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Opposite-view digital holographic microscopy with autofocusing capability.

ABSTRACT: Digital holographic microscopy (DHM) has its intrinsic ability to refocusing a sample by numerically propagating an object wave from its hologram plane to its image plane. In this paper opposite-view digital holographic microscopy (OV-DHM) is demonstrated for autofocusing, namely, digitally determining the location of the image plane, and refocusing the object wave without human intervention. In OV-DHM, a specimen is illuminated from two sides in a 4?-alike configuration, and two holograms are generated and recorded by a CCD camera along two orthogonal polarization orientations. The image plane of the sample is determined by finding the minimal variation between the two object waves, and consequently refocusing is performed by propagating the waves to the image plane. Furthermore, the field of view (FOV) of OV-DHM can be extended by combining the two object waves which have an angle in-between. The proposed technique also has the potential to reduce speckle noise and out-of-focus background.

SUBMITTER: Zheng J

PROVIDER: S-EPMC5484697 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Opposite-view digital holographic microscopy with autofocusing capability.

Zheng Juanjuan J Gao Peng P Shao Xiaopeng X

Scientific reports 20170626 1

Digital holographic microscopy (DHM) has its intrinsic ability to refocusing a sample by numerically propagating an object wave from its hologram plane to its image plane. In this paper opposite-view digital holographic microscopy (OV-DHM) is demonstrated for autofocusing, namely, digitally determining the location of the image plane, and refocusing the object wave without human intervention. In OV-DHM, a specimen is illuminated from two sides in a 4π-alike configuration, and two holograms are g ...[more]

PMID: 28652591

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Project description:BackgroundMany biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope.MethodsA modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features.ResultsBoth cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration.ConclusionDigital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

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Opposite-view digital holographic microscopy with autofocusing capability.

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Opposite-view digital holographic microscopy with autofocusing capability.

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