Digital Holographic Microscopy Technique Research Articles

Improving resolution in two orthogonal orientations from a single-shot digital holographic microscopy

We demonstrate a single-shot digital holographic microscopy technique to improve the diffraction-limited resolution in two orthogonal orientations simultaneously by dual-channel orthogonal polarization multiplexing approach. Orthogonally polarized two oblique beams are employed to illuminate the sample and two reference beams with mutually orthogonal polarization are interfered with the object beams in a custom-designed Mach–Zehnder configuration. This technique is potentially beneficial to encode high frequency sample information from two orthogonal directions simultaneously in a single recorded hologram where the high frequency spectra is synthesized by the selective spectral stitching. Thus, the resolution is enhanced along two orientations from a single-shot hologram in this work. Both the simulation and experimental results are shown for the proposed technique with around 2-fold resolution enhancement over the diffraction limit.

An Efficient Method of Observing Diatom Frustules via Digital Holographic Microscopy.

Herein, we propose a convenient method to enable pretreatment of target objects using digital holographic microscopy (DHM). As a test sample, we used diatom frustules (Nitzschia sp.) as the target objects. In the generally used sample preparation method, the frustule suspension is added dropwise onto a glass substrate or into a glass chamber. While our work confirms good observation of purified frustules using the typical sample preparation method, we also demonstrate a new procedure to observe unseparated structures of frustules prepared by baking them on a mica surface. The baked frustules on the mica surface were transferred to a glass chamber with 1% sodium dodecyl sulfate solution. In this manner, the unseparated structures of the diatom frustules were clearly observed. Furthermore, metal-coated frustules prepared by sputtering onto them on a mica surface were also clearly observed using the same procedure. Our method can be applied for the observation of any target object that is pretreated on a solid surface. We expect our proposed method to be a basis for establishing DHM techniques for microscopic observations of biomaterials.

Fast and pure phase-shifting off-axis holographic microscopy with a digital micromirror device.

We present a phase-shifting digital holographic microscopy technique, where a digital micromirror device enables to perform a precise phase-only shift of the reference wave. By coupling the beam into a monomode fiber, we obtain a laser mode with a constant phase shift, equally acting on all pixels of the hologram. This method has the advantage of being relatively simple and compatible with high frame rate cameras, which makes it of great interest for the observation of fast phenomena. We demonstrate the validity of the technique in an off-axis configuration by imaging living paramecia caudata.

Spatially multiplexed interferometric microscopy: principles and applications to biomedical imaging

Digital holographic microscopy (DHM) applied to quantitative phase imaging (QPI) has been successfully demonstrated as a powerful label-free method to analyse the optical properties of cells. Spatially multiplexed interferometric microscopy (SMIM) is a DHM technique that implements a common-path interferometric layout in the embodiment of a standard microscope to achieve QPI. More concretely, SMIM introduces three minimal modifications: (a) replaces the broadband illumination of the microscope by a coherent or partially coherent light source, (b) divides the input plane into two or three regions for transmission in parallel of both imaging and reference beams, and (c) includes a one-dimensional (1D) diffraction grating or a beam splitter cube for holographic recording. Hence, SMIM is a cost-effective, extremely simple, and highly stable manner of converting a standard bright field microscope into a holographic one. The goal of this contribution is to present the SMIM approaches implemented using a 1D diffraction grating, and highlight vast range of capabilities for biomedical imaging including super-resolved, reflective, transflective, noise-reduced and single-shot slightly off-axis amplitude and phase imaging.

Polychromatic digital holographic microscopy: a quasicoherent-noise-free imaging technique to explore the connectivity of living neuronal networks.

.Significance: Over the past decade, laser-based digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has become a significant label-free modality for live-cell imaging and used particularly in cellular neuroscience. However, coherent noise remains a major drawback for DHM, significantly limiting the possibility to visualize neuronal processes and precluding important studies on neuronal connectivity.Aim: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes.Approach: By combining a wavelength-tunable light source with the advantages of hologram numerical reconstruction of DHM, an approach called polychromatic DHM (P-DHM), providing OPD images with drastically decreased coherent noise, was developed.Results: When applied to cultured neuronal networks with an air microscope objective (, 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the -law, allowing to readily visualize the -wide thin neuronal processes with a signal-to-noise ratio of .Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.

Wavelength-Tuning Common-Path Digital Holographic Microscopy for Quantitative Phase Imaging of Functional Micro-Optics Components

This study proposes a novel wavelength-tuning common-path digital holographic microscopy technique for quantitative phase imaging of functional micro-optics components. The proposed technique is immune to vibration and can reduce system error. In the proposed configuration, a parallel glass plate was inserted into the light path to create two identical test beams, which passed through a specially designed window filter. In this process, one beam serves as the object beam, while the other is diffracted to produce an ideal spherical wave front (the reference beam). A wavelength tunable laser was used as the light source to generate phase-shifting digital holograms. Structural information for the functional micro-optics components was then extracted using a classical four-step phase-shift algorithm. The viability of the proposed technique was assessed by measuring a micro-optics array.

Realistic modeling of digital holographic microscopy

A self-contained platform for realistic modeling of digital holographic microscopy (DHM) is presented. Amplitude and phase samples, imaged in different architectures, can be modeled to produce numerical DHM holograms that include all the parameters that are present in real experiments, providing an accessible way for newcomers to have a first approach to this research field. The platform is based on considering the imaging arm of the DHM that produces the object wave as the result of the convolution process between the geometrical-optics image prediction of the sample with the point spread function introduced by diffraction. The DHM hologram is produced by the amplitude superposition of complex-valued object wave with a reference wave of arbitrary description. The sampling of the analytically produced DHM holograms is set from the input discretized image according to the specifications of the digital camera aimed to be used. The feasibility of the realistic platform is exemplified by contrasting the wrong results of a nonrealistic simulation with experimental results to show the need for using a complete realistic simulation like the one presented; further applications of the platform to numerical modeling speckle noise reduction over samples with controlled levels of roughness, and phase-shifting DHM techniques, are included.

Ferroelectric domain pattern in barium titanate single crystals studied by means of digital holographic microscopy

In this article, we report on the observation of a ferroelectric domain pattern in the whole volume of the ferroelectric barium titanate single crystal by means of digital holographic microscopy (DHM). Our particular implementation of DHM is based on the Mach–Zehnder interferometer and the numerical processing of data employs the angular spectrum method. A modification of the DHM technique, which allows a fast and accurate determination of the domain walls, i.e. narrow regions separating the antiparallel domains, is presented. Accuracy and sensitivity of the method are discussed. Using this approach, the determination of important geometric parameters of the ferroelectric domain patterns (such as domain spacing or the volume fraction of the anti-parallel domains) is possible. In addition to the earlier DHM studies of domain patterns in lithium niobate and lithium tantalate, our results indicate that the DHM is a convenient method to study a dynamic evolution of ferroelectric domain patterns in all perovskite single crystals.

Digital holographic microscopy as screening tool for cervical cancer preliminary study.

The liquid-based cytology Papanicolaou (Pap) test has gradually replaced the conventional Pap test, as it seems to be more efficient, in part because it uses computer assistance. However, Pap test diagnoses are still subjective and are cost-consuming or time-consuming. The authors describe a new digital holographic microscopy instrument and accompanying software. This technology provides an instantaneous, 3-dimensional image reconstruction of cells directly from the uterine cervical sample vial. Analyses were performed using the proprietary digital holographic microscopy (DHM) instrument, computer, and software. Residual materials from 3 randomly chosen laboratories were analyzed and subjected to DHM, and the sensitivity and specificity of DHM were calculated for the detection neoplasia. In 41 specimens that yielded normal Pap test results, 1333 cells were analyzed using DHM; and, in 30 specimens that yielded abnormal Pap test results, 494 cells were analyzed using DHM. Available histologic correlation was as follows: 4 histologic samples were diagnosed as grade 1 cervical intraepithelial neoplasia (corresponding to 2 cytologic diagnoses of low-grade squamous intraepithelial lesion and 2 cytologic diagnoses of atypical squamous cells of undetermined significance), 4 histologic samples were diagnosed as grade 3 cervical intraepithelial neoplasia (corresponding to 2 cytologic diagnoses of low-grade squamous intraepithelial lesion and 2 cytologic diagnosis of atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion), and 11 lesions were diagnosed as benign. Receiver operating characteristic curve analysis indicated that the best criteria were the nuclear-to-cytoplasmic ratio (area under the curve, 0.88; 95% confidence interval, 0.68-1.00) and the greatest nuclear dimension (area under the curve, 0.85; 95% confidence interval, 0.66-1.00). This preliminary study demonstrated for the first time that the DHM technique is suitable for the processing of gynecologic cervical samples. Nevertheless, DHM criteria and parameters could be better defined. Hopefully, holographic analysis will be performed automatically and will provide an instantaneous, cost-effective diagnosis from a closed vial with the preservation of all cellular material. Cancer Cytopathol 2016;124:573-80. © 2016 American Cancer Society.

Precise measurement of three-dimensional positions of transparent ellipsoidal particles using digital holographic microscopy.

The dynamic motions of various particles suspended in microscale flows are essential phenomena in the scientific and engineering fields. These motions can be precisely measured by using 3D quantitative flow visualization techniques. Moreover, most cells and particles in nature possess a nonspherical shape. Digital holographic microscopy (DHM) is employed to measure the 3D positional information of transparent ellipsoidal particles. Both in-plane and out-of-plane positional information are obtained by analyzing the distinctive light scattering from the microsized ellipsoidal particles. The performance of the 3D position measurement method is experimentally verified for ellipsoidal particles seeded in a planar surface and a microtube. This DHM technique exhibits promising potential in the dynamic analysis of ellipsoidal particles and cells suspended in various microscale fluid flows.

Improving Phase Contrast of Digital Holographic Microscope using Spatial Light Modulator

We propose a new method for improving the phase contrast of a multiphase digital holographic microscope using a spatial light modulator (SLM). Using the SLM as the annulus, our method improves the light contrast of the object edge to achieve higher accuracy. We demonstrate a digital holographic microscopy technique that provides a 30% improvement in the phase contrast compared to conventional microscopy, which utilizes a mechanical annulus. The phase-contrast improvement allows the 3D reconstructed hologram to be determined more precisely.

Review of digital holographic microscopy for three-dimensional profiling and tracking

Digital holographic microscopy (DHM) is a potent tool to perform three-dimensional imaging and tracking. We present a review of the state-of-the-art of DHM for three-dimensional profiling and tracking with emphasis on DHM techniques, reconstruction criteria for three-dimensional profiling and tracking, and their applications in various branches of science, including biomedical microscopy, particle imaging velocimetry, micrometrology, and holographic tomography, to name but a few. First, several representative DHM configurations are summarized and brief descriptions of DHM processes are given. Then we describe and compare the reconstruction criteria to obtain three-dimensional profiles and four-dimensional trajectories of objects. Details of the simulated and experimental evidences of DHM techniques and related reconstruction algorithms on particles, biological cells, fibers, etc., with different shapes, sizes, and conditions are also provided. The review concludes with a summary of techniques and applications of three-dimensional imaging and four-dimensional tracking by DHM.

Quantitative phase microscopy using dual-plane in-line digital holography

We present detailed theoretical evaluation and thorough experimental investigation of quantitative phase imaging using our previously demonstrated dual-plane in-line digital holographic microscopy technique [Opt. Lett. 35, 3426 (2010)]. This evaluation is based on the recording of two interferograms at slightly different planes and numerically reconstructing the object information. The zero-order diffracted wave is eliminated by using the method of subtraction of average intensity of the entire hologram, and the twin-image diffracted wave is removed by Fourier domain processing of the two recorded holograms. Experiments are performed using controlled amplitude and phase objects and human muscle cells to demonstrate the potential of this technique.

Automated imaging, identification, and counting of similar cells from digital hologram reconstructions

This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values.

Three-dimensional motion measurements of free-swimming microorganisms using digital holographic microscopy

A digital holographic microscope is employed to measure the 3D motion of free-swimming microorganisms. The focus function used to quantify image sharpness provides a better depth-directional accuracy with a smaller depth-of-focus compared with the intensity method in determining the depth-directional position of spherical particles of various diameters. The focus function is then applied to measure the 3D positions of free-swimming microorganisms, namely dinoflagellates C. polykrikoides and P. minimum. Both automatic segmentation and proper selection of a focus function for a selected segment are important processes in measuring the positional information of two free-swimming microorganisms of different shapes with various width-to-length ratios. The digital holographic microscopy technique improved in this work is useful for measuring 3D swimming trajectories, velocities and attitudes of hundreds of microorganisms simultaneously. It also exhibits exceptional depth-directional accuracy.

Transflective digital holographic microscopy and its use for probing plasmonic light beaming

We present a novel digital holographic microscopy technique termed transflective digital holographic microscopy in order to probe plasmonic beaming fields and to view their platform structures. Here, we borrow the term, 'transflective', a portmanteau meaning a blend of transmission and reflection according to the light-collecting condition, which is conventionally used in liquid crystal display systems. Incorporating the transmission type holographic microscopy with the reflection type, achieved by the utilization of polarization property of coherent light waves, we propose an application of the system to probing the beam path and its corresponding structure in plasmonic beaming phenomena.

Lateral and cross-lateral focusing of spherical particles in a square microchannel

The inertial migration of particles in micro-scale flows has received much attention due to its promising applications, such as the membrane-free passive separation of particles or cells. The particles suspended in rectangular channels are known to be focused near the center of each channel face as the channel Reynolds number (R(C)) increases due to the lift force balance and the hydrodynamic interactions of the particles with the wall. In this study, the three-dimensional positions of neutrally buoyant spherical particles inside a square microchannel are measured using the digital holographic microscopy technique, and a transition from the lateral tubular pinch to the cross-lateral focusing with increasing R(C) is reported. The particles are found to migrate first in the lateral direction and then cross-laterally toward the four equilibrium positions. A general criterion that can be used to secure the fully developed state of particle focusing in Lab-on-a-Chip applications is also derived. This criterion could be helpful for the accurate estimation of the design parameters of inertial microfluidic devices, such as R(C), channel length and width, and particle diameter.

Phase alteration compensation in reflection digital holography

The phase maps obtained from digital holographic microscopy techniques carry information about the axial lengths of the object under study. Additionally, these phase maps have information of tilt and curvatures with origin in the off-axis geometry and the magnification lenses system, respectively. Only a complete compensation of these extra phases allows a correct interpretation of the phase information. In this article a numerical strategy to compensate for these alterations is designed, using a phase mask located in different planes. This strategy is applied in the measurement of a phase steps plate using a digital holography setup.

Dual plane in-line digital holographic microscopy

We report a dual plane in-line digital holographic microscopy technique that exploits the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. Two interferograms are recorded at different planes to eliminate the conjugate image. The experimental results demonstrate successful reconstruction of phase objects as well as of amplitude objects. The two interferograms can be recorded simultaneously, using two CCD or CMOS sensors, in order to increase the acquisition rate. This enhanced acquisition rate, together with the improved reconstruction capability of the proposed method, may find applications in biomedical research for visualization of rapid dynamic processes at the cellular level.