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High-resolution quantitative phase-contrast microscopy by digital holography

Christopher J. Mann, Lingfeng Yu, Chun-Min Lo, and Myung K. Kim

Open Access Open Access

Abstract

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating holographic optical field is seen to have significant advantages including tight control of spurious noise components. Holographic phase images are obtained with 0.5 μm diffraction-limited lateral resolution and largely immune from the coherent noise common in other holographic techniques. The phase profile is accurate to about 30 nm of optical thickness. Images of SKOV-3 ovarian cancer cells display intracellular and intranuclear organelles with clarity and quantitative accuracy.

©2005 Optical Society of America

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Figures (4)
Fig. 1.
Fig. 1. Apparatus for digital holography experiments. See text for details

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Fig. 2.
Fig. 2. Holography of a resolution target. The image area is 25 × 25 μm2 (452 × 452 pixels) and the image is at z = 7 μm from the hologram: (a) hologram; (b) angular spectrum; (c) amplitude and (d) phase images by angular spectrum method; (e) amplitude and (f) phase images by Huygens convolution method; (g) amplitude and (h) phase images by Fresnel transform method; (i) 3D pseudocolor rendering of (d). The individual bars are 2.2 μm wide.

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Fig. 3.
Fig. 3. Holography of confluent SKOV-3 ovarian cancer cells. The image area is 60 × 60 μm2 (404 × 404 pixels) and the image is at z = 10 μm from the hologram: (a) Zernike phase contrast image; (b) holographic amplitude and (c) phase images; (d) unwrapped phase image; (e) 3D pseudocolor rendering of (d).

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Fig. 4.
Fig. 4. Holography of non-confluent SKOV-3 cells. The image area is 60 × 60 μm2 (404 × 404 pixels) and the image is at z = 5 μm from the hologram: (a) Zernike phase contrast image; (b) holographic amplitude and (c) phase images; (d) unwrapped phase image; (e) 3D pseudocolor rendering of (d).

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