In-line Holographic Microscopy Research Articles

Large scale calculation for holography using SSD

Computer based holography such as electro-holography and digital holography advances because the resolution of an imaging display and a sensing device such as a liquid crystal display (LCD) and a charge coupled device (CCD) becomes higher. A higher resolution of imaging device requires the more powerful computing system. On the other hand, a solid state drive (SSD) develops as a storage device of computers. We studied the effectiveness of SSD for a large-scale digital holography calculation. When the calculation data scale exceeds the main memory capacity, the SSD system showed better performance compared with the hard disk drive (HDD) system at computational speed and stability. Full Text: PDF References M. Stanley et al., 100-megapixel computer-generated holographic images from Active Tiling: a dynamic and scalable electro-optic modulator system, Proc. SPIE 5005, Practical Holography XVII and Holographic Materials IX, 247 (2003) CrossRef H. Sasaki, K. Yamamoto, Y. Ichihashi, T. Senoh, Large size three-dimensional video by electronic holography using multiple spatial light modulators, Scientific Reports 4, Article number 04000 (2014) CrossRef T. Kozacki, G. Finke, P. Garbat, W. Zaperty, M. Kujawinska, Wide angle holographic display system with spatiotemporal multiplexing, Opt. Express 20, 27473-27481 (2012) CrossRef T. Shimobaba et al., In-line digital holographic microscopy using a consumer scanner, Scientific Reports 3, Article number 02664 (2013). CrossRef S.O.Isikman, A.Greenbaum, W.Luo,A.F.Coskun, A.Ozcan, Giga-Pixel Lensfree Holographic Microscopy and Tomography Using Color Image Sensors, PloS one, 7(9), e45044 (2012) CrossRef T. Shimobaba et al., Computational wave optics library for C++: CWO++ library, Comput. Phys. Commun. 183, 1124-1138 (2012) CrossRef

Evaporation-induced flows inside a confined droplet of diluted saline solution.

Flow patterns inside a droplet of diluted aqueous NaCl solution confined by two flat substrates under natural evaporation were investigated both experimentally and numerically. We focused on natural convection-driven flows inside confined droplets at high Rayleigh numbers (i.e., the ratio of buoyancy to diffusion, Ra), where the convection of solutes is strongly dominant, compared to diffusion. The evaporated water at the free surface of the droplet builds up a concentration gradient inside the solution, which induces the Rayleigh convection flow. Three-dimensional trajectories of tracer particles in the droplet were tracked, and axisymmetric flow motions induced by the Rayleigh convection were experimentally measured by using a digital in-line holographic microscopy technique. In addition, the effects of the confined droplet's aspect ratio and the liquid's molar concentration on the evaporation-induced flows were investigated. The convection velocity is found to be increased as molar concentration increases, because Rayleigh convection becomes significant at high the molar concentration is high (i.e. high Ra). Our numerical simulation based on the Boussinesq approximation fairly well predicted the velocity profiles of evaporating confined droplets at low concentrations. Consequently, evaporation kinetics inside the confined droplets can be controlled with varying droplet's aspect ratio and the liquid's molar concentration, which provides helpful information for the design of biochemical microplating with limited resources and for tuning self-assembly micro/nanoparticle clusters.

Tomography by point source digital holographic microscopy.

We propose a tomographic method for point source inline holographic microscopy. By recording a set of holograms at different illumination angles, shadowing effects are eliminated resulting in three-dimensional images with the same precision at the micrometer-scale in all directions. The advantage of our tomographic approach is that it works for both absorbing and phase objects, regardless of the change of refractive index at interfaces. We develop the method with computer simulations and demonstrate its strength by presenting experimental results for micrometer-sized polystyrene beads and a cotton fiber.

Inertial migration of spherical elastic phytoplankton in pipe flow

Inertial migration of spherical elastic phytoplankton in a microscale pipe was investigated in Reynolds number range of 10–100. Three-dimensional position of migrating cells was obtained using digital in-line holographic microscopy. Characteristics of inertial migration were studied by analyzing the spatial distribution of cells. As Reynolds number increases, Segre–Silberberg annulus clearly appeared at the same measurement location. The effect of elastic shell compliance was experimentally investigated by comparing the probability density function of normal and hardened cells. As Re increases, the equilibrium positions of both normal and hardened cells drift toward the tube wall as the result of balanced lifts. The degree of inertial-migration development of elastic cells is slower than that of hardened cells. These results will be useful for better understanding of dynamic behaviors of phytoplankton, especially inertial migration in pipe flow.

Partially coherent digital in-line holographic microscopy in characterization of a microscopic target.

Digital holographic microscopy enables the capture of large three-dimensional volumes. Instead of using a laser as an illumination source, partially coherent alternatives can be used, such as light-emitting diodes, which produce parasitic reflection and speckle-free holograms. Captured high-contrast holograms are suitable for the characterization of micrometer-sized particles. As the reconstructed phase is not usable in the case of multiple overlapping objects, depth extraction can be conducted on a reconstructed intensity. This work introduces a novel depth extraction algorithm that takes into consideration the possible locations of multiple objects at various depths in the imaged volume. The focus metric, the Tamura coefficient, is applied for each pixel in the reconstructed amplitude throughout the volume. This work also introduces an optimized version of the algorithm, which is run in two stages. During the first stage, coarse positions of the objects are extracted by applying the Tamura coefficient to nonoverlapping window blocks of intensity reconstructions. The second stage produces high-precision characterizations of the objects by calculating the Tamura coefficient with overlapping window blocks around axial positions extracted in the first stage. Experimental results with real-world microscopic objects show the effectiveness of the proposed method.

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 µm x 200 µm x 200 µm using LED illumination, to 5 mm x 5 mm x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 µmx 200 µmx 200 µm using LED illumination, to 5 mmx 5 mmx 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Ultrafast electron and material dynamics following femtosecond filamentation induced excitation of transparent solids

We examine the spatiotemporal dynamics of filamentation and subsequent material changes in two transparent media, fused silica and poly(methyl methacrylate) (PMMA), using inline holographic microscopy. We are able to dynamically observe the nonlinear propagation of femtosecond laser pulses and the consequent evolution of the electronic excitatio n and trapping inside the bulk of both materials. In the case of fused silica we reveal the physical conditions for the formation of nanogratings, measuring excited electron densities well below the critical density while for PMMA we show that excited electrons with densities exceeding 1018 cm−3, exhibit complex trapping dynamics in a 200 fs time scale. The clear demonstration of ultrafast sub-ps photochemical processes that take place during the irradiation of PMMA with femtosecond pulses will have a strong impact on the laser microprocessing of polymers and nanosurgery applications of bio-related materials.

In-line digital holographic microscopy using a consumer scanner.

We demonstrate an in-line digital holographic microscopy using a consumer scanner. The consumer scanner can scan an image with 4,800 dpi. The pixel pitch is approximately 5.29 μm. The system using a consumer scanner has a simple structure, compared with synthetic aperture digital holography using a camera mounted on a two-dimensional moving stage. In this demonstration, we captured an in-line hologram with 23, 602 × 18, 023 pixels (≈0.43 gigapixels). The physical size of the scanned hologram is approximately 124 mm × 95 mm. In addition, to accelerate the reconstruction time of the gigapixel hologram and decrease the amount of memory for the reconstruction, we applied the band-limited double-step Fresnel diffraction to the reconstruction.

One-shot dual-wavelength in-line digital holographic microscopy

In this work, we propose a dual-wavelength in-line digital holographic microscopy (DHM) configuration in order to eliminate the conjugate image and reach the maximum resolution of CCD. By using this configuration, two holograms are acquired in one shot. Our method is based not only on the recordings of two holograms at slightly different planes, but also on the diffraction patterns formed with two wavelengths. With this experimental setup, we are able to analyze fast dynamic processes at the microscopic scale in real time. Theoretical evaluation, computer simulations and experimental results validate our proposal. The experimental results are obtained using a phase-amplitude object and compared with those calculated from the well-established phase-shifting interferometry technique. As far as we know, in-axis configuration with a single exposure has not been used in DHM, as we present in this paper.

Calibration of a digital in-line holographic microscopy system: depth of focus and bioprocess analysis

Digital in-line holographic microscopy (DIHM) allows access to both intensity and phase information with conventional microscopic lateral resolutions. Such imaging techniques can, however, be used to increase the depth of focus compared to conventional compound microscopes. We present a simple DIHM capable of imaging weakly scattering 10 μm diameter microspheres as well as Hs578T cells over a depth of 1 mm; i.e., we demonstrate an increase by a factor of 100 over the depth of focus of a conventional microscope.

Application of inverse Abel techniques in in-line holographic microscopy

In this paper, we compare two well-established techniques, namely the BASEX and the Fourier–Hankel, as regards their efficiency of retrieving the three-dimensional distribution of cylindrically symmetric objects in the presence of noise. This situation is commonly encountered in pump–probe experiments where the refractive index profile of elongated structures, such as plasma strings or transient refractive index changes, is under study. We performed numerical experiments for a variety of objects, with respect to the spatial distribution and size, and for various statistical distributions and levels of noise. In this area of application, the BASEX can surpass the Fourier–Hankel technique in respect of reconstruction quality, spatial resolution and robustness to noise especially after proper tuning of the basis set functions width.

Development and deployment of a point‐source digital inline holographic microscope for the study of plankton and particles to a depth of 6000 m

A point‐source digital inline holographic microscope (DIHM) was designed for the imaging of particles from 50 µm to several millimeters in size. The DIHM operates autonomously without connection to external recording devices or power sources and delivers 4.2 megapixel images at a rate of approximately 7 images s−1, each image representing a snapshot of 1.8 mL seawater. Reliance on largely off‐the‐shelf components, and simplification in its construction makes this camera system adaptable to various particle size ranges and environments, and easy‐to‐operate for nonexpert users. The DIHM produced sharp images of protists with skeletal structures (e.g., acantharians, tintinnids, dinoflagellates), mesoplankton (e.g., copepods, appendicularians, medusae), Trichodesmium colonies and marine snow particles while descending in the water column at 1 m s−1, a typical velocity for deployments of tethered instruments and samplers from oceanographic vessels. To validate the usefulness of the new instrument in an oceanographic context, data are presented of the surface distribution of Trichodesmium spp., and of the vertical frequency distribution of fecal pellets and other particles in the deep sea. The point‐source DIHM has the potential to become a standard instrument on the CTD rosette (i.e., on the basic oceanographic instrument and sampling frame) in the future providing a permanent archival record of the water column that can be mined for specific target particles in the future.

Superresolved common-path phase-shifting digital inline holographic microscopy using a spatial light modulator

Common-path phase-shifting lensless holographic microscopy has been recently proposed as a novel approach capable of high numerical aperture imaging in a lensless digital inline holographic microscopy layout [Opt. Lett.35, 3919 (2010)]. Here we present proof-of-concept validation for improving the resolution limit imposed by diffraction in such a setup. This is accomplished by shifting the phase lens displayed at the spatial light modulator, which moves the illumination point source to different off-axis positions. For each off-axis position, a set of inline phase-shifted holograms are recorded by the digital sensor and stored at the computer's memory for later digital postprocessing. As a consequence, each recording allows the recovery of different spatial frequency content of the object's diffracted wavefront meaning a superresolved image of the input object. Experimental results are reported validating the proposed method.

Digital holographic microscope with low-frequency attenuation filter for position measurement of a nanoparticle

We propose a new method for three-dimensional (3D) position measurement of nanoparticles using an in-line digital holographic microscope. The method improves the signal-to-noise ratio of the amplitude of the interference fringes to achieve higher accuracy in the position measurement by increasing weak scattered light from a nanoparticle relative to the reference light by using a low spatial frequency attenuation filter. We demonstrated the improvements of signal-to-noise ratio of the optical system and contrast of the interference fringes, allowing the 3D positions of nanoparticles to be determined more precisely.

Phase shifting technique for extended inline holographic microscopy with a pinhole array

Digital inline holographic microscopy using a pinhole for sample illumination allows lensless imaging. To overcome restrictions of the sample size and density in the setup additional reference waves are generated by extending the single pinhole to a regular 2D pinhole array illumination. A technique is presented that uses phase shifting between the pinhole waves. Multiple foci with stable phase differences and a phase error (rms) of 0.027 rad generate pinhole waves which illuminate an undiluted, dense blood smear sample. Amplitude and phase images of the blood sample were successfully reconstructed.

Holographic microrefractometer

In-line holographic microscopy of micrometer-scale colloidal spheres yields heterodyne scattering patterns that may be interpreted with Lorenz-Mie theory to obtain precise time-resolved information on the refractive index of the suspending medium. We demonstrate this approach to spatially resolved refractometry with measurements on calibrated refractive index standards and use it to monitor chemical concentration in a microfluidic channel. Using commercially available colloidal spheres as probe particles and a standard video camera for detection yields values for the fluid’s refractive index at the position of each probe particle in each holographic snapshot with a demonstrated resolution of 2×10−3 refractive index units (RIU) and a potential resolution surpassing 10−4 RIU. The combination of spatial resolution, temporal resolution, multi-point in situ access, and technical simplicity recommends this technique for cost-effective lab-on-a-chip applications.

Inertial migration of erythrocytes in low-viscosity and high-shear rate microtube flows: Application of simple digital in-line holographic microscopy

A simple digital in-line holographic microscopy technique is applied to investigate the inertial migration of human erythrocytes in low-viscosity and high-shear rate microtube flows. The technique provides radial distribution statistics of the erythrocytes, as well as their orientation information in the tube flow. The radial distribution of human erythrocytes is found to be similar to that of spherical particles of 7μm in diameter. The results show that both the erythrocytes and the spherical particles are fully focused at similar radial positions as the shear-rate of the flow increases. The erythrocytes require a higher shear rate than the spherical particle to establish the fully focused state. As the radial migration of erythrocytes fully develops, more than half of the cells have symmetry axes orthogonal to the main stream and wall-normal directions. This orientation demonstrates similar hydrodynamic effects of disk-shaped biconcave erythrocytes to those of the spherical particles, and consequently results in similar radial equilibrium positions.

Simultaneous holographic, ellipsometric, and optical imaging of pitting corrosion on SS 316LVM stainless steel

Digital in-line holographic microscopy, a three-dimensional imaging technique, is incorporated for simultaneous usage with the previously established surface sensitive optical imaging techniques, ellipsomicroscopy and microscopy. This addition allows volume reconstructions of the solution in the vicinity of the working electrode with microscopic resolution, and particle tracking. Holography is also sensitive to local variations in the solution concentrations; estimations of the index of refraction can be made using the interference patterns alone. These three optical techniques complement each other and simultaneously provide information about changes in the oxide layer, pit developments, and local solution changes during electrochemical testing of stainless steel.

Hologram reconstruction corrected for measurements through layers with different refractive indices in digital in-line holographic microscopy

Digital in-line holographic microscopy (DIHM) using point sources has been shown to be a versatile technique, especially for three-dimensional tracking of particles or microorganisms. However, the spherical source wave is altered when measurements are performed through layers with different refractive indices, such as water cuvettes. The situations where a layer of medium with a refractive index different than that of the predominant surrounding propagation medium (usually air) is situated behind or in front of the plane to be reconstructed are analyzed in detail, and a general approach for reconstruction under such circumstances is developed. The proposed refractive index correction is tested experimentally and compared to conventional reconstruction algorithms. Using 3D traces of swimming algal spores, the influence on the velocity calculation is also shown.