Search Results (85)

Non-contact, non-destructive testing of surface microgeometry plays a key role in such industries as microelectronics, additive manufacturing, and precision engineering. This paper presents the development and experimental testing of a digital holographic system based on a low-coherence laser diode operating at two close wavelengths, designed to measure height differences in the micrometer range. The method is based on a Michelson interferometer and reconstruction of the complex amplitude of the object wave, which allows phase measurements with subsequent phase conversion into heights. The tests were carried out on micrometer roughness standards with a trapezoidal profile with a groove depth from 24.5 μm to 100 μm and a profile width from 65 μm to 150 μm, as well as on reference strokes with a width from 25 to 200 μm. The obtained data demonstrate the possibility of three-dimensional and two-dimensional visualization of the objects under study with a relative error in height from 5.3% to 11.6% and in width up to 18.6%. It is shown that the system allows reliable measurement of defects of metal surfaces in the range from 25 to 100 μm both vertically and horizontally. Thus, the developed method can be used for high-precision, non-destructive testing in a wide range of technological tasks. Full article

This study presents a prism-based self-referencing digital holographic microscopy (PSDHM) system that utilizes a wedge prism. The front and rear surfaces of the prism have a wedge angle of 2°, which can reflect the parallel incident light, respectively, to generate a lateral displacement that varies with the propagation distance of the optical path. Focusing on the quantitative analysis of droplets, this innovative system effectively images water droplets and their dynamic evaporation processes. Results show that the evaporation process of water droplets undergoes three stages, each stage corresponding to a theoretical model. These are the constant contact radius (CCR) mode, the stick-slip (SS) mode, and the stick-jump (SJ) mode. Furthermore, by comprehensively analyzing the contact angle and the specific morphology of the droplet’s contact area, we revealed that the hydrophilicity of the cover glass influences the droplet morphology, contact area, and the evaporation process. Full article

Digital holographic microscopes provide a microscopy solution with a resolution in the low-micrometre range that offers similar performance to optical microscopy, but as a relatively low-cost alternative. The most significant cost saving is due to the ability to reconstruct microscopic images from holograms using low-cost components without the need for an optical stack. The cost saving opens up the avenue towards a feasible solution for geographically distributed in situ microscopic sensing in rural areas for problems like air and water pollution monitoring. The most significant contributors to cost are the camera sensor module, the pinhole, and the processing platform. The latter two components are addressed, at least in part, in this work. We successfully manufactured sub-100 $\mu$m diameter pinholes using ultraviolet (UV) laser cutting with an LPKF printed circuit board (PCB) prototyping platform and present the low-cost micromachining method. The pinholes were utilised within a prototype field-programmable gate array (FPGA) demonstrator that successfully reconstructed the holographic images. The choice for the FPGA approach as the initial step, albeit more complex, lends itself towards the easier development of a dedicated reconstructed application-specific integrated circuit (ASIC) to ultimately drive the cost down even further. Full article

Digital holographic microscopy (DHM) provides numerous advantages, such as noninvasive sample analysis, real-time dynamic detection, and three-dimensional (3D) reconstruction, making it a valuable tool in fields such as biomedical research, cell mechanics, and environmental monitoring. To achieve more accurate and comprehensive imaging, it is crucial to capture detailed information on the microstructure and 3D morphology of samples. Phase processing of holograms is essential for recovering phase information, thus making it a core component of DHM. Traditional phase processing techniques often face challenges, such as low accuracy, limited robustness, and poor generalization. Recently, with the ongoing advancements in deep learning, addressing phase processing challenges in DHM has become a key research focus. This paper provides an overview of the principles behind DHM and the characteristics of each phase processing step. It offers a thorough analysis of the progress and challenges of deep learning methods in areas such as phase retrieval, filtering, phase unwrapping, and distortion compensation. The paper concludes by exploring trends, such as ultrafast 3D holographic reconstruction, high-throughput holographic data analysis, multimodal data fusion, and precise quantitative phase analysis. Full article

The concurrent dissolution and early hydration of tricalcium aluminate (C₃A) and tetracalcium aluminoferrite (C₄AF) critically govern early-stage reaction dynamics in Portland cement systems. However, their mutual kinetic interactions during reaction, particularly sulfate-dependent modulation mechanisms, remain poorly understood. Using in-situ digital holographic microscopy (DHM), this study resolved their interaction mechanisms during co-dissolution in aqueous and sulfate-bearing environments. Results reveal asymmetric modulation: while C₄AF’s dissolution exhibited limited sensitivity to C₃A’s presence, C₃A’s kinetics were profoundly altered by C₄AF through sulfate-concentration-dependent pathways, which originated from two competing C₄AF-mediated mechanisms: (1) suppression via common-ion effects, and (2) acceleration through competitive sulfate species adsorption. These mechanistic insights would provide a roadmap for optimizing cementitious materials through optimized reaction pathways. Full article

Label-free, high-throughput, and 3D morphological analysis of blood cells remains a major challenge in biomedical optics. In this study, we investigate this issue using flow cytometry with digital holographic microscopy (DHM) to enable real-time, label-free imaging of red blood cells (RBCs) and blood coagulation structures (BCSs) without the need for staining or chemical pretreatment. We demonstrate an approach for the automated quantification and statistical characterization of these cells using quantitative phase information reconstructed from digital holograms. Although established image processing techniques such as phase unwrapping and segmentation are used, this study presents, to the best of our knowledge, the first statistical characterization of the 3D morphological features of BCSs. This is particularly useful in analyzing the heterogeneous and complex 3D structures of BCSs, which are difficult to assess using conventional microscopy. The results suggest that this DHM-based flow cytometry system provides a promising platform for non-invasive, real-time morphological evaluation of blood samples and has potential applications in hematological diagnostics and research related to blood coagulation. Full article

Automatic focusing is a crucial research issue for achieving high-quality reconstructed images in digital holographic microscopy. This paper proposes an automatic focusing method that combines the discrete cosine transform (DCT) sparse dictionary with edge preservation index (EPI) criteria for off-axis digital holographic microscopy. Specifically, within a predefined search range, Fresnel transform is utilized to reconstruct the off-axis digital hologram, yielding reconstruction images at various reconstruction distances. Synchronously, the DCT sparse dictionary is employed to reduce speckle noise, and the EPI is calculated between the denoised image and original image. The value of EPI is used as an indicator for assessing the focal position. A single-peak focusing curve is obtained within the search range 10 mm, with a step size of 0.1 mm. Once the optimal focus position is determined, a focused and noise-reduced reconstructed image can be simultaneously achieved. Full article

We propose a pulsed modulated digital holographic microscopy (PM-DHM) technique for the dynamic measurement of flowing microparticles in microfluidic systems. By digitally tuning the pulse width and the repetition rate of a laser source within a single-frame exposure, this method enables the recording of multiple images of flowing microparticles at different time points within a single hologram, allowing the quantification of velocity and acceleration. We demonstrate the feasibility of PM-DHM by measuring the velocity, acceleration, and forces exerted on PMMA microspheres and red blood cells flowing in microfluidic chips. Compared to traditional frame-sampling-based imaging methods, this technique has a much higher time resolution (in a range of microseconds) that is limited only by the pulse duration. This method demonstrates significant potential for high-throughput label-free flow cytometry detection and offers promising applications in drug development and cell analysis. Full article

We present a single-capture multimodal bright-field (BF) and quantitative phase imaging (QPI) approach that enables the analysis of large, connected, or extended samples, such as confluent cell layers or tissue sections. The proposed imaging concept integrates a fiber-optic Mach–Zehnder interferometer-based off-axis digital holographic microscopy (DHM) with an inverted commercial optical BF microscope. Utilizing 8-bit grayscale dynamic range multiplexing, we simultaneously capture both BF images and digital holograms, which are then demultiplexed numerically via Fourier filtering, phase aberration compensation, and weighted image subtraction procedures. Compared to previous BF-DHM systems, our system avoids synchronization challenges caused by multiple image recording devices, improves acquisition speed, and enhances versatility for fast imaging of large, connected, and rapidly moving samples. Initially, we perform a systematic characterization of the system’s multimodal imaging performance by optimizing numerical as well as coherent and incoherent illumination parameters. Subsequently, the application capabilities are evaluated by multimodal imaging of living cells. The results highlight the potential of single-capture BF-DHM for fast biomedical imaging. Full article

Holographic detection technology has found extensive applications in biomedical imaging, surface profilometry, vibration monitoring, and defect inspection due to its unique phase detection capability. However, the accuracy of quantitative holographic phase imaging is significantly affected by the interference from direct current and twin image terms. Traditional methods, such as multi-exposure phase shifting and off-axis holography, have been employed to mitigate these interferences. While off-axis holography separates spectral components by introducing a tilted reference beam, it inevitably induces phase disturbances that compromise measurement accuracy. This study provides a computational explanation for the incomplete phase compensation issue in existing algorithms and establishes precision criteria for phase compensation based on theoretical formulations. We propose two novel phase compensation methods—the non-iterative compensation approach and the multi-iteration compensation technique. The principles and applicable conditions of these methods are thoroughly elucidated, and their superiority is demonstrated through comparative experiments. The results indicate that the proposed methods effectively compensate for phase disturbances induced by the tilted reference beam, offering enhanced precision and reliability in quantitative holographic phase measurements. Full article

Digital Holographic Microscopy (DHM) is a method of converting hologram images into three-dimensional (3D) images by image processing, which enables us to obtain the detailed shapes of the objects to be observed. Three-dimensional imaging of the microscopic objects by DHM can contribute to the early diagnosis and the detection of the diseases in the medical field by observing the shape of the cells. DHM requires several experimental components. One of them is the laser, which is a problem because its high power may cause the deformation and the destruction of the cells and the death of the microorganisms. Since the greatest advantage of DHM is the detailed geometrical information of the object by 3D measurement, the loss of such information is a serious problem. To solve this problem, a Neutral Density (ND) filter has been used to reduce power after the laser irradiation. However, the image acquired by the image sensor becomes too dark to obtain sufficient information, and the effect of noise increased due to the decrease in the amount of light. Therefore, in this paper, we propose the Frame-Stacking Method (FSM) for dark DHM for reproducing 3D profiles that enable us to observe the shape of the objects from the images taken in low-power environments when the power is reduced. The proposed method realizes highly accurate 3D profiles by the frame decomposition of the low-power videos into images and superimposing and rescaling the obtained low-power images. On the other hand, the continuous irradiation of the laser beam for a long period may destroy the shape of the cells and the death of the microorganisms. Therefore, we conducted experiments to investigate the relationship between the number of superimposed images corresponding to the irradiation time and the 3D profile, as well as the characteristics of the power and the 3D profile. Full article

Digital Holographic Microscopy (DHM) is a technique that uses the phase information of light to generate a three-dimensional (3D) profile of an object. Recently, it has been utilized in various fields such as disease diagnosis and research on microorganisms. In the process in DHM, a narrow region around one of the sidebands from the frequency domain is windowed to avoid noise caused by the direct current (DC) term. However, it may not obtain the high-frequency information about the object. On the other hand, windowing a wide region increases the noise caused by the DC term, and generates the noise in the 3D profile. To solve this trade-off, we propose a noise reduction method using Kalman filter. From the recorded hologram image, we can create the frequency domain. It obtains multiple windowed sidebands centered on multiple pixels at random from the frequency domain. This creates a group of data in which noise is generated randomly. This is regarded as frequency series data, and Kalman filtering is performed. This method can reduce the noise caused by the DC term while acquiring high-frequency information. In addition, this method has the advantage that only one image is needed for frequency series data in the Kalman filter. The effectiveness of the proposed method is verified by comparison with conventional filtering methods and general image processing methods. The validation results prove the usefulness of the proposed method, and the proposed method is expected to have a significant effect on improving the accuracy of disease diagnosis techniques using DHM. Full article

Lens-free on-chip microscopy (LFOCM) is a powerful computational imaging technology that combines high-throughput capabilities with cost efficiency. However, in LFOCM, the phase recovered by iterative phase retrieval techniques is generally wrapped into the range of −$\pi$ to $\pi$, necessitating phase unwrapping to recover absolute phase distributions. Moreover, this unwrapping process is prone to errors, particularly in areas with large phase gradients or low spatial sampling, due to the absence of reliable initial guesses. To address these challenges, we propose a novel biplane phase retrieval (BPR) method that integrates phase unwrapping results obtained at different propagation distances to achieve accurate absolute phase reconstruction. The effectiveness of BPR is validated through live-cell imaging of HeLa cells, demonstrating improved quantitative phase imaging (QPI) accuracy when compared to conventional off-axis digital holographic microscopy. Furthermore, time-lapse imaging of COS-7 cells in vitro highlights the method’s robustness and capability for long-term quantitative analysis of large cell populations. Full article

For several years, the determination of a differential cell count of a raw milk sample has been proposed as a more accurate tool for monitoring the udder health of dairy cows compared with using the absolute somatic cell count. However, the required sample preparation and staining process can be labor- and cost-intensive. Therefore, the aim of our study was to demonstrate the feasibility of analyzing unlabeled blood and milk leukocytes from dairy cows by means of digital holographic microscopy (DHM). For this, we trained three different machine learning methods, i.e., k-Nearest Neighbor, Random Forests, and Support Vector Machine, on sorted leukocyte populations (granulocytes, lymphocytes, and monocytes/macrophages) isolated from blood and milk samples of three dairy cows by using fluorescence-activated cell sorting. Afterward, those classifiers were applied to differentiate unlabeled blood and milk samples analyzed by DHM. A total of 70 blood and 70 milk samples were used. Those samples were collected from five clinically healthy cows at 14-time points within a study period of 26 days. The outcome was compared with the results of the same samples analyzed by flow cytometry and (in the case of blood samples) also to routine analysis in an external laboratory. Moreover, a standard vaccination was used as an immune stimulus during the study to check for changes in cell morphology or cell counts. When applied to isolated leukocytes, Random Forests performed best, with a specificity of 0.93 for blood and 0.84 for milk cells and a sensitivity of 0.90 and 0.81, respectively. Although the results of the three analytical methods differed, it could be demonstrated that a DHM analysis is applicable for blood and milk leukocyte samples with high reliability. Compared with the flow cytometric results, Random Forests showed an MAE of 0.11 (SD = 0.04), an RMSE of 0.13 (SD = 0.14), and an MRE of 1.00 (SD = 1.11) for all blood leukocyte counts and an MAE of 0.20 (SD = 0.11), an RMSE of 0.21 (SD = 0.11) and an MRE of 1.95 (SD = 2.17) for all milk cell populations. Further studies with larger sample sizes and varying immune cell compositions are required to establish method-specific reference ranges. Full article

Imaging of microscopic objects is of fundamental importance, especially in life sciences. Recent fast progress in electronic detection and control, numerical computation, and digital image processing, has been crucial in advancing modern microscopy. Digital holography is a new field in three-dimensional imaging. Digital reconstruction of a hologram offers the remarkable capability to refocus at different depths inside a transparent or semi-transparent object. Thus, this technique is very suitable for biological cell studies in vivo and could have many biomedical and biological applications. A comprehensive review of the research carried out in the area of digital holographic microscopy (DHM) for live-cell imaging is presented. The novel microscopic technique is non-destructive and label-free and offers unmatched imaging capabilities for biological and bio-medical applications. It is also suitable for imaging and modelling of key metabolic processes in living cells, microbial communities or multicellular plant tissues. Live-cell imaging by DHM allows investigation of the dynamic processes underlying the function and morphology of cells. Future applications of DHM can include real-time cell monitoring in response to clinically relevant compounds. The effect of drugs on migration, proliferation, and apoptosis of abnormal cells is an emerging field of this novel microscopic technique. Full article