Study on the simplified phase-shifting digital holographic microscopy

In this paper, a new simplified technique for effectively eliminating the zero order and the conjugate virtual image in digital holographic microcopy, which makes use of two-step phase-shifting method of just recording two holograms and an intensity image of object wave, is proposed. Meanwhile, combined with the principle of making full use of spatial bandwidth of the CCD sensor by in-line lens-less Fourier holographic recording geometry, the theory and experimental methods to increase the resolution of the reconstructed image in digital holography by using phase-shifting technique are detailedly analyzed. At end, the validity and availability of this technique has been demonstrated through the off-axis and in-line Fourier transform recording geometry. The study provides some theoretical and experimental guidance for the design and operation of a digital holographic microscopy system.     

Phase reconstruction in lensless digital in-line holographic microscopy

The phase reconstruction in a digital in-line holographic microscopy is compared using two numerical reconstruction methods. The first method uses one Fourier transform and second one uses three Fourier transforms. It is shown that the latter method gives improved object phase reconstruction as compared to the former.     

Polarization phase shifting in digital holographic microscopy

In the present work we have made use of polarization phase shifting in digital holographic microscopy (DHM) for three dimensional phase profiling of transmissive and reflecting microscopic samples. The Mach–Zehnder arrangement with proper polarizing elements (polarizer-masked cube beam splitter, quarter wave plate and a linear polarizer) is used for recording the phase-shifted digital holograms. The suggested procedure is simple and accurate and obviates the need of piezo devices for phase shifting. The phase profile of the specimen is reconstructed from the holograms by using standard phase shifting algorithms.     

An experimental investigation of three-dimensional particle aggregation using digital holographic microscopy

The tendency of particles to aggregate depends on particle-particle and particle-fluid interactions. These interactions can be characterized but it requires accurate 3D measurements of particle distributions. We introduce the application of an off-axis digital holographic microscopy for measuring distributions of dense micrometer (2 μm) particles in a liquid solution. We demonstrate that digital holographic microscopy is capable of recording the instantaneous 3D position of particles in a flow volume. A new reconstruction method that aids identification of particle images was used in this work. About 62% of the expected number of particles within the interrogated flow volume was detected. Based on the 3D position of individual particles, the tendency of particle to aggregate is investigated. Results show that relatively few particles (around 5–10 of a cohort of 1500) were aggregates. This number did not change significantly with time.     

Wave front reconstruction of Fresnel off-axis holograms with compensation of aberrations by means of phase-shifting digital holography

Off-axis holograms recorded with a CCD camera are numerically reconstructed in amplitude by calculating through the Fresnel–Kirchhoff integral. A phase-shifting Mach–Zehnder interferometer is used for recording four-quadrature phase-shifted off-axis holograms. The basic principle of this technique and its experimental verification are described. We show that the application of this algorithm allows for the suppression of the zero order of diffraction and of the twin image and that the contrast of the reconstructed images can be further enhanced by digital compensation of the aberrations introduced by the holographic recording system     

基于涡旋光照明的暗场数字全息显微方法研究

提出了一种基于涡旋光照明的暗场数字全息显微方法. 从理论上阐述了涡旋光环形照明原理和暗场数字全息显微原理, 分析了涡旋光的准无衍射特性对成像的影响; 搭建了相应的数字全息显微成像系统, 采用690 nm的聚苯乙烯小球作为实验样品; 最后通过对小球明暗场下数字全息显微再现像的分析对比, 证明该方法可以有效地提高数字全息系统的分辨率, 同时增强了再现像的对比度. 关键词： 全息 暗场数字全息显微 涡旋光 分辨率     

Wave optics analysis by phase-shifting real-time holographic interferometry

The purpose of this work is to study the potentialities in the phase-shifting real-time holographic interferometry using photorefractive crystals as the recording medium for wave-optics analysis in optical elements and non-linear optical materials. This technique was used for obtaining quantitative measurements from the phase distributions of the wave front of lens and lens systems along the propagation direction with in situ visualization, monitoring and analysis in real time.     

Reconstruction algorithms applied to in-line Gabor digital holographic microscopy

This paper investigates the application of Fresnel based numerical algorithms for the reconstruction of Gabor in-line holograms. We focus on the two most widely used Fresnel approximation algorithms, the direct method and the angular spectrum method. Both algorithms involve calculating a Fresnel integral, but they accomplish it in fundamentally different ways. The algorithms perform differently for different physical parameters such as distance, CCD pixel size, and so on. We investigate the constraints for the algorithms when applied to in-line Gabor digital holographic microscopy. We show why the algorithms fail in some instances and how to alter them in order to obtain useful images of the microscopic specimen. We verify the altered algorithms using an optically captured digital hologram.     

Error evaluation for Zernike polynomials fitting based phase compensation of digital holographic microscopy

Combining the experimental research with the simulation calculation, the error evaluation for Zernike polynomials fitting (ZPF) based phase compensation of digital holographic microscopy (DHM) is performed. The obtained results show that the reconstructed phase with high precision can be obtained by ZPF phase compensation algorithm. Moreover, the phase error for ZPF based phase compensation algorithm increases with both the variation of object height and object transverse area, the larger variation of object height, the larger of phase error, and the larger of object transverse area, the faster increase of RMS phase error. To decrease the error of ZPF phase compensation algorithm, it is required to ensure one of the variations of object height and object transverse area to be a small value. Importantly, the proposed method supplies a useful tool for the error evaluation of phase compensation algorithm.     

Optical diffraction tomography microscopy with transport of intensity equation using a light-emitting diode array

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of ±37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ∼0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.     

基于相移技术的显微数字全息重构细胞相位

介绍了用显微镜物镜、压电陶瓷和CCD建立的一套测量细胞相位的显微数字全息光路,基于相移技术,给出了重构相位的理论分析,并用洋葱磷片叶细胞作为测试样品,完成了测量细胞相位的实验.结果表明：该系统可以完成细胞相位重构,系统分辨率不低于1 μm.     

4-D imaging of fluid flow with digital in-line holographic microscopy

The large depth of field of digital in-line holographic microscopy (DIHM) with numerical reconstruction provides an ideal tool for the study of microfluidic phenomena. As indicators of the flow patterns we use latex microspheres and also red blood cells whose three-dimensional trajectories and velocities can easily be measured as a function of time with subsecond and micron resolution. We demonstrate the efficiency of DIHM by showing 3-D views of the flow patterns around big spheres in various geometric arrangements.     

Common-path phase-shifting digital holographic microscopy: A way to quantitative phase imaging and superresolution

We present an experimental setup useful for complex amplitude evaluation and phase image quantification of three-dimensional (3-D) samples in digital holographic microscopy (DHM). It is based on a common-path interferometric configuration performed by dividing the input plane in two contiguous regions and by placing a translation grating near to the Fourier plane. Then, complex amplitude distribution of the sample under test is recovered with phase-shifting standard method obtained by moving the grating using a linear motion stage. Some experimental results of an USAF resolution test are presented for different numerical aperture (NA) microscope lenses. In a second part, the proposed setup is tested under superresolution purposes. Based on the object’s spectrum shift produced by off-axis illumination, we use time multiplexing to generate a synthetic aperture enlargement that improves the final image resolution. Experimental results for the case of a biosample (human red blood cells) and a commercial low NA microscope lens validates the suggested superresolution approach.     

Measuring and calculating geometrical parameters of marine plankton using digital laser holographic imaging

The purpose of this paper is to develop an approach using digital laser holographic imaging for measuring and calculating geometrical parameters of marine plankton. It is very important for study of marine plankton and it facilitates the analysis of marine plankton. Using this approach, we obtained geometrical parameters of marine plankton including perimeter, equivalent diameter, lateral size and so on. It is found that a measurement aberration is less than 8% for copepods sample by using collimating reference wave.     

Design of the spatial filter window for digital holographic convolution reconstruction of object beam field

In the convolution reconstruction process of digital holographic object beam field, the object beam fields with different magnifications can be obtained when the reconstructing beams are spherical waves with different wave curvature radii. This paper presents a theoretical and experimental discussion on the useful spatial frequency spectrum of the hologram numerically illuminated by the spherical wave. The result shows that there would be an image in the spatial frequency spectrum of digital hologram, which is completely the same as the object, if the wave surface radius of the spherical wave is equal to the distance from the object to the CCD sensor. Taking this image as a reference and designing a filter, the position of the reconstructed images with different magnifications can be predicted correctly in the reconstructed plane. Additionally, since the spectrum distribution of zero-order diffraction can be forecasted accurately in theory, its contribution can be effectively eliminated through changing the spatial filter shape; then a reconstructed object field containing more high-frequency information can be obtained.     

Objective evaluation of images reconstructed from digital in-line Fresnel holograms by using coherent background elimination

Quality of images reconstructed from in-line Fresnel holograms using elimination of a coherent background is quantitatively studied through computer simulation and experiment. The coherent background is digitally calculated by averaging an intensity of a recorded hologram. The results show that in spite of the fact that virtual image is still intact, the background elimination can resolve images with quality comparable to the phase-shifting technique.     

Complex-amplitude-based phase unwrapping method for digital holographic microscopy

A complex-amplitude-based phase unwrapping approach for digital holographic microscopy is proposed in this paper. A quality map is derived directly from the reconstructed complex amplitude distribution of object wave to evaluate noise influence and phase reliability in the wrapped phase image. Quality-guided phase unwrapping algorithm is then implemented with the quality map to retrieve continuous phase profile. Unwrapping errors caused by unreliable phase data are successfully suppressed. The effectiveness of this method is demonstrated with simulation and experimental results.     

Design of adaptive spatial filter at uniform standard for automatic analysis of digital holographic microscopy

The spatial filtering method is a useful technique for the dynamic and automatic analysis in digital holographic microscopy. It has been shown that the proper selection of the spatial filter would improve the quality of the reconstructed image. However different results would be obtained by employing the spatial filter with different parameter threshold decided at different standard. This paper, according to the histogram analysis of the distribution of the +1 term spectrum of each hologram, gropes for the uniform standard for the decision of the threshold of the adaptive filter. It helps the adaptive spatial filtering method to be more advantageous for the dynamic and automatic analysis.     

Rough surface characterization using off-axis digital holographic microscopy compensated with self-hologram rotation

In this paper, an off-axis digital holographic microscopy compensated with self-hologram rotation is presented. The process is implemented via subtracting the unwrapped phase maps of the off-axis parabolic hologram and its rotation 180° to eliminate the tilt induced by the angle between the spherical object wave O and the plane reference wave R. Merit of the proposed method is that it can be done without prior knowledge of physical parameters and hence can reconstruct a parabolic hologram of 1024 × 768 pixels within tens of milliseconds since it doesn't require a digital reference wave. The method is applied to characterize rough gold bumps and the obtained results were compared with those extracted from the conventional reconstruction method. The comparison showed that the proposed method can characterize rough surfaces with excellent contrast and in real-time. Merit of the proposed method is that it can be used for monitoring smaller biological cells and micro-fluidic devices.     

Method for eliminating zero-order diffraction in lensless Fourier transform digital holography

A method to induce phase shifting in lensless Fourier of digital holography system is presented. In this method, by computer simulation and theoretical analysis on the technology to eliminate the influence of zero-order diffraction in force, it can be found that, a reference light induced in a random non-2π integral number of phase shifting shooting to get the second hologram, and wave reconstruction can be got by the difference value image of the two holograms. And, the method of different digital holography record system with different phase shifting should be used. In this paper, theoretical analyses have been done in detail to discuss the problems that exist in the unsuitable phase-shifting methods. Furthermore, some experiments have been done to prove the reliability of this method. This method can significantly improve the image quality and give better resolution of the reconstructed image.