Digital holographic microscope for measuring three-dimensional particle distributions and motions

Better understanding of particle-particle and particle-fluid interactions requires accurate 3D measurements of particle distributions and motions. We introduce the application of in-line digital holographic microscopy as a viable tool for measuring distributions of dense micrometer (3.2 microm) and submicrometer (0.75 microm) particles in a liquid solution with large depths of 1-10 mm. By recording a magnified hologram, we obtain a depth of field of approximately 1000 times the object diameter and a reduced depth of focus of approximately 10 particle diameters, both representing substantial improvements compared to a conventional microscope and in-line holography. Quantitative information on depth of field, depth of focus, and axial resolution is provided. We demonstrate that digital holographic microscopy can resolve the locations of several thousand particles and can measure their motions and trajectories using cinematographic holography. A sample trajectory and detailed morphological information of a free-swimming copepod nauplius are presented.     

Digital holographic microscopy with reduced spatial coherence for three-dimensional particle flow analysis

We investigate the use of a digital holographic microscope working in partially coherent illumination to study in three dimensions a micrometer-size particle flow. The phenomenon under investigation rapidly varies in such a way that it is necessary to record, for every camera frame, the complete holographic information for further processing. For this purpose, we implement the Fourier-transform method for optical amplitude extraction. The suspension of particles is flowing in a split-flow lateral-transport thin separation cell that is usually used to separate the species by their sizes. Details of the optical implementation are provided. Examples of reconstructed images of different particle sizes are shown, and a particle-velocity measurement technique that is based on the blurred holographic image is exploited.     

Digital shearing method for three-dimensional data extraction in holographic particle image velocimetry

We report a new digital shearing method for extracting the three-dimensional displacement vector data from double-exposure holograms. With this method we can manipulate both the phase and the amplitude of the recorded signal, which, like optical correlation analysis, is inherently immune to imaging aberration. However, digital shearing is not a direct digital implementation of optical correlation, and a considerable saving in computation time results. We demonstrate the power of the method by MATLAB simulation and discuss its performance with reference to optical analysis.     

Digital in-line holographic microscopy

We first briefly review the state of the art of digital in-line holographic microscopy (DIHM) with numerical reconstruction and then discuss some technical issues, such as lateral and depth resolution, depth of field, twin image, four-dimensional tracking, and reconstruction algorithm. We then present a host of examples from microfluidics and biology of tracking the motion of spheres, algae, and bacteria. Finally, we introduce an underwater version of DIHM that is suitable for in situ studies in an ocean environment that show the motion of various plankton species.     

Aberration compensation of holographic particle images using digital holographic microscopy

Characterisation of small and large-scale vortices in turbulent flows demands a system with high spatial resolution. The measurement of high spatial resolution, three-dimensional vector displacements in fluid mechanics using holography, is usually hampered by aberration. Aberration poses some problems in particle image identification due to low fidelity of real image reconstruction. Phase mismatch between the recording and the reconstruction waves was identified as the main source of aberration in this study. This paper demonstrates how aberration compensation can be achieved by cross-correlating the complex amplitude of an aberrated reconstructed object with the phase conjugate of a known reference object in the plane of the hologram (frequency space). Results favourably show significant increase in Strehl ratio and suppression of background noise that are more pronounced for particle images of 10 and 5 microns. It is clear from the work conducted that wavefront aberration measurement and compensation of holographic microscopic objects are now possible with the use of a variant digital holographic microscope.     

Single beam two-views holographic particle image velocimetry

Holographic particle image velocimetry (HPIV) is presently the only method that can measure at high resolution all three components of the velocity in a finite volume. In systems that are based on recording one hologram, velocity components parallel to the hologram can be measured throughout the sample volume, but elongation of the particle traces in the depth direction severely limits the accuracy of the velocity component that is perpendicular to the hologram. Previous studies overcame this limitation by simultaneously recording two orthogonal holograms, which inherently required four windows and two recording systems. This paper introduces a technique that maintains the advantages of recording two orthogonal views, but requires only one window and one recording system. Furthermore, it enables a quadruple increase in the spatial resolution. This method is based on placing a mirror in the test section that reflects the object beam at an angle of 45 degrees. Particles located in the volume in which the incident and reflected beams from the mirror overlap are illuminated twice in perpendicular directions. Both views are recorded on the same hologram. Off-axis holography with conjugate reconstruction and high-pass filtering is used for recording and analyzing the holograms. Calibration tests show that two views reduce the uncertainty in the three-dimensional (3-D) coordinates of the particle centroids to within a few microns. The velocity is still determined plane-by-plane by use of two-dimensional particle image velocimetry procedures, but the images are filtered to trim the elongated traces based on the 3-D location of the particle. Consequently, the spatial resolution is quadrupled. Sample data containing more than 200 particles/mm3 are used for calculating the 3-D velocity distributions with interrogation volumes of 220 x 154 x 250 microm, and vector spacing of 110 x 77 x 250 microm. Uncertainty in velocity is addressed by examining how well the data satisfies the continuity equation. The results show significant improvements compared with previous procedures. Limitations of the technique are also discussed.     

Digital quasi-phase-matched two-color nonvolatile holographic storage

Unwanted erasure during readout of holographic data can be reduced or eliminated by use of a different wavelength for reading than that which was used for writing. To prevent distortion and Bragg mismatch that would be unacceptable for digital data storage, one can format data to account for the wavelength difference. Techniques to format data and the results of this formatting are presented. Varying the formatting parameters is investigated to optimize diffraction efficiency.     

Digital watermarking by a holographic technique

A holographic technique is applied for digital watermarking by a computer. A digital-watermark image to be hidden is phase modulated in a random fashion, and its Fourier-transformed hologram is superposed on a content image. The watermark is reconstructed by means of a holographic-reconstruction technique from the bit-map image that hides it. In the study the processes of constructing and reconstructing a digital hologram are described on the basis of the theory of Fourier optics. The conditions for superposing the hologram onto the content images are investigated in detail. The validity of the present method is verified by changing the weighting of the hologram relative to that of the content image. The effect of image size is also discussed with respect to reconstruction of the watermark, and it is shown that watermark information in a form of a diffuse-type Fourier-transform hologram cannot be removed by cutting it out of the host image.     

Evanescent wave holographic recording in a photopolymer

We present a study of holographic recording with an evanescent reference and a homogeneous (plane) object wave. The grating step was 324 nm. The dependence of the diffraction efficiency on exposure was investigated. The maximum value obtained was 0.01% at 5 mJ cm^?2 exposure. The optimal pre-exposure, needed for grating adhesion to the glass substrate, was 1 μJ cm^?2.     

Airborne digital holographic system for cloud particle measurements

An in-line holographic system for in situ detection of atmospheric cloud particles [Holographic Detector for Clouds (HOLODEC)] has been developed and flown on the National Center for Atmospheric Research C-130 research aircraft. Clear holograms are obtained in daylight conditions at typical aircraft speeds of 100 m s(-1). The instrument is fully digital and is interfaced to a control and data-acquisition system in the aircraft via optical fiber. It is operable at temperatures of less than -30 degrees C and at typical cloud humidities. Preliminary data from the experiment show its utility for studies of the three-dimensional spatial distribution of cloud particles and ice crystal shapes.     

In-line holographic particle image velocimetry for turbulent flows

A holographic system has been developed to measure the velocity field in three-dimensional flow regions. The system records the position of small tracer particles on two in-line holograms of the flow obtained simultaneously. Two exposures are recorded on each hologram. The flow velocity is derived from the displacement of the particles between exposures. A general design procedure is described for selecting the particle diameter and the concentration on the basis of the configuration of the flow facility and the resolution characteristics of the holographic imaging system. The system was implemented in a 2 ft x 2 ft (1 ft = 30.48 cm) water channel to measure the velocity field in a turbulent free-surface jet. The spatial resolution of the system is 1 mm, and the field of view is 100 mm, approximately. Measurements performed with this system are compared with results reported in the literature and are found to be in good agreement.     

Optical alignment-induced errors in holographic particle image velocimetry

A ray-tracing analysis of point-source imaging in the presence of optical misalignment is used to analyze relative image shift as a source of measurement error in holographic particle image velocimetry (HPIV). Although single-reference-beam HPIV is relatively insensitive to optical misalignment, dual-reference-beam systems may suffer substantial errors because of misalignments of the order of microradians. These systems are particularly sensitive to rotations of the hologram about an axis perpendicular to the film and to reconstruction beam misalignment. In a swirling flow experiment, a proposed error-compensation scheme was able to reduce uncertainty from 130% to 10% of the mean measured velocity.     

Automatic threshold technique for holographic particle field characterization

The 3D distribution of a particle field by digital holography is obtained by 3D numerical reconstruction of a 2D hologram. The proper identification of particles from the background during numerical reconstruction influences the overall effectiveness of the technique. The selection of a suitable threshold value to segment particles from the background of reconstructed images during 3D holographic reconstruction process is a critical issue, which influences the accuracy of particle size and number density of reconstructed particles. The object particle field parameters, such as depth of sample volume and density of object particles, influence the optimal threshold value. The present study proposes a novel technique for the determination of the optimal threshold value of a reconstructed image. The effectiveness of the proposed technique is demonstrated using both simulated and experimental data. The proposed technique is robust to variation in optical properties of particle and background, depth of sample volume, and number density of object particle field. The particle diameter obtained from the proposed threshold technique is within 5% of that obtained from the particle size analyzer. There is a maximum ten times increase in reconstruction effectiveness by using the proposed automatic threshold technique in comparison with the fixed manual threshold technique.     

Digital holography of particle fields: reconstruction by use of complex amplitude

Digital holography appears to be a strong contender as the next-generation technology for holographic diagnostics of particle fields and holographic particle image velocimetry for flow field measurement. With the digital holographic approach, holograms are directly recorded by a digital camera and reconstructed numerically. This not only eliminates wet chemical processing and mechanical scanning, but also enables the use of complex amplitude information inaccessible by optical reconstruction, thereby allowing flexible reconstruction algorithms to achieve optimization of specific information. However, owing to the inherently low pixel resolution of solid-state imaging sensors, digital holography gives poor depth resolution for images, a problem that severely impairs the usefulness of digital holography especially in densely populated particle fields. This paper describes a technique that significantly improves particle axial-location accuracy by exploring the reconstructed complex amplitude information, compared with other numerical reconstruction schemes that merely mimic traditional optical reconstruction. This novel method allows accurate extraction of particle locations from forward-scattering particle holograms even at high particle loadings.     

Digital holographic microtomography with few angle data-sets

In this paper, we report the experimental implementation of handling the reconstruction problem from few angle data-sets for digital holographic microtomography. First, the digital holographic microscopy with sample-rotating scheme is established and few holograms with regularly spaced angle steps are recorded. Then, an algebraic iterative reconstruction algorithm with non-positivity constraint and a smoothing operator is applied to reconstruct three-dimensional refractive index distribution of the measured sample from the few angle data-sets. The experimental results demonstrate that the algebraic iterative technique can accurately reconstruct refractive index distribution from few angle data-sets in digital holographic microtomography. The technique is easy to implement and reduces greatly the required recording times.     

Digital holographic microscopy with dual-wavelength phase unwrapping

We apply the techniques of digital holography to obtain microscopic three-dimensional images of biological cells. The optical system is capable of microscopic holography with diffraction-limited resolution by projecting a magnified image of a microscopic hologram plane onto a CCD plane. Two-wavelength phase-imaging digital holography is applied to produce unwrapped phase images of biological cells. The method of three-wavelength phase imaging is proposed to extend the axial range and reduce the effect of phase noise. These results demonstrate the effectiveness of digital holography in high-resolution biological microscopy.     

Encrypted holographic memory using an encoded reference wave

A secure holographic memory system is proposed by use of an encoded reference beam. The reference beam is encrypted by a fiber-optic faceplate, which serves as a phase mask. There are seven keys in the system including the position and direction of the fiber bundle and the direction of the incident beam. The experiment shows that the total key length is larger than 1.8 x 1019. The method can be used directly in a shift-multiplexing system with high selectivity.     

Four-dimensional dynamic flow measurement by holographic particle image velocimetry

The ultimate goal of holographic particle image velocimetry (HPIV) is to provide space- and time-resolved measurement of complex flows. Recent new understanding of holographic imaging of small particles, pertaining to intrinsic aberration and noise in particular, has enabled us to elucidate fundamental issues in HPIV and implement a new HPIV system. This system is based on our previously reported off-axis HPIV setup, but the design is optimized by incorporating our new insights of holographic particle imaging characteristics. Furthermore, the new system benefits from advanced data processing algorithms and distributed parallel computing technology. Because of its robustness and efficiency, for the first time to our knowledge, the goal of both temporally and spatially resolved flow measurements becomes tangible. We demonstrate its temporal measurement capability by a series of phase-locked dynamic measurements of instantaneous three-dimensional, three-component velocity fields in a highly three-dimensional vortical flow-the flow past a tab.     

Two-dimensional wavelet transform and application to holographic particle velocimetry

The goal of holographic particle velocimetry is to infer fluid velocity patterns from images reconstructed from doubly exposed holograms of fluid volumes seeded with small particles. The advantages offered by in-line holography in this context usually make it the method of choice, but seeding densities sufficient to achieve high spatial resolution in the sampling of the velocity fields cause serious degradation, through speckle, of the signal-to-noise ratio in the reconstructed images. The in-line method also leads to a great depth of field in paraxial viewing of reconstructed images, making it essentially impossible to estimate particle depth with useful accuracy. We present here an analysis showing that these limitations can be circumvented by variably scaled correlation, or wavelet transformation. The shift variables of the wavelet transform are provided automatically by the optical correlation methodology. The variable scaling of the wavelet transform derives, in this case, directly from the need to accommodate varying particle depths. To provide such scaling, we use a special optical system incorporating prescribed variability in spacings and focal length of lenses to scan through the range of particle depths.

Calculation shows, among other benefits, improvement by approximately two orders of magnitude in depth resolution. A much higher signal-to-noise ratio together with faster data extraction and processing should be attainable.

     

Digital holographic microscopy with pure-optical spherical phase compensation

Telecentric architecture is proposed for circumventing, by the pure-optical method, the residual parabolic phase distortion inherent to standard configuration of digital holographic microscopy. This optical circumvention produces several important advantages. One is that there is no need for computer compensation of the parabolic phase during the phase map recovering procedure. The other is that in off-axis configuration, the spatial frequency useful domain is enlarged. The validity of the method is demonstrated by performing quantitative measurement of depth differences with high axial resolution.