Fitzpatrickʼs Dermatology in General Medicine

Digital holography is an emerging field of new paradigm in general imaging applications. We present a review of a subset of the research and development activities in digital holography, with emphasis on microscopy techniques and applications. First, the basic results from the general theory of holography, based on the scalar diffraction theory, are summarized, and a general description of the digital holographic microscopy process is given, including quantitative phase microscopy. Several numerical diffraction methods are described and compared, and a number of representative configurations used in digital holography are described, including off-axis Fresnel, Fourier, image plane, in-line, Gabor, and phase-shifting digital holographies. Then we survey numerical techniques that give rise to unique capabilities of digital holography, including suppression of dc and twin image terms, pixel resolution control, optical phase unwrapping, aberration compensation, and others. A survey is also given of representative application areas, including biomedical microscopy, particle field holography, micrometrology, and holographic tomography, as well as some of the special techniques, such as holography of total internal reflection, optical scanning holography, digital interference holography, and heterodyne holography. The review is intended for students and new researchers interested in developing new techniques and exploring new applications of digital holography.

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Principles and techniques of digital holographic microscopy

Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the marker-free analysis of living cells. The digital holographic feature of (subsequent) numerical focus adjustment makes possible applications for multifocus imaging. An overview of digital holographic microscopy methods is described. Applications of digital holographic microscopy are demonstrated by results obtained from livings cells and engineered surfaces.

Digital holographic microscopy for live cell applications and technical inspection

A technique to perform two-wavelengths digital holographic microscopy (DHM) measurements with a single hologram acquisition is presented. The vertical measurement range without phase ambiguity is extended to the micron-range, thanks to the resulting synthetic wavelength defined by the beating of two wavelengths with a separation of about 80nm. Real-time dual-wavelength imaging is made possible by using two reference waves having different wavelengths and propagation directions for the hologram recording. The principle of the method is exposed and experimental results concerning a 1.2μm m high test sample as well as a moving micro-mirror are presented. To the extent of our knowledge, this is the first time that real-time synthetic beat-wavelength digital holography measurements are reported.

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Real-time dual-wavelength digital holographic microscopy with a single hologram acquisition

A novel method is proposed for simulating free-space propagation. This method is an improvement of the angular spectrum method (AS). The AS does not include any approximation of the propagation distance, because the formula thereof is derived directly from the Rayleigh-Sommerfeld equation. However, the AS is not an all-round method, because it produces severe numerical errors due to a sampling problem of the transfer function even in Fresnel regions. The proposed method resolves this problem by limiting the bandwidth of the propagation field and also expands the region in which exact fields can be calculated by the AS. A discussion on the validity of limiting the bandwidth is also presented.

Band-limited angular spectrum method for numerical simulation of free-space propagation in far and near fields.

In microscopy, high magnifications are achievable for investigating micro-objects but the paradigm is that higher is the required magnification, lower is the depth of focus. For an object having a three-dimensional (3D) complex shape only a portion of it appears in good focus to the observer who is essentially looking at a single image plane. Actually, two approaches exist to obtain an extended focused image, both having severe limitations since the first requires mechanical scanning while the other one requires specially designed optics. We demonstrate that an extended focused image of an object can be obtained through digital holography without any mechanical scanning or special optical components. The conceptual novelty of the proposed approach lies in the fact that it is possible to completely exploit the unique feature of DH in extracting all the information content stored in hologram, amplitude and phase, to extend the depth of focus.

Extended focused image in microscopy by digital Holography.

A method for controlling the size of amplitude and phase images reconstructed from digital holograms by the Fresnel-transform method is proposed and demonstrated. The method can provide a constant reconstruction pixel width in the reconstructed image plane, independent of the recording and reconstruction distance. The proposed method makes it possible to maintain the size of an object for a sequence of digital holograms recorded at different distances and, therefore, to subtract phase maps for an object recorded at different distances. Furthermore, the method solves the problem of superimposition in multiwavelength digital holography for color display and holographic interferometry applications.

Controlling image size as a function of distance and wavelength in Fresnel-transform reconstruction of digital holograms

Aberrations and the distortions due to the imaging optics can be compensated in quantitative phase microscopy of thin phase objects by digital holography using a single hologram. The reconstructed quantitative phase microscopy phase distribution map can be directly corrected in the reconstructed image plane by a numerical method. To remove this unwanted aberration, in the special case of thin objects, the authors perform a two-dimensional fit with the Zernike polynomials of the reconstructed unwrapped phase. Subtraction of the fitted polynomial from the original phase map gives quantitative phase microscopy phase map free of aberrations.

Direct full compensation of the aberrations in quantitative phase microscopy of thin objects by a single digital hologram

We present a new method for numerically reconstructing digital holograms on tilted planes. The method is based on the angular spectrum of plane waves. Fast Fourier transform algorithm is used twice and coordinate rotation in the Fourier domain enables to reconstruct the object field on the tilted planes. Correction of the anamorphism resulting from the coordinate transformation is performed by suitable interpolation of the spectral data. Experimental results are presented to demonstrate the method for a singleaxis rotation. The algorithm is especially useful for tomographic image reconstruction.

Angular spectrum method with correction of anamorphism for numerical reconstruction of digital holograms on tilted planes

We demonstrate experimentally that correct phase imaging without 2? ambiguity is obtainable in digital holography by using a multiwavelength approach in the microscope configuration. We describe a general approach for removing chromatic aberrations and for controlling the pixel size of the reconstructed phase image in multiwavelength digital holography when the Fourier transform method is adopted for the numerical reconstruction of digital holograms. The retrieved phase is affected by the unavoidable, unwanted chromatic aberration. The correct phase can be obtained by evaluating the phase from the reference holograms reconstructed at different wavelengths to compensate for the chromatic aberration.

Domenico Alfieri

Papers

Extended focused image in microscopy by digital Holography.

Controlling image size as a function of distance and wavelength in Fresnel-transform reconstruction of digital holograms

Direct full compensation of the aberrations in quantitative phase microscopy of thin objects by a single digital hologram

Angular spectrum method with correction of anamorphism for numerical reconstruction of digital holograms on tilted planes

Recovering correct phase information in multiwavelength digital holographic microscopy by compensation for chromatic aberrations.