In-line Holographic Microscopy Research Articles

3D image distortion problem in digital in-line holographic microscopy and its effective solution.

Digital in-line holographic microscopy (DIHM) has attracted attention because of its simple but powerful three-dimensional (3D) imaging capability. To improve the spatial resolution, 3D image reconstruction algorithms use numerical magnification, which generates distortions in the generated images. We propose a method to overcome this problem by using the simple relation between the object and image positions in 3D space. Several holograms were taken while translating a resolution target at different axial positions by a motorized stage. We demonstrated the effectiveness of our method by reconstructing the 3D positions of 3-μm-diameter polymer beads on a tilted slide glass from a single measured hologram.

Label-Free Sensing and Classification of Old Stored Blood.

Transfusion is crucial in surgical operation and anemia treatment. However, several hemorheological properties of blood are adversely altered during blood storage. After transfusion, these adverse alterations are related with decrease of oxygen and ion transport in blood circulation, which increase the mortality of transfused patients. Therefore, accurate sensing of whether a blood supply is still viable for transfusion or not is extremely important. In this study, a H-shaped microfluidic device and digital in-line holographic microscopy were employed to measure temporal variations of blood viscosity and the optical focusing property of erythrocytes during blood storage. Stored rat blood samples separately preserved in citrate phosphate dextrose adenine-1 (CPDA-1) and ethylenediaminetetraacetic acid (EDTA) underwent considerable changes in their biophysical parameters after 2weeks. Compared with EDTA, CPDA-1 preserves the hemorheological properties of stored blood more effectively. We propose new criteria for depository period of stored blood and indexes, such as viscosity and focal length of erythrocytes, to determine its appropriateness for transfusion. These criteria and indexes can be effectively used for high-throughput prescreening to reduce the risk of transfusion of aged blood or diagnose hematological diseases.

Enhanced resolution in lensless in-line holographic microscope by data interpolation and iterative reconstruction

In a compact lensless in-line holographic microscope, the imaging resolution is generally limited by the sensor pixel size because of the short sample-to-sensor distance. To overcome this problem, we propose to use data interpolation based on iteration with only two intensity measurements to enhance the resolution in holographic reconstruction. We did numerical simulations using the U.S. air force target as the sample and showed that data interpolation in the acquired in-line hologram can be used to enhance the reconstruction resolution. The imaging resolution and contrast can be further improved by combining data interpolation with iterative holographic reconstruction using only two hologram measurements acquired by slightly changing the sample-to-sensor distance while recording the in-line holograms. The two in-line hologram intensity measurements were used as a priori constraint in the iteration process according to the Gerchberg–Saxton algorithm for phase retrieval. The iterative reconstruction results showed that the iteration between the sample plane and the sensor planes can refine the interpolated data and thus further improve the resolution as well as the imaging contrast. Besides numerical simulation, we also experimentally demonstrated the enhancement of imaging resolution and contrast by imaging the U.S. air force target and a microscope slide of filamentous algae.

UmUTracker: A versatile MATLAB program for automated particle tracking of 2D light microscopy or 3D digital holography data

We present a versatile and fast MATLAB program (UmUTracker) that automatically detects and tracks particles by analyzing video sequences acquired by either light microscopy or digital in-line holographic microscopy. Our program detects the 2D lateral positions of particles with an algorithm based on the isosceles triangle transform, and reconstructs their 3D axial positions by a fast implementation of the Rayleigh–Sommerfeld model using a radial intensity profile. To validate the accuracy and performance of our program, we first track the 2D position of polystyrene particles using bright field and digital holographic microscopy. Second, we determine the 3D particle position by analyzing synthetic and experimentally acquired holograms. Finally, to highlight the full program features, we profile the microfluidic flow in a 100μm high flow chamber. This result agrees with computational fluid dynamic simulations. On a regular desktop computer UmUTracker can detect, analyze, and track multiple particles at 5 frames per second for a template size of 201 ×201 in a 1024 × 1024 image. To enhance usability and to make it easy to implement new functions we used object-oriented programming. UmUTracker is suitable for studies related to: particle dynamics, cell localization, colloids and microfluidic flow measurement. Program summaryProgram title : UmUTrackerProgram Files doi : http://dx.doi.org/10.17632/fkprs4s6xp.1Licensing provisions : Creative Commons by 4.0 (CC by 4.0)Programming language : MATLABNature of problem: 3D multi-particle tracking is a common technique in physics, chemistry and biology. However, in terms of accuracy, reliable particle tracking is a challenging task since results depend on sample illumination, particle overlap, motion blur and noise from recording sensors. Additionally, the computational performance is also an issue if, for example, a computationally expensive process is executed, such as axial particle position reconstruction from digital holographic microscopy data. Versatile robust tracking programs handling these concerns and providing a powerful post-processing option are significantly limited.Solution method: UmUTracker is a multi-functional tool to extract particle positions from long video sequences acquired with either light microscopy or digital holographic microscopy. The program provides an easy-to-use graphical user interface (GUI) for both tracking and post-processing that does not require any programming skills to analyze data from particle tracking experiments. UmUTracker first conduct automatic 2D particle detection even under noisy conditions using a novel circle detector based on the isosceles triangle sampling technique with a multi-scale strategy. To reduce the computational load for 3D tracking, it uses an efficient implementation of the Rayleigh–Sommerfeld light propagation model. To analyze and visualize the data, an efficient data analysis step, which can for example show 4D flow visualization using 3D trajectories, is included. Additionally, UmUTracker is easy to modify with user-customized modules due to the object-oriented programming styleAdditional comments: Program obtainable from https://sourceforge.net/projects/umutracker/

Aliasing, coherence, and resolution in a lensless holographic microscope.

We have shown that the maximum achievable resolution of an in-line lensless holographic microscope is limited by aliasing and, for collimated illumination, cannot exceed the camera pixel size. This limit can be achieved only when the optimal conditions on the spatial and temporal coherence state of the illumination are satisfied. The expressions defining the configuration, delivering maximum resolution with given spatial and temporal coherence of the illumination, are obtained. The validity of these conditions is confirmed experimentally.

High efficiency and flexible working distance digital in-line holographic microscopy based on Fresnel zone plate

Traditional digital in-line holography suffers from twin-image noise problems and extremely short working distances between the object and light source. Here, we propose lensless Fourier transform digital in-line holographic microscopy based on a single optical element. A Fresnel zone plate is used to split the incident light into two parts: one is scattered along the original direction, the other is gathered at a focal point and the sample is put behind the focus. The interference fringe pattern, formed by the two beams, is recorded digitally by a CCD camera. A novel reconstruction algorithm is proposed to present the object image. The proof-of-concept experiments demonstrate that the proposed technique can eliminate the twin-image noise problem, improving the image contrast with high efficiency, and increasing the flexibility of the working distance. Furthermore, a wide field of view and no contact make it a promising tool for the study of materials science, biology and microelectronics.

Focusing and alignment of erythrocytes in a viscoelastic medium

Viscoelastic fluid flow-induced cross-streamline migration has recently received considerable attention because this process provides simple focusing and alignment over a wide range of flow rates. The lateral migration of particles depends on the channel geometry and physicochemical properties of particles. In this study, digital in-line holographic microscopy (DIHM) is employed to investigate the lateral migration of human erythrocytes induced by viscoelastic fluid flow in a rectangular microchannel. DIHM provides 3D spatial distributions of particles and information on particle orientation in the microchannel. The elastic forces generated in the pressure-driven flows of a viscoelastic fluid push suspended particles away from the walls and enforce erythrocytes to have a fixed orientation. Blood cell deformability influences the lateral focusing and fixed orientation in the microchannel. Different from rigid spheres and hardened erythrocytes, deformable normal erythrocytes disperse from the channel center plane, as the flow rate increases. Furthermore, normal erythrocytes have a higher angle of inclination than hardened erythrocytes in the region near the side-walls of the channel. These results may guide the label-free diagnosis of hematological diseases caused by abnormal erythrocyte deformability.

Sparsity-based multi-height phase recovery in holographic microscopy.

High-resolution imaging of densely connected samples such as pathology slides using digital in-line holographic microscopy requires the acquisition of several holograms, e.g., at >6–8 different sample-to-sensor distances, to achieve robust phase recovery and coherent imaging of specimen. Reducing the number of these holographic measurements would normally result in reconstruction artifacts and loss of image quality, which would be detrimental especially for biomedical and diagnostics-related applications. Inspired by the fact that most natural images are sparse in some domain, here we introduce a sparsity-based phase reconstruction technique implemented in wavelet domain to achieve at least 2-fold reduction in the number of holographic measurements for coherent imaging of densely connected samples with minimal impact on the reconstructed image quality, quantified using a structural similarity index. We demonstrated the success of this approach by imaging Papanicolaou smears and breast cancer tissue slides over a large field-of-view of ~20 mm2 using 2 in-line holograms that are acquired at different sample-to-sensor distances and processed using sparsity-based multi-height phase recovery. This new phase recovery approach that makes use of sparsity can also be extended to other coherent imaging schemes, involving e.g., multiple illumination angles or wavelengths to increase the throughput and speed of coherent imaging.

Tracking E. coli runs and tumbles with scattering solutions and digital holographic microscopy.

We use in-line digital holographic microscopy to image freely swimming E. coli. We show that fitting a light scattering model to E. coli holograms can yield quantitative information about the bacterium's body rotation and tumbles, offering a precise way to track fine details of bacterial motility. We are able to extract the cell's three-dimensional (3D) position and orientation and recover behavior such as body angle rotation during runs, tumbles, and pole reversal. Our technique is label-free and capable of frame rates limited only by the camera.

Reversible Adsorption Kinetics of Near Surface Dimer Colloids.

We investigate the effect of shape on reversible adsorption kinetics using colloidal polystyrene dimers near a solid glass surface as a model system. The interaction between colloid and wall is tuned using electrostatic, depletion, and gravity forces to produce a double-well potential. The dwell time in each of the potential wells is measured from long duration particle trajectories. The height of each monomer relative to the glass surface is measured to a resolution of <20 nm by in-line holographic microscopy. The measured transition probability distributions are used in kinetic equations to describe the flux of particles to and from the surface. The dimers are compared to independent isolated monomers to determine the effects of shape on adsorption equilibria and kinetics. To elucidate these differences, we consider both mass and surface coverage and two definitions of surface coverage. The results show that dimers with single coverage produce slower adsorption, lower surface coverage, and higher mass coverage in comparison to those of monomers, while dimers with double coverage adsorb faster and result in higher surface coverage.

Lensless in-line holographic microscope with Talbot grating illumination

We have developed a wide field-of-view lensless in-line holographic microscope (LIHM) capable of acquiring microscopic images with a compact design. In our imaging system, a Ronchi grating was illuminated by a collimated laser beam to generate a Talbot self-imaging grating illumination on the sample, and the in-line holograms were recorded by a CMOS imaging sensor behind the sample. An iterative reconstruction algorithm was then applied to reconstruct the sample image while eliminating the twin-image background that appears in traditional in-line holography. In the algorithm, the dark areas of the illumination grating were used as a known constraint to define the sample support that led to convergence of the iteration. The whole-sample image can be acquired by laterally shifting the grating. We demonstrated the performance of our iteration algorithm and imaging system by successfully acquiring images of polystyrene microspheres with 5 μm diameter and the wing of a green lacewing.

Single-shot and phase-shifting digital holographic microscopy using a 2-D grating.

We demonstrate digital holographic microscopy that, while being based on phase-shifting interferometry, is capable of single-shot measurements. A two-dimensional (2-D) diffraction grating placed in a Fourier plane of a standard in-line holographic phase microscope generates multiple copies of a sample image on a camera sensor. The identical image copies are spatially separated with different overall phase shifts according to the diffraction orders. The overall phase shifts are adjusted by controlling the lateral position of the grating. These phase shifts are then set to be multiples of π/2. Interferograms composed of four image copies combined with a parallel reference beam are acquired in a single shot. The interferograms are processed through a phase-shifting algorithm to produce a single complex image. By taking advantage of the higher sampling capacity of the in-line holography, we can increase the imaging information density by a factor of 3 without compromising the imaging acquisition speed.

Single-exposure super-resolved interferometric microscopy by RGB multiplexing in lensless configuration

Single-Exposure Super-Resolved Interferometric Microscopy (SESRIM) reports on a way to achieve one-dimensional (1-D) superresolved imaging in digital holographic microscopy (DHM) by a single illumination shot and digital recording. SESRIM provides color-coded angular multiplexing of the accessible sample׳s range of spatial frequencies and it allows their recording in a single CCD (color or monochrome) snapshot by adding 3 RGB coherent reference beams at the output plane. In this manuscript, we extend the applicability of SESRIM to the field of digital in-line holographic microscopy (DIHM), that is, working without lenses. As consequence of the in-line configuration, an additional restriction concerning the object field of view (FOV) must be imposed to the technique. Experimental results are reported for both a synthetic object (USAF resolution test target) and a biological sample (swine sperm sample) validating this new kind of superresolution imaging method named as lensless SESRIM (L-SESRIM).

Domain structure investigation of triglycine sulfate by digital in-line holographic microscopy

abstractThe domain structure is an important characteristic of ferroelectrics. One of the most intensively studied ferroelectrics is triglycine sulfate (TGS). The domains in TGS cannot be observed directly by usual optical methods. Many methods have been developed for the observation of the domain structure in TGS like: the etching method, the powder deposition method, the decoration method, the nematic liquid crystal method, the pyroelectric probe technique, the cleavage method, the scanning electron microscopy method, and others. Despite of all these efforts, the domain structure in TGS still cannot be visualized routinely. We suggest a new method for observation of the domains in TGS, in particular digital in-line holographic microscopy (DILHM). DILHM allows direct visualization of the domain structure, without any preliminary preparation of the samples. A big advantage of the new method is its ability to extract information about the change of the domain structure in depth from one digital hologram only.

Quantitative phase measurements of tendon collagen fibres.

Collagen is the main component of structural mammalian tissues. In tendons, collagen is arranged into fibrils with diameters ranging from 30 nm to 500 nm. These fibrils are further assembled into fibres several micrometers in diameter. Upon excessive thermal or mechanical stress, damage may occur in tendons at all levels of the structural hierarchy. At the fibril level, reported damage includes swelling and the appearance of discrete sites of plastic deformation that are best observed at the nanometer-scale using, for example, scanning electron microscopy. In this paper, digital in-line holographic microscopy is used for quantitative phase imaging to measure both the refractive index and diameter of collagen fibres in a water suspension in the native state, after thermal treatments, and after mechanical overload. Fibres extracted from tendons and subsequently exposed to 70°C for 5, 15, or 30 minutes show a significant decrease in refractive index and an increase in diameter. A significant increase in refractive index is also observed for fibres extracted from tendons that were subjected to five tensile overload cycles.

Holographic characterization of colloidal fractal aggregates.

In-line holographic microscopy images of micrometer-scale fractal aggregates can be interpreted with an effective-sphere model to obtain each aggregate's size and the population-averaged fractal dimension. We demonstrate this technique experimentally using model fractal clusters of polystyrene nanoparticles and fractal protein aggregates composed of bovine serum albumin and bovine pancreas insulin.

Co-design of an in-line holographic microscope with enhanced axial resolution: selective filtering digital holography.

Common-path digital in-line holography is considered as a valuable 3D diagnostic technique for a wide range of applications. This configuration is cost effective and relatively immune to variation in the experimental environment. Nevertheless, due to its common-path geometry, the signal-to-noise ratio of the acquired hologram is weak as most of the detector (i.e., CCD/CMOS sensor) dynamics are occupied by the reference field signal, whose energy is orders of magnitude higher than the field scattered by the imaged object. As it is intrinsically impossible to modify the ratio of energy of reference to the object field, we propose a co-design approach (optics/data processing) to tackle this issue. The reference to the object field ratio is adjusted by adding a 4-f device to a conventional in-line holographic setup, making it possible to reduce the weight of the reference field while keeping the object field almost constant. Theoretical analysis of the Crámer-Rao lower bounds of the corresponding imaging model illustrates the advantages of this approach. These lower bounds can be asymptotically reached using a parametric inverse problem reconstruction. This implementation results in a 60% gain in axial localization accuracy (for 100μm diameter spherical objects) compared to a classical in-line holography setup.

Estimation of parameters for evaluating subsurface microcracks in glass with in-line digital holographic microscopy.

In this work, a new method for imaging subsurface damage (SSD) is proposed, which is, to the best of our knowledge, the first application of the in-line digital holographic microscopy (IDHM) to the reconstruction of the subsurface damage in glass. By combination of the in-line arrangement and an objective lens to image the hologram on the CCD surface, the method is characterized by its high resolution in both the lateral and depth directions. Then the three-dimensional reconstruction of the microcracks within the glass was realized by numerically focusing en-face images at different depths, and the sizes of SSD along the transversal and depth directions were estimated. Based on the experimental results, the cracks can be divided into two categories: one is that the cracks begin from the surface of optical elements, the other is totally within the components. To indicate the propagation or development trajectory of the cracks and predict the magnitude of the laser-induced damage threshold, the relative intensity distributions of the light scattered by the cracks compared with the ones without cracks were also reconstructed. In this case all the required parameters for evaluating SSD are obtained with our IDHM system, so that the SSD produced in the manufacturing process can be reduced or removed more easily to optimize the performance of the optical component and extend its lifetime. These results provide the guidance for the optical system design of precision measurements.

Nano-level position resolution for particle tracking in digital in-line holographic microscopy.

Three-dimensional particle tracking in biological systems is a quickly growing field, many techniques have been developed providing tracking characters. Digital in-line holographic microscopy is a valuable technique for particle tracking. However, the speckle noise, out-of-focus signals and twin image influenced the particle tracking. Here an adaptive noise reduction method based on bidimensional ensemble empirical mode decomposition is introduced into digital in-line holographic microscopy. It can eliminate the speckle noise and background of the hologram adaptively. Combined with the three-dimensional deconvolution approach in the reconstruction, the particle feature would be identified effectively. Tracking the fixed beads on the cover-glass with piezoelectric stage through multiple holographic images demonstrate the tracking resolution, which approaches 2 nm in axial direction and 1 nm in transverse direction. This would facilitate the development and use in the biological area such as living cells and single-molecule approaches.

Lens-free inline holographic microscopy with numerical correction of layers with different refractive index.

Digital inline holographic microscopy is applied for lens-free imaging with high lateral resolution. Microfluidic chambers for the imaging of cells in water-like or native solutions, e.g., thick layers of glass and other materials with different refractive index, cause aberrations that limit the spatial resolution and change the magnification scale. In this Letter, a fast reconstruction technique considering parallel layer systems of different refractive indices is presented. In the experiments, properly scaled images of microbeads and red human blood cells with an optical resolution corresponding to a numerical aperture of about 0.62 were reconstructed.