Digital Holographic Microscopy Setup Research Articles

Nanoscale surface profile measurement using state space approach in digital holographic microscopy

Surface profile measurement at the nanoscale level has important applications in non-destructive testing and evaluation. The paper proposes a robust method for surface profilometry using digital holographic microscopy setup. The proposed method relies on extracting the phase map encoded in the hologram signal, which directly corresponds to the surface profile, using state space approach. The main advantage of the proposed method is high robustness against noise, which is demonstrated using numerical simulations. For designing the experimental system, a combination of Raspberry-Pi computer and camera module is used for hologram acquisition and processing, which is a step towards low cost imaging. For surface profile measurement, the experimental system has both temporal and spatial sensitivity parameters within 5 nanometers, which indicates robust design. In addition, experimental results show that the proposed method shows superior performance compared to the existing methods for measuring 100 nanometers surface profile features corresponding to different micro-structure regions of a standard calibration test target. Overall, the proposed method allows for single shot, non-contact and full-field measurement of nanoscale surface profile with additional benefits of noise robustness and low cost imaging design.

PyDHM: A Python library for applications in digital holographic microscopy.

pyDHM is an open-source Python library aimed at Digital Holographic Microscopy (DHM) applications. The pyDHM is a user-friendly library written in the robust programming language of Python that provides a set of numerical processing algorithms for reconstructing amplitude and phase images for a broad range of optical DHM configurations. The pyDHM implements phase-shifting approaches for in-line and slightly off-axis systems and enables phase compensation for telecentric and non-telecentric systems. In addition, pyDHM includes three propagation algorithms for numerical focusing complex amplitude distributions in DHM and digital holography (DH) setups. We have validated the library using numerical and experimental holograms.

Measurement of morphology thickness and refractive index in a melanoma A375 cell line using digital holographic microscopy.

Digital holographic microscopy (DHM) is a technique that has high potential for analyzing biological samples and has been successfully applied to the study of cells and cell lines providing information about important parameters such as refractive index, morphology, and dry mass, among others; it has also found applicability to study the effects of therapeutic treatments. Finding the size and shape of cells is important since they tend to change in the presence of some pathologies. In this research work, we obtain the morphology thickness and refractive index of the A375 melanoma cell line through a slight tilting of the cell in a DHM setup. Further, the development of a novel mathematical expression based on this tilt and in the optical phase difference is presented. We show images of melanoma cells with the refractive index information included, and their morphology thickness as rendered from the holographic phase maps recorded with DHM.

Erythrocyte volumetric measurements in imaging flow cytometry using simultaneous three-wavelength digital holographic microscopy.

We report a cross-talk free simultaneous three-wavelength digital holographic microscopy setup for spectroscopic imaging of biological cells during flow. The feasibility of the proposed measurement technique is demonstrated on erythrocytes, due to their unique morphology and dependency of hemoglobin (Hb) molecule absorption on wavelength. From the spectroscopic quantitative phase profiles of cells acquired during flow in a microfluidic device, we decoupled the refractive index and the physical thickness. We then used our quantitative phase imaging results to dynamically calculate the mean cell volume (MCV), mean corpuscular Hb concentration (MCHC), mean corpuscular Hb content (MCH) and sphericity index.

Changes in optical properties of electroporated cells as revealed by digital holographic microscopy.

Changes in optical and shape-related characteristics of B16F10 cells after electroporation were investigated using digital holographic microscopy (DHM). Bipolar rectangular pulses specific for electrochemotherapy were used. Electroporation was performed in an "off-axis" DHM set-up without using exogenous markers. Two types of cell parameters were monitored seconds and minutes after pulse train application: parameters addressing a specifically defined area of the cell (refractive index and cell height) and global cell parameters (projected area, optical phase shift profile and dry mass). The biphasic behavior of cellular parameters was explained by water and mannitol dynamics through the electropermeabilized cell membrane.

Compact and field-portable 3D printed shearing digital holographic microscope for automated cell identification.

We propose a low-cost, compact, and field-portable 3D printed holographic microscope for automated cell identification based on a common path shearing interferometer setup. Once a hologram is captured from the portable setup, a 3D reconstructed height profile of the cell is created. We extract several morphological cell features from the reconstructed 3D height profiles, including mean physical cell thickness, coefficient of variation, optical volume (OV) of the cell, projected area of the cell (PA), ratio of PA to OV, cell thickness kurtosis, cell thickness skewness, and the dry mass of the cell for identification using the random forest (RF) classifier. The 3D printed prototype can serve as a low-cost alternative for the developing world, where access to laboratory facilities for disease diagnosis are limited. Additionally, a cell phone sensor is used to capture the digital holograms. This enables the user to send the acquired holograms over the internet to a computational device located remotely for cellular identification and classification (analysis). The 3D printed system presented in this paper can be used as a low-cost, stable, and field-portable digital holographic microscope as well as an automated cell identification system. To the best of our knowledge, this is the first research paper presenting automatic cell identification using a low-cost 3D printed digital holographic microscopy setup based on common path shearing interferometry.

Image plane digital holographic microscope for the inspection of ferroelectric single crystals

One of the most important characteristics in the research and application of ferroelectric materials is the appearance of the domain patterns, which take place in phenomena such as ferroelectric phase transitions or ferroelectric switching. The ability to visualize domains is the key factor that enables the progress in the research of these extremely interesting phenomena. However, the three-dimensional visualization of the ferroelectric domain patterns in the whole volume of the ferroelectric single crystal is not a straightforward task. We present the optical method, which allows the acquisition of quantitative and qualitative data substantial for the ferroelectric domain research. The principle of the method is based on image plane digital holographic microscopy (DHM). We used DHM setup outcomes from a Mach–Zehnder type of interferometer and phase-shifting digital holography. The studied specimen is a single crystal of barium titanate. It is demonstrated that the use of solid-state thin-film transparent electrodes of indium tin oxide greatly reduces the unwanted wavefront distortions, which are frequently produced in liquid electrodes. Using this approach, it is possible to greatly improve the DHM measurements in low applied electric fields. Thanks to the properties of the setup, real-time observation of domain walls growth or existing patterns of the ferroelectric crystal is possible.

Photorefractive digital holographic microscopy: an application in the microdevices surfaces

In this work, we present a Photorefractive Digital Holographic Microscopy (PRDHM) technique based on the writing-reading holographic process in photorefractive Bi12TiO20 (BTO) crystal using the numerical reconstruction of the phase and amplitude image performed by a Digital Holographic Microscopy (DHM) method. We demonstrate that a holographic reconstructed image by diffraction process in photorefractive BTO crystal can be combined with a second reference beam to form a hologram in CCD plane in a DHM setup. The experimental results in a glass slide resolution target and thin film structure was obtained, where, amplitude, phase and 3D phase images are calculated performing digital reconstruction. This technique presents potentials possibilities to obtain 3D phase image of the microdevices for dynamic holography applications.

Application of three-dimensional spatial correlation properties of coherent noise in phase noise suppression for digital holographic microscopy

The inherited coherent noise degrades the phase imaging quality in digital holographic microscopy (DHM). To overcome the problem, an experimental investigation on the three-dimensional (3D) spatial correlation properties of coherent noise is carried out. Multiple blank holograms are recorded without any specimen in DHM setup by consecutively shifting camera along the optical axis, and a series of phase distribution of coherent noise can be obtained by numerical reconstruction. Then, based on the phase distributions, the lateral and longitudinal correlation properties of coherent noise are analyzed by a discrete correlation algorithm. Furthermore, a method for reducing phase noise is proposed by use of multiple holograms. Firstly, a series of holograms are recorded by shifting the camera longitudinally with the step more than longitudinal correlation length of coherent noise field. Secondly, the reconstruction of the holograms leads to a series of phase images of object, in which the coherent noise has different patterns. Consequently, by averaging the phase images, the reductions of phase noise are achieved. The applicability of the method is demonstrated by imaging of the resolution targets and the grating.

Autofocusing method for tilted image plane detection in digital holographic microscopy

A rapid autofocusing technique is a fundamental element for a vast range of applications of digital holography. In this paper we propose a new autofocusing method that permits to detect a tilted optimal focus plane. The method is based on estimation of focusing condition of the optical field by evaluating the sharpness in its amplitude distribution. The developed algorithm is fully automated. It consists of two major steps, first the rotation axis is localized from the map of local sharpness and then the angular orientation of the image plane is derived by maximizing the focus of optical fields reconstructed in many subsequent tilted planes. In our study six image sharpness quantification methods are investigated and the numerical propagation between inclined planes is performed with an efficient band limited angular spectrum algorithm. The utility of the proposed method for autofocusing to a tilted image plane is experimentally confirmed in a digital holographic microscopy setup by carrying out a successful reconstruction of holograms of the exemplary amplitude (the 1951 USAF positive target) and phase (a matrix of microlenses) objects.

Quantitative phase-contrast imaging with compact digital holographic microscope employing Lloyd’s mirror

Digital holographic microscopy (DHM) is one of the most effective techniques used for quantitative phase imaging of cells. Here we present a compact, easy to implement, portable, and very stable DHM setup employing a self-referencing Lloyd's mirror configuration. The microscope is constructed using a diode laser source and a CMOS sensor, making it cost effective. The reconstruction of recorded holograms yields the amplitude and phase information of the object. The temporal stability of the presented technique was found to be around 0.9nm without any vibration compensation, which makes it ideal for studying cell profile changes. This aspect of the technique is demonstrated by studying membrane fluctuations of red blood cells.

Digital holographic microscopy long-term and real-time monitoring of cell division and changes under simulated zero gravity

The long-term and real-time monitoring the cell division and changes of osteoblasts under simulated zero gravity condition were succeed by combing a digital holographic microscopy (DHM) with a superconducting magnet (SM). The SM could generate different magnetic force fields in a cylindrical cavity, where the gravitational force of biological samples could be canceled at a special gravity position by a high magnetic force. Therefore the specimens were levitated and in a simulated zero gravity environment. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet's bore. The results presented the first-phase images of living cells undergoing dynamic divisions and changes under simulated zero gravity environment for a period of 10 hours. The experiments demonstrated that the SM-compatible DHM setup could provide a highly efficient and versatile method for research on the effects of microgravity on biological samples.

Microscopy image resolution improvement by deconvolution of complex fields

Based on truncated inverse filtering, a theory for deconvolution of complex fields is studied. The validity of the theory is verified by comparing with experimental data from digital holographic microscopy (DHM) using a high-NA system (NA=0.95). Comparison with standard intensity deconvolution reveals that only complex deconvolution deals correctly with coherent cross-talk. With improved image resolution, complex deconvolution is demonstrated to exceed the Rayleigh limit. Gain in resolution arises by accessing the objects complex field - containing the information encoded in the phase - and deconvolving it with the reconstructed complex transfer function (CTF). Synthetic (based on Debye theory modeled with experimental parameters of MO) and experimental amplitude point spread functions (APSF) are used for the CTF reconstruction and compared. Thus, the optical system used for microscopy is characterized quantitatively by its APSF. The role of noise is discussed in the context of complex field deconvolution. As further results, we demonstrate that complex deconvolution does not require any additional optics in the DHM setup while extending the limit of resolution with coherent illumination by a factor of at least 1.64.

Suppression of the zero-order term in off-axis digital holography through nonlinear filtering

We present experimental validation of a new reconstruction method for off-axis digital holographic microscopy (DHM). This method effectively suppresses the object autocorrelation, namely, the zero-order term, from holographic data, thereby improving the reconstruction bandwidth of complex wavefronts. The algorithm is based on nonlinear filtering and can be applied to standard DHM setups with realistic recording conditions. We study the robustness of the technique under different experimental configurations, and quantitatively demonstrate its enhancement capabilities on phase signals.

Quasi-physical phase compensation in digital holographic microscopy

In digital holographic microscopy, if an optical setup is well aligned, the phase curvature introduced by the microscope objective (MO) together with the illuminating wave to the object wave is a spherical phase curvature. It can be physically compensated by introducing the same spherical phase curvature in the reference beam. Digital holographic microscopy setups based on the Michelson interferometric configuration with MO and an adjustable lens are presented, which can well perform the quasi-physical phase compensation during the hologram recording. In the reflection mode, the adjustable lens serves as both the condensing lens and the compensation lens. When the spatial frequency spectra of the hologram become a point spectrum, one can see that the phase curvature introduced by imaging is quasi-physically compensated. A simple plane numerical reference wavefront used for the reconstruction can give the correct quantitative phase map of the test object. A theoretical analysis and experimental demonstration are given. The simplicity of the presented setup makes it easy to align it well at lower cost.