Search Results (73)

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

Reducing side effects in non-cancerous tissue is a key aim of modern radiotherapy. Here, we assessed whether the use of the antioxidants hydroxytyrosol (HT) and thioredoxin-mimetic peptide CB3 (TMP) attenuated radiation-induced normal tissue toxicity in vitro. We used primary human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (HaCaT) as normal tissue models. Cells were treated with HT and TMP 24 h or immediately prior to irradiation. Reactive oxygen species (ROS) were assessed via luminescent- and fluorescence-based assays, migration was investigated using digital holographic microscopy, and clonogenic survival was quantified by colony formation assays. Angiogenesis and wound healing were evaluated via time-dependent microscopy. Secreted cytokines were validated in quantitative polymerase chain reaction (qPCR) studies. Treatment with HT or TMP was well tolerated by cells. The application of either antioxidant before irradiation resulted in reduced ROS formation and a distinct decrease in cytokines compared to similarly irradiated, but otherwise untreated, controls. Antioxidant treatment also increased post-radiogenic migration and angiogenesis while accelerating wound healing. HT or TMP treatment immediately before radiotherapy increased clonogenic survival after radiotherapy, while treatment 24 h before radiotherapy enhanced baseline proliferation. Both antioxidants may decrease radiation-induced normal tissue toxicity and deserve further pre-clinical investigation. Full article

With the development of digital holography, the accuracy requirements for the reconstruction phase are becoming increasingly high. The transfer function of the double fast transform (D-FFT) algorithm is distorted when the diffraction distance is larger than the criterion distance ${\mathrm{d}}_{\mathrm{t}}$, which reduces the accuracy of solving the phase. In this paper, the Fresnel diffraction integration algorithm is improved by using the low-pass Tukey window to obtain more accurate reconstructed phases. The improved algorithm is called the D-FFT (Tukey) algorithm. The D-FFT (Tukey) algorithm adjusts the degree of edge smoothing of the Tukey window, using the peak signal-to-noise ratio (PSNR) and the structural similarity (SSIM) to remove the ringing effect and obtain a more accurate reconstructed phase. In a simulation of USAF1951, the longitudinal resolution of the reconstructed phase obtained by D-FFT (Tukey) reached 1.5 $\mathsf{\mu }\mathrm{m}$, which was lower than the 3 $\mathsf{\mu }\mathrm{m}$ obtained by the T-FFT algorithm. The results of Fresnel holography experiments on lung cancer cell slices also demonstrated that the phase quality obtained by the D-FFT (Tukey) algorithm was superior to that of the T-FFT algorithm. D-FFT (Tukey) algorithm has potential applications in phase correction, structured illumination digital holographic microscopy, and microscopic digital holography. Full article

In 3D microsphere tracking, unlike in-plane motion that can be measured directly by a microscope, axial displacements are resolved by optical interference or a diffraction model. As a result, the axial results are affected by the environmental noise. The immunity to environmental noise increases with measurement accuracy and the signal-to-noise ratio (SNR). In compound digital holography microscopy (CDHM)-based measurements, precise identification of the tracking marker is critical to ensuring measurement precision. The reconstruction centering method (RCM) was proposed to suppress the drawbacks caused by installation errors and, at the same time, improve the correct identification of the tracking marker. The reconstructed center is considered to be the center of the microsphere, rather than the center of imaging in conventional digital holographic microscopy. This method was verified by simulation of rays tracing through microspheres and axial moving experiments. The axial displacements of silica microspheres with diameters of 5 μm and 10 μm were tested by CDHM in combination with the RCM. As a result, the SNR of the proposed method was improved by around 30%. In addition, the method was successfully applied to axial displacement measurements of overlapped microspheres with a resolution of 2 nm. Full article

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC₅₀) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays. Full article

We demonstrate an experimental assessment of a high-Q, high-angle piezoelectric (2 µm PZT) MEMS scanning micromirror featuring distributed backside reinforcement, suitable for applications demanding energy-efficient and high-quality image projection. Frequency response measurements at 10 different vacuum levels ranging from atmospheric pressure to 10⁻⁶ mbar allow for the quantitative separation of damping mechanisms (air and structural). Stroboscopic digital holographic microscopy was used to assess the static and dynamic deformation of the mirror surface. The experimental results are in good agreement with simulations and models. Full article

Digital Holographic Microscopy (DHM) is a 3D imaging technology widely applied in biology, microelectronics, and medical research. However, the noise generated during the 3D imaging process can affect the accuracy of medical diagnoses. To solve this problem, we proposed several frequency domain filtering algorithms. However, the filtering algorithms we proposed have a limitation in that they can only be applied when the distance between the direct current (DC) spectrum and sidebands are sufficiently far. To address these limitations, among the proposed filtering algorithms, the HiVA algorithm and deep learning algorithm, which effectively filter by distinguishing between noise and detailed information of the object, are used to enable filtering regardless of the distance between the DC spectrum and sidebands. In this paper, a combination of deep learning technology and traditional image processing methods is proposed, aiming to reduce noise in 3D profile imaging using the Improved Denoising Diffusion Probabilistic Models (IDDPM) algorithm. Full article

Quantitative phase imaging by digital holographic microscopy (DHM) is a nondestructive and label-free technique that has been playing an indispensable role in the fields of science, technology, and biomedical imaging. The technique is competent in imaging and analyzing label-free living cells and investigating reflective surfaces. Herein, we introduce a new configuration of a wide field-of-view single-shot common-path off-axis reflective DHM for the quantitative phase imaging of biological cells that leverages several advantages, including being less-vibration sensitive to external perturbations due to its common-path configuration, also being compact in size, simple in optical design, highly stable, and cost-effective. A detailed description of the proposed DHM system, including its optical design, working principle, and capability for phase imaging, is presented. The applications of the proposed system are demonstrated through quantitative phase imaging results obtained from the reflective surface (USAF resolution test target) as well as transparent samples (living plant cells). The proposed system could find its applications in the investigation of several biological specimens and the optical metrology of micro-surfaces. Full article

Digital holographic microscopy is an important measurement method for micro-nano structures. However, when the structured features are of high-slopes, the interference fringes can become too dense to be recognized. Due to the Nyquist’s sampling limit, reliable wavefront restoration and phase unwrapping are not feasible. To address this problem, the interference fringes are proposed to be sparsified by tilting the reference wavefronts. A data fusion strategy including region extraction and tilt correction is developed for reconstructing the full-area surface topographies. Experimental results of high-slope elements demonstrate the validity and reliability of the proposed method. Full article

In this paper, we propose a measurement method of micro-optical elements with parallel phase-shifting digital holographic phase imaging. This method can record four phase-shifting holograms with a phase difference of π/2 in a single shot and correct the pixel mismatch error of the polarization camera using a bilinear interpolation algorithm, thereby producing high-resolution four-step phase-shifting holograms. This method reconstructs the real phase information of the object to be measured through a four-step phase-shifting algorithm. The reproduced image eliminates the interference of zero-order images and conjugate images, overcoming the problem that traditional phase-shifting digital holography cannot be measured in real time. A simulation analysis showed that the relative error of this measurement method could reach 0.0051%. The accurate surface topography information of the object was reconstructed from an experimental measurement through a microlens array. Multiple measurements yielded a mean absolute error and a mean relative error for the vertical height of the microlens array down to 5.9500 nm and 0.0461%, respectively. Full article

Recently, research on disease diagnosis using red blood cells (RBCs) has been active due to the advantage that it is possible to diagnose many diseases with a drop of blood in a short time. Representatively, there are disease diagnosis technologies that utilize deep learning techniques and digital holographic microscope (DHM) techniques. However, three-dimensional (3D) profile obtained by DHM has a problem of random noise caused by the overlapping DC spectrum and sideband in the Fourier domain, which has the probability of misjudging diseases in deep learning technology. To reduce random noise and obtain a more accurate 3D profile, in this paper, we propose a novel image processing method which randomly selects the center of the high-frequency sideband (RaCoHS) in the Fourier domain. This proposed algorithm has the advantage of filtering while using only recorded hologram information to maintain high-frequency information. We compared and analyzed the conventional filtering method and the general image processing method to verify the effectiveness of the proposed method. In addition, the proposed image processing algorithm can be applied to all digital holography technologies including DHM, and in particular, it is expected to have a great effect on the accuracy of disease diagnosis technologies using DHM. Full article

Digital holographic microscopy (DHM) is a valuable technique for investigating the optical properties of samples through the measurement of intensity and phase of diffracted beams. However, DHMs are constrained by Lagrange invariance, compromising the spatial bandwidth product (SBP) which relates resolution and field of view. Synthetic aperture DHM (SA-DHM) was introduced to overcome this limitation, but it faces significant challenges such as aberrations in synthesizing the optical information corresponding to the steering angle of incident wave. This paper proposes a novel approach utilizing deep neural networks (DNNs) for compensating aberrations in SA-DHM, extending the compensation scope beyond the numerical aperture (NA) of the objective lens. The method involves training a DNN from diffraction patterns and Zernike coefficients through a circular aperture, enabling effective aberration compensation in the illumination beam. This method makes it possible to estimate aberration coefficients from the only part of the diffracted beam cutoff by the circular aperture mask. With the proposed technique, the simulation results present improved resolution and quality of sample images. The integration of deep neural networks with SA-DHM holds promise for advancing microscopy capabilities and overcoming existing limitations. Full article

Urinary tract infections are among the most frequent infectious diseases and require screening a great amount of urine samples from patients. However, a high percentage of samples result as negative after urine culture plate tests (CPTs), demanding a simple and fast preliminary technique to screen out the negative samples. We propose a digital holographic microscopy (DHM) method to inspect fresh urine samples flowing in a glass capillary for 3 min, recording holograms at 2 frames per second. After digital reconstruction, bacteria, white and red blood cells, epithelial cells and crystals were identified and counted, and the samples were classified as negative or positive according to clinical cutoff values. Taking the CPT as reference, we processed 180 urine samples and compared the results with those of urine flow cytometry (UFC). Using standard evaluation metrics for our screening test, we found a similar performance for DHM and UFC, indicating DHM as a suitable and fast screening technique retaining several advantages. As a benefit of DHM, the technique is label-free and does not require sample preparation. Moreover, the phase and amplitude images of the cells and other particles present in urine are digitally recorded and can serve for further investigation afterwards. Full article