Digital Holography

Techniques of digital holography are improved in order to obtain highresolution, 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.

We applied phase-shifting digital holography to microscopy by deriving the complex amplitude of light scattered from microscopic three-dimensional objects through a microscope objective by video camera recording, phaseshifting analysis, and computer reconstruction. This method requires no mechanical movement and provides a f lexible display and quantitative evaluation of the reconstructed images. A theory of image formation and experimental verification with specimens are described.

An information security method that uses a digital holographic technique is presented. An encrypted image is stored as a digital hologram. The decryption key is also stored as a digital hologram. The encrypted image can be electrically decrypted by use of the digital hologram of the key. This security technique provides secure storage and data transmission. Experimental results are presented to demonstrate the proposed method.

We present a technique for performing three-dimensional (3D) pattern recognition by use of in-line digital holography. The complex amplitude distribution generated by a 3D object at an arbitrary plane located in the Fresnel diffraction region is recorded by phase-shifting interferometry. The digital hologram contains information about the 3D object's shape, location, and orientation. This information allows us to perform 3D pattern-recognition techniques with high discrimination and to measure 3D orientation changes. Experimental results are presented. 

An approach is proposed for removing the wave front curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes. The unwanted curvature is compensated by evaluating a correcting wave front at the hologram plane with no need for knowledge of the optical parameters, focal length of the imaging lens, or distances in the setup. Most importantly it is shown that a correction effect can be obtained at all reconstruction planes. Three different methods have been applied to evaluate the correction wave front and the methods are discussed in detail. The proposed approach is demonstrated by applying digital holography as a method of coherent microscopy for imaging amplitude and phase contrast of microstructures.

We report on a method to achieve real-time dual-wavelength digital holographic microscopy with a single hologram acquisition. By recording both interferograms from two laser sources at different wavelengths in only one spatially-multiplexed digital hologram, we are able to independently propagate and apply numerical corrections on both wavefronts in order to obtain a beat-wavelength phase map of the specimen. This beat-wavelength being up to 10-100 times larger than the original wavelengths, we are in a situation where the 2π phase ambiguity of conventional DHM is removed and the phase measurement range of the technique is extended up to several tens of microns in height. The unique capability to perform such an operation with a single acquisition unables real-time dual-wavelength DHM measurements. Results on a moving micro-mirror are presented in this paper. We think that such a real-time dual-wavelength method represents a strong improvement to the current DHM state-of-the-art, and that it opens a whole new field of applications for this technique.

We propose and test a focus plane determination method that computes the digital refocus distance of an object investigated by digital holographic microscopy working in transmission. For this purpose we analyze the integrated amplitude modulus as a function of the digital holographic reconstruction distance. It is shown that when the focus distance is reached, the integrated amplitude is minimum for pure amplitude object and maximum for pure phase object. After a theoretical analysis, the method is demonstrated on actual digital holograms for the refocusing of pure amplitude and of pure phase microscopic samples.

The concept of numerical parametric lenses (NPL) is introduced to achieve wavefront reconstruction in digital holography. It is shown that operations usually performed by optical components and described in ray geometrical optics, such as image shifting, magnification, and especially complete aberration compensation (phase aberrations and image distortion), can be mimicked by numerical computation of a NPL. Furthermore, we demonstrate that automatic one-dimensional or two-dimensional fitting procedures allow adjustment of the NPL parameters as expressed in terms of standard or Zernike polynomial coefficients. These coefficients can provide a quantitative evaluation of the aberrations generated by the specimen. Demonstration is given of the reconstruction of the topology of a microlens.

| This paper presents an overview of the potential of free space optical technology in information security, encryption, and authentication. Optical waveform posses many degrees of freedom such as amplitude, phase, polarization, spectral content, and multiplexing which can be combined in different ways to make the information encoding more secure. This paper reviews optical techniques for encryption and security of two-dimensional and three-dimensional data.

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 threedimensional (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.

In this paper, we have investigated on the potentialities of digital holography for whole reconstruction of wavefields. We show that this technique can be efficiently used for obtaining quantitative information from the intensity and the phase distributions of the reconstructed field at different locations along the propagation direction. The basic concept and procedure of wavefield reconstruction for digital in-line holography is discussed. Numerical reconstructions of the wavefield from digitally recorded in-line hologram patterns and from simulated test patterns are presented. The potential of the method for analysing aberrated wave front has been exploited by applying the reconstruction procedure to astigmatic hologram patterns.

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.

We present a new method for variable tomographic scanning based on the wavelength scanning digital interference holography (WSDIH). A series of holograms are generated with a range of scanned wavelengths. The object field is reconstructed in a number of selected tilted planes from each hologram, and the numerical superposition of all the tilted object fields results in a variable tomographic scanning. The scanning direction can be arbitrary angles in 3D space but not limited in a 2D plane, thus the proposed algorithm offers more flexibility for acquiring and observing randomly orientated features of a specimen in a WSDIH system. Experiments are performed to demonstrate the effectiveness of the method.

Compressive sensing is a relatively new measurement paradigm which seeks to capture the "essential" aspects of a high-dimensional object using as few measurements as possible. In this work we demonstrate successful application of compressive sensing framework to digital Fresnel holography. It is shown that when applying compressive sensing approach to Fresnel fields a special sampling scheme should be adopted for improved results.

This paper proposes a new micro-particles localization scheme in digital holography. Most conventionnal digital holography methods, are based on Fresnel transform and have several issues such as twin-image, border effects... To avoid these difficulties, we propose an inverse problem approach, which yields the optimal particles set which best models the observed hologram image. We resolve this global optimization problem by conventional particle detection followed by a local refinement for each particle. Results on both simulated and real digital holograms show strong improvements in the localization of the particles, particularly along the depth dimension. In our simulations, the position precision is about or better than 1 µm rms. Our results also show that the localization precision does not deteriorate for particles near the edges of the field of view.

We present what we believe to be a new digital holographic imaging method that is able to determine simultaneously the distributions of intensity, phase, and polarization state at the surface of a specimen on the basis of a single image acquisition. Two reference waves with orthogonal polarization states interfere with the object wave to create a hologram that is recorded on a CCD camera. Two wave fronts, one for each perpendicular polarization state, are numerically reconstructed in intensity and phase. Combining the intensity and the phase distributions of these two wave fronts permits the determination of all the components of the Jones vector of the object-wave front. We show that this method can be used to image and measure the distribution of the polarization state at the surface of a specimen, and the obtained results indicate that precise quantitative measurements of the polarization state can be achieved. An application of the method to image the birefringence of a stressed polymethyl methacrylate sample is presented.

Phase-only holographic projection has prompted
a great deal of research and has often been cited as a desirable
method of 2-D image formation, since such a technique offers
a number of advantages over conventional imaging projection
technology [1], [2]. Although holographic image formation was
demonstrated some forty years ago [3], efforts at realizing a
real-time 2-D video projection system based on this technique
have not been successful, principally due to the computational
complexity of calculating diffraction patterns in real time and
the poor quality of the resultant images. In this paper, a new
approach to hologram generation and display is presented which
overcomes both of these problems, enabling—for the first time—a high-quality real-time holographic projector.

We present a phase-imaging technique to quantitatively study the three-dimensional structure of cells. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of SKOV-3 ovarian cancer cells are presented.

An approach that uses an electro-optically tunable two dimensional phase grating to enhance the resolution in digital holographic microscopy is proposed. We show that, by means of a flexible hexagonal phase grating, it is possible to increase the numerical aperture of the imaging system, thus improving the spatial resolution of the images in two dimensions. The augment of the numerical aperture of the optical system is obtained by recording spatially multiplexed digital holograms. The grating tuneability allows one to adjust the intensity among the spatially multiplexed holograms maximizing the grating diffraction efficiency. Furthermore we demonstrate that the flexibility of the numerical reconstruction allows one to use selectively the diffraction orders carrying useful information for increasing the spatial resolution. The proposed approach can improve the capabilities of digital holography in threedimensional imaging and microscopy.

In this paper, we demonstrate a fully digital technique using double random Fourier, and fractional plane encryption. The input image to be encrypted is multiplied by a phase mask, and either its Fourier or fractional Fourier transform is obtained. Using interference with a wave from another random phase mask, the encrypted data (Fourier or fractional hologram) is recorded digitally. The decryption key is also recorded as a digital hologram, called the key hologram. An electronic key generated in the PC is multiplied with the encrypted hologram, and a Fourier or fractional Fourier transform (encrypted image) is obtained. This decryption key hologram, the electronically generated key (random phase code), and the encrypted image can be transmitted through communication channels. The retrieval is carried out by all-digital means.

We present a digital signal processing technique that reduces the speckle content in reconstructed digital holograms. The method is based on sequential sampling of the discrete Fourier transform of the reconstructed image field. Speckle reduction is achieved at the expense of a reduced intensity and resolution, but this tradeoff is shown to be greatly superior to that imposed by the traditional mean and median filtering techniques. In particular, we show that the speckle can be reduced by half with no loss of resolution (according to standard definitions of both metrics).

We report on the fabrication, by a 26 MHz stretched-cavity femtosecond Ti:sapphire oscillator, of optical waveguides in different glass substrates, and their optical characterization. Operation of these waveguides in the telecom range at 1.55 µm is demonstrated. Digital holography microscopy is used to measure their refractive index profile. The results evidence a strong dependence of the fabrication process on the glass matrix.

Holographic imaging offers a reliable and fast method to capture the complete 3-D information of the scene from a single perspective. We review our recently proposed single-channel optical system for generating digital Fresnel holograms of 3-D real-existing objects illuminated by incoherent light. In this motionless holographic technique, light is reflected, or emitted, from a 3-D object, propagates through a spatial light modulator (SLM), and is recorded by a digital camera. The SLM is used as a beam-splitter of the single-channel incoherent interferometer, such that each spherical beam originated from each object point is split into two spherical beams with two different curve radiuses. Incoherent sum of the entire interferences between all the couples of spherical beams creates the Fresnel hologram of the observed 3-D object. When this hologram is reconstructed in the computer, the 3D properties of the object are revealed.

We present an optical method for information watermarking of three-dimensional (3D) objects by digital holography. A hidden image is embedded by double phase encoding in a phase-shift digital hologram of the 3D object. We decode the watermarked hologram to reconstruct the hidden image and the 3D object. We use either the entire hologram or a part of it to decode the hidden image. Experiments are presented to illustrate the ability to recover both the 3D object and the decoded hidden image. Digital holograms of the 3D object are obtained by optical experiments. The watermarking process, 3D object reconstruction, and hidden image recovery are performed digitally. To the best of our knowledge, this is the first report of 3D object watermarking by use of a phase encoding technique and digital holography.

One of the main drawbacks of Digital Holography (DH) is the coherent nature of the light source, which severely corrupts the quality of holographic reconstructions. Although numerous techniques to reduce noise in DH have provided good results, holo-graphic noise suppression remains a challenging task. We propose a novel framework that combines the concepts of encoding multiple uncorrelated digital holograms, block grouping and collaborative filtering to achieve quasi noise-free DH reconstructions. The optimized joint action of these different image-denoising methods permits the removal of up to 98% of the noise while preserving the image contrast. The resulting quality of the hologram reconstructions is comparable to the quality achievable with non-coherent techniques and far beyond the current state of art in DH. Experimental validation is provided for both single-wavelength and multi-wavelength DH, and a comparison with the most used holographic denoising methods is performed.

We present a digital holographic microscope that permits one to image polarization state. This technique results from the coupling of digital holographic microscopy and polarization digital holography. The interference between two orthogonally polarized reference waves and the wave transmitted by a microscopic sample, magnified by a microscope objective, is recorded on a CCD camera. The off-axis geometry permits one to reconstruct separately from this single hologram two wavefronts that are used to image the object-wave Jones vector. We applied this technique to image the birefringence of a bent fiber. To evaluate the precision of the phase-difference measurement, the birefringence induced by internal stress in an optical fiber is measured and compared to the birefringence profile captured by a standard method, which had been developed to obtain high-resolution birefringence profiles of optical fibers.

A technique based on superresolution by digital holographic microscopic imaging is presented. We used a two dimensional (2-D) vertical-cavity self-emitting laser (VCSEL) array as spherical-wave illumination sources. The method is defined in terms of an incoherent superposition of tilted wavefronts. The tilted spherical wave originating from the 2-D VCSEL elements illuminates the target in transmission mode to obtain a hologram in a Mach-Zehnder interferometer configuration. Superresolved images of the input object above the common lens diffraction limit are generated by sequential recording of the individual holograms and numerical reconstruction of the image with the extended spatial frequency range. We have experimentally tested the approach for a microscope objective with an exact 2-D reconstruction image of the input object. The proposed approach has implementation advantages for applications in biological imaging or the microelectronic industry in which structured targets are being inspected.

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.

The presence of speckle noise in the reconstruction process of digital hologram reduces the signal to noise ratio (SNR) in the reconstructed images. In this paper we present wavelet filtering to improve SNR in the reconstructed images. Experimental results are presented.

Features offered by the combination of time averaging and digital Fresnel holography are investigated. In particular, we introduce the concept of the zero-crossing phase of Bessel fringes, which allows a highly contrasted determination of the dark fringes in the hologram. We discuss some particularities of the digital reconstruction and show how time-averaged digital holography can be used to study vibration drifts. Experiment results are presented in the case of a loudspeaker under a sinusoidal excitation; digital and analogical holography are also compared.

Many applications in non-destructive testing at a microscopic level and in live cell imaging require automated focusing due to unstable environmental conditions, moving specimen or the limited depth of field of the applied optical imaging systems. Digital holography permits the recording and the numerical reconstruction of optical wave fields in amplitude and phase. This enables imaging of multiple focal planes from a single recorded hologram without mechanical realignment. The combination of numerical refocusing with image sharpness quantification algorithms yields subsequent autofocusing. With calibrated optical imaging systems this feature can be used also to determine the position and axial displacements of a sample. In order to show the application potential of digital holographic autofocusing in microscopy the method and results from investigations on several amplitude and phase objects are reviewed. This includes a demonstration of the reliability of automated refocusing, multi-focus quantitative phase contrast imaging of suspended cells, refocusing of quantitative phase contrast images during the analysis of the temporal dependency of cell spreading on surfaces and the quantification of toxin mediated morphological cell alterations during long-term observations. It is also shown for the example of sedimenting red blood cells that the method can be applied for minimally-invasive tracking of multiple particles. Finally, the usage of numerical autofocus for quantitative migration analysis of arbitrary shaped cells in a threedimensional collagen matrix is demonstrated.

We discuss quantization effects of hologram recording on the quality of reconstructed images in phase-shifting digital holography. We vary bit depths of phase-shifted holograms in both numerical simulation and experiments and then derived the complex amplitude, which is subjected to Fresnel transformation for the image reconstruction. The influence of bit-depth limitation in quantization has been demonstrated in a numerical simulation for spot-array patterns with linearly varying intensities and a continuous intensity object. The objects are provided with uniform and random phase modulation. In experiments, digital holograms are originally recorded at 8 bits and the bit depths are changed to deliver holograms at bit depths of 1 to 8 bits for the image reconstruction. The quality of the reconstructed images has been evaluated for the different quantization levels.

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the shortcoherence length of light source. A metallic object, covered by a 150᎑m-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 m underneath the eye surface.

We propose a variant of the heterodyne holography scheme, which combines the properties of off-axis and phase-shifting holography. This scheme makes it possible filter-off numerically the zero order image alias, and the technical noise of the reference. It is then possible to record and reconstruct holographic images at an extremely low signal level. We show experimentally that the sensitivity of the method is limited only by the quantum nature of photons.

The ability to see behind flames is a key challenge for the industrial field and particularly for the safety field. Development of new technologies to detect live people through smoke and flames in fire scenes is an extremely desirable goal since it can save human lives. The latest technologies, including equipment adopted by fire departments, use infrared bolometers for infrared digital cameras that allow users to see through smoke. However, such detectors are blinded by flame-emitted radiation. Here we show a completely different approach that makes use of lensless digital holography technology in the infrared range for successful imaging through smoke and flames. Notably, we demonstrate that digital holography with a cw laser allows the recording of dynamic human-size targets. In this work, easy detection of live, moving people is achieved through both smoke and flames, thus demonstrating the capability of digital holography at 10.6 μm.

This work reveals an experimental microscopy acquisition scheme successfully combining Compressed Sensing (CS) and digital holography in off-axis and frequency-shifting conditions. CS is a recent data acquisition theory involving signal reconstruction from randomly undersampled measurements, exploiting the fact that most images present some compact structure and redundancy. We propose a genuine CS-based imaging scheme for sparse gradient images, acquiring a diffraction map of the optical field with holographic microscopy and recovering the signal from as little as 7% of random measurements. We report experimental results demonstrating how CS can lead to an elegant and effective way to reconstruct images, opening the door for new microscopy applications. OCIS : 070.0070, 180.3170

Terahertz digital off-axis holography is demonstrated using a Mach-Zehnder interferometer with a highly coherent, frequency tunable, continuous wave terahertz source emitting around 0.7 THz and a single, spatially-scanned Schottky diode detector. The reconstruction of amplitude and phase objects is performed digitally using the angular spectrum method in conjunction with Fourier space filtering to reduce noise from the twin image and DC term. Phase unwrapping is achieved using the dual wavelength method, which offers an automated approach to overcome the 2π phase ambiguity. Potential applications for nondestructive test and evaluation of visually opaque dielectric and composite objects are discussed.

In this paper we present a method to watermark a 3D object with another hidden 3D object using digital holography. The watermark or the hidden information is a 3D object that is embedded in the digital hologram of a 3D host object. The digital holograms are obtained optically by phase shift interferometery. The hologram of the hidden 3D object is double phase encoded before embedding it to the host 3D object hologram. Then, the watermarked hologram is double phase encoded again using different set of codes. The resultant watermarked hologram is very secure because of the multi-key nature of the watermarking process. We discuss the effect of distortion caused by hologram quantization and occlusion of some of the hologram pixels. We present tests to illustrate the effect of using a window of the hologram to reconstruct the hidden 3D object and the host 3D object. Both mathematical analysis and simulations are presented to illustrate the system performance. To the best of our knowledge, this is the first report of embedding a 3D objects within another 3D object.

We develop a deterministic algorithm for coherent diffractive imaging (CDI) that employs a modified Fourier transform of a Fraunhofer diffraction pattern to quantitatively reconstruct the complex scalar wavefield at the exit surface of a sample of interest. The sample is placed in a uniformly-illuminated rectangular hole with dimensions at least two times larger than the sample. For this particular scenario, and in the far-field diffraction case, our non-iterative reconstruction algorithm is rapid, exact and gives a unique analytical solution to the inverse problem. The efficacy and stability of the algorithm, which may achieve resolutions in the nanoscale range, is demonstrated using simulated X-ray data.

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the
intracellular refractive index and to cell thickness.
DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes.
In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple
automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

It is demonstrated for the first time to the authors' knowledge that digital holographic image reconstruction can be applied to correction of image reconstruction in the presence of severe anamorphism and aberrations. It is shown that anamorphism and aberrations can be taken into account and compensated for separately to yield a correct-image reconstruction. Anamorphism and aberrations are introduced by a holographic recording system based on a ref lective grating interferometer (RGI). The strong anamorphic behavior of the RGI prevents reconstruction of a correct image unless that behavior is compensated for in the process of numerical holographic reconstruction.

Digital holography is an emerging technology that can generate complex light patterns for controlling the excitability of neurons and neural circuits. The strengths of this technique include a high efficiency with which available light can be effectively utilized and the ability to deliver highly focused light to multiple locations simultaneously. Here we demonstrate another strength of digital holography: the ability to generate instantaneous three-dimensional light patterns. This capability is demonstrated with the photolysis of caged glutamate on the dendritic arbor of hippocampal neurons, to study the nature of the integration of inputs arriving on multiple dendritic branches.

Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.