image of the sample. (d) Image contrast is extremely poor when the reflected light within the interior region of the exit pupil (i.e., the test beam) is blocked, and only the annular ring is allowed to reach the image plane.

Fig. 5
Fig. 5

(a) Simulated phase objects (marks) on a uniform background. The reflectivity is the same over the entire area of this object, and the marks impart to the incident beam a 180° phase shift in double path on reflection. (b) Phase-contrast image obtained when the reference is phase shifted by 90° and its amplitude is attenuated by 70%. (c) When no phase-shift or attenuation is applied to the reflected beam, a conventional bright-field image of the sample is obtained. (d) When the reflected light within the annular ring (i.e., the reference beam) is blocked, one obtains the dark-field image of the sample. (e) Image contrast is extremely poor when the reflected light within the interior region of the exit pupil (i.e., the test beam) is blocked, and only the annular ring is allowed to reach the image plane.

Fig. 6
Fig. 6

(a) Conventional image of a CD surface, obtained by removal of the annular mask in the illumination path and blockage of the reference beam. (b) Dark-field image of the disk with the annular mask present in the illumination path but with the reference beam blocked in the return path. (c) Image of the disk obtained with the annular mask present in the illumination path but with the test beam passing through the mirror pair (M3, M4) blocked. (d) Same as (c) but with a somewhat wider annular mask.

Fig. 7
Fig. 7

White-light interference images of the CD obtained with two different phase shifts between the reference beam and the test beam. Both (a) and (b) are phase contrast images, but the reference beam passing through the mirror pair (M1, M2) has been tilted to introduce a continuously varying 4π linear phase shift over the image area.

Fig. 8
Fig. 8

Intensity variations monitored by the CCD camera at two fixed pixels, when the mirror air (M1, M2) moves a distance of 5 µm to impart a continuously varying phase shift to the reference beam. (a) The monitored pixel is at the center of a pit on the CD surface. (b) The monitored pixel is in a flat region of the disk in the vicinity of the pit monitored in (a).

Fig. 9
Fig. 9

(a) The intensity variations depicted in Fig. 8 are normalized and plotted on the same set of axes. (b) Theoretical fits to the normalized intensity variation curves shown in (a). The solid curve represents the pixel at the center of a pit, and the dotted curve represents the pixel in a flat region.

Fig. 10
Fig. 10

(a) Conventional image of a biological sample. (b) Dark-field image obtained in the presence of the annular mask in the illumination path and when the reference beam is blocked. (c) The image obtained in the presence of the annular ring but with the test beam blocked. (d) Phase-contrast image obtained when both the reference beam and the test beam are allowed to interfere in the image plane. The reference beam is slightly tilted in this case, giving rise to faint vertical fringes.

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