In-line Holograms Research Articles

Multipoint-projection x-ray microscopy

Point projection is a mature geometry of x-ray imaging that is implemented in scientific and industrial applications. Objects to be imaged are placed near a microscopic x-ray source, and the magnification is accomplished by x-ray propagation towards a distant detector. The source size is a trade-off between the signal level and the spatial resolution. In this work, we demonstrate multipoint-projection x-ray imaging realized with an x-ray tube and compound structured microcapillary optics that generates nearly one thousand submicrometer secondary x-ray sources. The generated microbeams are multiplexed at the object. Demultiplexing of the transmitted beams, magnification, and phase contrast are achieved by the free-space propagation. A massive improvement in the signal-to-noise ratio, relative to a single secondary source, was achieved without a loss in spatial resolution. Hence, x-ray projections of highly absorbing samples at submicrometer spatial resolution could be recorded with only a few photons per detector pixel. For weakly absorbing samples, the multipoint-projection method enabled us to record, in a parallel way, replicated in-line x-ray holograms with incoherent radiation from an x-ray tube. Our method may enrich the panel of x-ray nanoscale imaging techniques and may be adopted for on-chip x-ray photonic devices.

Compact lensless subpixel resolution large field of view microscope.

We report on a method to increase the spatial resolution in a compact lensless microscope. A compact side illumination is fabricated to illuminate the sample with a collimated beam by diffraction from a volume phase grating. The wavelength of a semi-conductor laser source (vertical-cavity surface-emitting laser) is tuned with the injection current to alter the illumination direction by wavelength selective diffraction from the volume phase grating. The angle tuning is such that several subpixel shifted digital inline holograms are obtained. The stack of holograms is then processed in a pixel super-resolution reconstruction algorithm. The amplitude of the sample is reconstructed with subpixel resolution over a large field of view (FOV). The technique is demonstrated on a 1951 USAF test target. A resolution of ∼2.76 μm, over a FOV of ∼28 mm2, is demonstrated for a device of <2 cm height. The original pixel size was 5.2 μm demonstrating the subpixel resolution.

Regularized reconstruction of absorbing and phase objects from a single in-line hologram, application to fluid mechanics and micro-biology.

Reconstruction of phase objects is a central problem in digital holography, whose various applications include microscopy, biomedical imaging, and fluid mechanics. Starting from a single in-line hologram, there is no direct way to recover the phase of the diffracted wave in the hologram plane. The reconstruction of absorbing and phase objects therefore requires the inversion of the non-linear hologram formation model. We propose a regularized reconstruction method that includes several physically-grounded constraints such as bounds on transmittance values, maximum/minimum phase, spatial smoothness or the absence of any object in parts of the field of view. To solve the non-convex and non-smooth optimization problem induced by our modeling, a variable splitting strategy is applied and the closed-form solution of the sub-problem (the so-called proximal operator) is derived. The resulting algorithm is efficient and is shown to lead to quantitative phase estimation on reconstructions of accurate simulations of in-line holograms based on the Mie theory. As our approach is adaptable to several in-line digital holography configurations, we present and discuss the promising results of reconstructions from experimental in-line holograms obtained in two different applications: the tracking of an evaporating droplet (size ∼ 100μm) and the microscopic imaging of bacteria (size ∼ 1μm).

Single-shot dual-wavelength in-line and off-axis hybrid digital holography

We propose an in-line and off-axis hybrid holographic real-time imaging technique. The in-line and off-axis digital holograms are generated simultaneously by two lasers with different wavelengths, and they are recorded using a color camera with a single shot. The reconstruction is carried using an iterative algorithm in which the initial input is designed to include the intensity of the in-line hologram and the approximate phase distributions obtained from the off-axis hologram. In this way, the complex field in the object plane and the output by the iterative procedure can produce higher quality amplitude and phase images compared to traditional iterative phase retrieval. The performance of the technique has been demonstrated by acquiring the amplitude and phase images of a green lacewing's wing and a living moon jellyfish.

Towards Real-Time Holographic Three-Dimensional Imaging with Machine Learning

Real-time visualization of cellular and molecular dynamics is critical in achieving a flexible, adaptive imaging platform capable of automatically identifying regions of interest and proactively modifying acquisition parameters In holographic microscopy, the time-intensive steps of sample and / or stage manipulation are replaced by the computational time required to accomplish the volume reconstruction. A key challenge in real time visualisation of time-lapse holographic microscopy arises when long-term studies are underway, such as the study of biofilm development, which may take place over hours or days. In this case, computing 3-D positions and reconstructions is a CPU-intensive process. To address this, efforts are underway to apply GP-GPU based approaches that allow for parallelization of the process. In the absence of GPU platform, an attractive alternative is to consider the use of artificial neural networks(ANN) to bypass the repeated matrix multiplication associated with each reconstructing distance. Here, we demonstrate how supervised ANN can be used by determining the algorithms necessary to estimate the z-position of particles from a single 2-dimensional in-line hologram. With only a small cost of one-time training, the newly-found algorithm would bypass the iterative calculations required to search through the dataset to identify the final position of objects in each newly acquired hologram. This in turn will help realise real-time visualisation of holographic imaging and achieving a adaptive imaging system where executive decisions on the speed and volume of imaging interest can be made during acquisition.

Improved accuracy in Wigner–Ville distribution-based sizing of rod-shaped particle using flip and replication technique

A method for improving accuracy in Wigner–Ville distribution (WVD)-based particle size measurements from inline holograms using flip and replication technique (FRT) is proposed. The FRT extends the length of hologram signals being analyzed, yielding better spatial-frequency resolution of the WVD output. Experimental results verify reduction in measurement error as the length of the hologram signals increases. The proposed method is suitable for particle sizing from holograms recorded using small-sized image sensors.

合成孔径同轴数字全息图的相位恢复再现

合成孔径同轴数字全息图的相位恢复再现

Multilayer pixel super-resolution lensless in-line holographic microscope with random sample movement

We report a multilayer lensless in-line holographic microscope (LIHM) with improved imaging resolution by using the pixel super-resolution technique and random sample movement. In our imaging system, a laser beam illuminated the sample and a CMOS imaging sensor located behind the sample recorded the in-line hologram for image reconstruction. During the imaging process, the sample was moved by hand randomly and the in-line holograms were acquired sequentially. Then the sample image was reconstructed from an enhanced-resolution hologram obtained from multiple low-resolution in-line holograms by applying the pixel super-resolution (PSR) technique. We studied the resolution enhancement effects by using the U.S. Air Force (USAF) target as the sample in numerical simulation and experiment. We also showed that multilayer pixel super-resolution images can be obtained by imaging a triple-layer sample made with the filamentous algae on the middle layer and microspheres with diameter of 2 μm on the top and bottom layers. Our pixel super-resolution LIHM provides a compact and low-cost solution for microscopic imaging and is promising for many biomedical applications.

Resolution enhancement method for lensless in-line holographic microscope with spatially-extended light source.

We propose a resolution enhancement method for lensless in-line holographic microscope (LIHM) with spatially-extended light source, where the resolution is normally deteriorated by the insufficient spatial coherence of the illumination. In our LIHM setup, a light-emitting diode (LED), which was a spatially-extended light source, directly illuminated the sample, and the in-line hologram were recorded by a CMOS imaging sensor located behind the sample. In our holographic reconstruction process, the in-line hologram was first deconvoled with a properly resized image of the LED illumination area, and then back-propagated with scalar diffraction formula to reconstruct the sample image. We studied the hologram forming process and showed that the additional deconvolution process besides normal scalar diffraction reconstruction in LIHM can effectively enhance the imaging resolution. The resolution enhancements capability was calibrated by numerical simulations and imaging experiments with the U.S. air force target as the sample. We also used our LIHM to image the wing of a green lacewing to further demonstrate the capability of our methods for practical imaging applications. Our methods provide a way for LIHM to achieve satisfactory resolution with less stringent requirement for spatial coherence of the source and could reduce the cost for compact imaging system.

Enhanced resolution for amplitude object in lensless inline holographic microscope with grating illumination

In a compact digital lensless inline holographic microscope (LIHM), where the sample-to-sensor distance is short, the imaging resolution is often limited by sensor pixel size instead of the system numerical aperture. We propose to solve this problem by applying data interpolation with an iterative holographic reconstruction method while using grating illumination in the LIHM system. In the system setup, the Talbot self-image of a Ronchi grating was used to illuminate the sample, and the inline hologram was recorded by a CMOS imaging sensor located behind the sample. The hologram was then upsampled by data interpolation before the reconstruction process. In the iterative holographic reconstruction, the sample support was defined by the bright areas of the grating illumination pattern and was used as constraint. A wide-field image can also be obtained by shifting the grating illumination pattern. Furthermore, we assumed that the sample was amplitude object, i.e., no obvious phase change was caused by the sample, which provided additional constraint to refine the interpolated data values. Besides improved resolution, the iterative holographic reconstruction also helped to reduce the twin-image background. We demonstrated the effectiveness of our method with simulation and imaging experiment by using the USAF target and polystyrene microspheres with 1 μm diameter as the sample.

Imaging the potential distribution of individual charged impurities on graphene by low-energy electron holography

While imaging individual atoms can routinely be achieved in high resolution transmission electron microscopy, visualizing the potential distribution of individually charged adsorbates leading to a phase shift of the probing electron wave is still a challenging task. Low-energy electrons (30 – 250 eV) are sensitive to localized potential gradients. We employed low-energy electron holography to acquire in-line holograms of individual charged impurities on free-standing graphene. By applying an iterative phase retrieval reconstruction routine we recover the potential distribution of the localized charged impurities present on free-standing graphene.

Experimental extractions of particle position from inline holograms using single coefficient of Wigner–Ville analysis

Extractions of particle positions from inline holograms using a single coefficient of Wigner–Ville distribution (WVD) are experimentally verified. WVD analysis of holograms gives local variation of fringe frequency. Regardless of an axial position of particles, one of the WVD coefficients has the unique characteristics of having the lowest amplitude and being located on a line with a slope inversely proportional to the particle position. Experimental results obtained using two image sensors with different resolutions verify the feasibility of the present method.

Compact lensless off-axis transmission digital holographic microscope.

Current compact lensless holographic microscopes are based on either multiple angle in-line holograms, multiple wavelength illumination or a combination thereof. Complex computational algorithms are necessary to retrieve the phase image which slows down the visualization of the image. Here we propose a simple compact lensless transmission holographic microscope with an off-axis configuration which simplifies considerably the computational processing to visualize the phase images and opens the possibility of real time phase imaging using off the shelf smart phone processors and less than $3 worth of optics and detectors, suitable for broad educational dissemination. This is achieved using a side illumination and analog hologram gratings to shape the reference and signal illumination beams from one light source. We demonstrate experimentally imaging of cells with a field of view (FOV) of ~12mm2, and a resolution of ~3.9μm.

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.

Quasi-three-dimensional particle imaging with digital holography.

In this work, approximate three-dimensional structures of microparticles are generated with digital holography using an automated focus method. This is done by stacking a collection of silhouette-like images of a particle reconstructed from a single in-line hologram. The method enables estimation of the particle size in the longitudinal and transverse dimensions. Using the discrete dipole approximation, the method is tested computationally by simulating holograms for a variety of particles and attempting to reconstruct the known three-dimensional structure. It is found that poor longitudinal resolution strongly perturbs the reconstructed structure, yet the method does provide an approximate sense for the structure's longitudinal dimension. The method is then applied to laboratory measurements of holograms of single microparticles and their scattering patterns.

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.

Effects of particle locations on reconstructed particle images in digital holography.

The intensity and phase reconstructed from digital in-line holograms by the convolution approach are analyzed. Distortions of particle images depending on their position in the plane transverse to the optical axis are identified. For this purpose, the object fields of numerically simulated particle holograms as well as of experimental data are reconstructed. The results of three-dimensional correlations of numerical and experimental data are superior when the numerically generated reference volumes are adapted to the transverse locations of the particle. Thus, proof is given that the characteristics of a particle image change distinctly with the transverse position of the particle and that the numerical model successfully simulates these changes. Hence, this knowledge can be integrated in future particle position detection algorithms.

Holographic tracking and sizing of optically trapped microprobes in diamond anvil cells.

We demonstrate that Digital Holographic Microscopy can be used for accurate 3D tracking and sizing of a colloidal probe trapped in a diamond anvil cell (DAC). Polystyrene beads were optically trapped in water up to Gigapascal pressures while simultaneously recording in-line holograms at 1 KHz frame rate. Using Lorenz-Mie scattering theory to fit interference patterns, we detected a 10% shrinking in the bead's radius due to the high applied pressure. Accurate bead sizing is crucial for obtaining reliable viscosity measurements and provides a convenient optical tool for the determination of the bulk modulus of probe material. Our technique may provide a new method for pressure measurements inside a DAC.

Bayesian approach to analyzing holograms of colloidal particles.

We demonstrate a Bayesian approach to tracking and characterizing colloidal particles from in-line digital holograms. We model the formation of the hologram using Lorenz-Mie theory. We then use a tempered Markov-chain Monte Carlo method to sample the posterior probability distributions of the model parameters: particle position, size, and refractive index. Compared to least-squares fitting, our approach allows us to more easily incorporate prior information about the parameters and to obtain more accurate uncertainties, which are critical for both particle tracking and characterization experiments. Our approach also eliminates the need to supply accurate initial guesses for the parameters, so it requires little tuning.

Digital in-line holography for the characterization of flowing particles in astigmatic optical systems

An extended Generalized Fresnel Transform (GFT) is proposed to account for the astigmatism introduced by optical elements described, in the paraxial approximation, with a ray transfer matrix analysis. Generalized impulse response and generalized Fresnel transfer function propagators as well as sampling conditions are derived to properly implement this transformation. As a test case, the near-field diffraction patterns and in-line holograms produced by droplets flowing in a tube with cylindrical interfaces have been simulated. A best fitting approach is introduced to retrieve, from the propagated holograms, the 3D position and size of the droplets. Several hologram focusing indicators based on the analysis of droplets focus region are also proposed to further improve the estimation of the droplets position along the optical axis. Numerical simulations and experimental results confirm the applicability and accuracy of the proposed methods.