Process engineering and failure analysis of MEMS and MOEMS by digital holography microscopy (DHM)

Motility, or co-ordinated movement, of cells is fundamental for the development of many processes, such as formation of the embryo, the immune response, inflammation, wound healing, and metastasis of cancer cells 1. Cells often migrate in response to specific external signals, but they can also move spontaneously. Aberrant cell motility can be fatal in cancer diseases when cells start to metastasize 2. Metastasis is strictly associated with tissue invasion of the primary tumor cells, and occurs as a multistep progress 3. The first phase of cancer cell metastasis involves the physical translocation of the cell to a distant organ, whereas the second phase implicates the ability of the cancer cell to develop into a metastatic lesion at that distant site 4. For skin cancer melanoma, the main cause of death is widespread metastases 5. Melanoma cells may travel along external vessel lattices. After settling in the metastatic sites, melanoma cells develop mechanisms that protect them against the attack of the immune system. It is, therefore, of great interest and importance to investigate, monitor, and understand the principles of tumor cell invasion and migration. The development of novel therapeutic applications that have the ability to block migratory processes is crucial. For in vitro studies, the cell culture wound-closure, and the transwell migration and invasion assays can provide data for an understanding how well a particular cell type can migrate 6. The wound healing assay is also called a scratch assay because it is performed by making a scratch in the cell monolayer (Fig. 1A). The transwell migration assay is a commonly used test to study the migratory response of endothelial cells to angiogenic inducers or inhibitors (Fig. 1B). This assay is also known as the Boyden or modified Boyden chamber assay. These assays have been frequently used to study movement of cells. Weckmann et al. developed an interesting system to monitor and quantify neutrophils on single cell level, by utilizing commercially available tissue culture hardware, simple video microscopic equipment, and standardized tracking 7. However, the interest for novel standardized real-time analyses using quantitative phase imaging (QPI) for cell tracking is growing (Fig. 2). More than 10 years ago, the digital holographic (DH) microscopy methodology of QPI became known to a larger audience and the technique has since then successfully been applied for label-free monitoring of live cells undergoing cell division, cell proliferation, cell death, and cell migration over time 8-10. By QPI, studies associated with both cellular thickness and refractive index fluctuations have been performed. The possibilities for utilizing QPI for cancer diagnostics have increased 11. Cellular behavior and phenotypes are other parameters that can be measured 12. For two human osteosarcoma cell types, the motility pattern, but not the proliferative pattern, was different. Early migration studies using DH microscopy showed fibroblast migration 13 and HT-1080 fibrosarcoma cells embedded in a 3D collagen gel 14. Alm et al. and Persson et al. also presented data on movement of different cell types and cell sizes 15 as well as demonstrated the correlation of cell movement with cell morphology 16. In the recent article published in the November 2018 issue of Cytometry Part A by Zhang et al., (pages 1125–1131) commercially available DH cytometry platforms equipped with semi-automated image acquisition, segmentation, and analysis software packages for assessing cell behavior are used and discussed. The motility data was obtained with QPI/cytometry using "Track cells module" and "Wound healing module," respectively, and with transwell migration assays determining migration and invasion, respectively. The different techniques assessing motility were compared. The two melanoma cell lines, WM793 and 1205Lu, used in the study were reported to have different metastatic capacity, resulting in differences in proliferation and motility. The motility measured with the "Track cells module" and "Wound healing module," using DH cytometry equipment housed in an incubator, produced highly correlated relative motilities, even when compared to the standard transwell migration and invasion assays. Images acquired with DH are from the same position in a cell culture vessel, making it possible to study individual cells. This is a strong advantage, since most studies of effects of new drugs on proliferation and migration of cancer cells in culture are performed on a population level. In a recent study by Kamlund et al., cell images were captured every fifth minute for a total of up to 72 h 17. For cell movement studies, the semi-automated algorithm attempts to find each tracked cell in the next frame by selecting the closest cell based on the centroid position. The advantages of the Track cells and Wound healing modules over the transwell migration and invasion assays in the paper by Zhang et al., included the possibility to use the cells in the experiment for other purposes after completing the measurement. DH microscopy also benefits from the fact that morphology analysis can be performed of each cell. Indeed, this opens up for almost unlimited possibilities to perform cell morphology analysis using the QPI methodology, since each image is very rich in cellular information. As discussed in a review by El-Schich et al., automated analysis and machine learning is one approach to handle the large amount of morphological data that can now be generated when analyzing cells with QPI 8. Only the computer resources and the training speed will set the limitations.

In this work, we present a patented procedure that generates three dimensional (3D) image of a microscopic object with high definition, obtained by merging Scanning Electron Microscopy (SEM) and Digital Holography Microscopy (DHM) data. A high spatial resolution (up to r z =λ/20, r yx ≅100nm) 3D model of the object is generated starting from two digital images, acquired separately, following standard characterization procedures. Then, the two set of data are combined using an original mathematical algorithm, which has been implemented with a commercially available software. The developed methodology is of broad interest and has been applied to elements of biological interest, in order to prove its features.

Super-resolved Mirau digital holography by structured illumination

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.

Digital holography (DH) has a big advantage to retrieve the three-dimensional (3D) information of the object from only one interference recording. Especially, the digital holographic microscope (DHM) using a microscope objective (MO) has been researched for 3D microscopy. The researches have progressed for compensation of aberrations and improvement of the resolution in the optical system in recent years. Most of small aberrations caused by a MO are compensated through various researches. However, the measured phase is distorted in the optical system, which has the significant wavefront deformation in illuminating wave larger than number of wavelengths. In this paper, the relation between illuminating wave and the reconstructed phase is studied based on the wave optics and the analysis is confirmed by the simulations. The analysis of the wavefront compensation is applied to a super-resolution DHM in theory and the technique for retrieving the distribution of the intensity and phase is demonstrated in simulation.

The electrically tunable lens (ETL) is a novel current-controlled adaptive optical component which can continuously tune its focus in a specific range via changing its surface curvature. To quantitatively characterize its tuning power, here we assume the ETL to be a pure phase object and present a novel calibration method to dynamically measure its wavefront by use of digital holographic microscopy (DHM). The least squares method is then used to fit the radius of curvature of the wavefront. The focal length is obtained by substituting the radius into the Zemax model of the ETL. The behavior curve between the focal length of the ETL and its driven current is drawn, and a quadratic mathematic model is set up to characterize it. To verify our model, an ETL and offset lens combination is proposed and applied to ETL-based transport of intensity equation (TIE) phase retrieval microscopy. The experimental result demonstrates the calibration works well in TIE phase retrieval in comparison with the phase measured by DHM.

An area of particular importance in developing advanced imaging techniques concerns 3D motion measurement in small-scale mechatronics and automated microscopy. One major drawback is related to complex motion measurement with 6 degrees of freedom. In the proposed work, the extraction of unknown metrics such as focusing distance, in plane and out-of-plane positioning from digital holograms is performed including real‐time constraints. This work explores extended computer micro-vision capabilities offered by combining digital holographic microscopy (DHM) and last generation of deep learning algorithms such as Vision Transformer (ViT) networks. Our experiments show that the reconstruction in-focus distance can be predicted in DHM with a high accuracy using tiny modified architectures of deep ViT networks and convolutional neural networks (CNN). We compare ViT and Tiny ViT models with deep CNN usually used in digital holography such as VGG16, LeNet and AlexNet.

In this work, an off-axis digital holographic microscope operating in reflection mode and a telecentric regimen to produce 3D topography of a microscopy sample is shown. The main characteristics of the proposed method, which make it different from the previous works in the field, are the possibility of producing the 3D topography by a single shot over the complete field of view with sensitivity of λ/100, without phase perturbations introduced by the illuminating-imaging system, and with no further numerical processing beyond that required for recovering the phase map of the sample. A complete analysis of the illuminating-imaging system of the digital holographic microscope is presented. The proposed digital holographic microscope is tested on imaging a USAF resolution test target and some micro-electromechanical systems (MEMs).

Three-dimensional profiling and tracking by digital holography microscopy (DHM) provide label-free and quantitative analysis of the characteristics and dynamic processes of objects, since DHM can record real-time data for microscale objects and produce a single hologram containing all the information about their three-dimensional structures. Here, we have utilized DHM to visualize suspended microspheres and microfibers in three dimensions, and record the four-dimensional trajectories of free-swimming cells in the absence of mechanical focus adjustment. The displacement of microfibers due to interactions with cells in three spatial dimensions has been measured as a function of time at subsecond and micrometer levels in a direct and straightforward manner. It has thus been shown that DHM is a highly efficient and versatile means for quantitative tracking and analysis of cell motility.

Digital Holography is a new technology that promises many applications in diverse areas. Using numerical reconstruction of digitally recorded hologram, the Reflection Digital Holographic Microscope (DHM) provides the intensity, phase images which give the 3D depth profile of micro-devices. These essential data provided by digital holography are very useful for new MEMS design and fabrication as well as thin film growth. Having advantages over other existing measurement methods, it can possibly serve as an easier alternative in future. In this study, we further investigate the accuracy and specifications of thickness measurement, topography and roughness analysis using the DHM. By comparing the DHM results with alpha-stepper (a form of stylus probing) and Atomic Force Microscope (AFM), we find that the accuracy of the DHM for thickness measurement and roughness analysis is about 10%. The thickness and roughness range is also found to be 50nm to 500nm. Our experimental study hence demonstrates that digital holography has the potential to be developed into a suitable tool for thickness measurement and roughness analysis due to it being non-invasive and being able to provide quick time and relatively accurate measurements.

Nano graphene-based materials offer interesting physicochemical and biological properties for biotechnological applications due to their small size, large surface area and ability to interact with cells/tissues. Among carbon-based nanomaterials, graphene oxide is one of the most used in biological field. There is an increasing interest in shedding light on the interaction mechanisms of nanographene oxide (nGO) with cells. In fact, the effects on human health of GO, and its toxicological profile, are still largely unknown. Here we show that, by minimizing the oxidation degree of GO, its toxicity is significantly reduced in NIH 3T3 cells. Moreover, we show that mild oxidation of graphene nanoplatelets produces nGO particles, which are massively internalized into the cell cytoplasm. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to analyze cell viability. Transmission electron microscopy (TEM) analysis was performed to evaluate nGO internalization mechanism into the cytoplasm under different oxidation degree and concentrations. For the first time, we evaluated quantitatively, the cell volume variation after nGO internalization in live fibroblasts through a label-free digital holography (DH) imaging technique and in quasi-real-time modality, thus avoiding the time-consuming and detrimental procedures usually employed by electron-based microscopy. In conclusion, here we have demonstrated that DH can be a viable tool to visualize and display 3D distributions of nano graphene oxide (nGO) uptake by fibroblast cells. DH opens the route for high-throughput investigation at single cell level for understanding how in different conditions nanoparticles aggregates distribute inside the cells.

This study aims to investigate the migration behaviour of individual JIMT-1 breast cancer cells during the cell cycle. Mechanisms behind cell migration are important to unravel in the context of cancer relapses caused by distant metastases. JIMT-1 cells were cultivated in normoxia or hypoxia and treated with 0.5 µM of the cancer stem cell inhibiting compound salinomycin. The cells were imaged in a digital holography microscope placed in a regular cell culturing CO2 incubator (normoxia) or in a hypoxia chamber (1% O2). In digital holography, a laser beam is split into two beams - the reference beam and the object beam. The object beam is directed through the sample, where the difference in refractive index between air, the cell medium, and the cell body induces a phase shift in the laser beam. After having passed the object, the beam is merged with the reference beam giving an interference pattern, which is used to construct a three-dimensional image of the cells in a software program. The laser is non-phototoxic and therefor it can be used for long-term studies. In our study, images were acquired every 5th minute during a 72-hour incubation period. After acquisition, the individual cells in the images were tracked throughout the time lapse. Cell movement is described by three parameters: motility, which is the length of the total cell movement path, migration, which is the shortest distance from the start to the end position, and migration directness, which is migration divided by motility. When cells were exposed to hypoxia and/or treated with salinomycin, the cell cycle time increased compared to non-treated cells in normoxia. When the cell cycle time increased, motility increased as well. However, migration directness decreased despite increase in motility implying increased local movement of cells treated with salinomycin compared to control cells. We have previously shown that salinomycin-treated JIMT-1 cells obtain an epithelia phenotype (Huang et al., 2016). Thus, we decided to investigate a breast cancer cell line with epithelial traits i.e, the MCF-7 cells. The MCF-7 cells behaved like the JIMT-1 cells treated with salinomycin i.e. the motility was high and the migration directness was low. Thus, we suggest that cells with mesenchymal and epithelial traits can be distinguished using digital holographic imaging based on motility and migration directness and that drug-induced phenotypic shifts can be studied using this technique.

Digital holographic (DH) microscopy has grown into a powerful nondestructive technique for the real-time study of living cells including dynamic membrane changes and cell fluctuations in nanometer and sub-nanometer scales. The conventional DH microscopy configurations require a separately generated coherent reference wave that results in a low phase stability and a necessity to precisely adjust the intensity ratio between two overlapping beams. In this work, we present a compact, simple, and very stable common-path DH microscope, employing a self-referencing configuration. The microscope is implemented by a diode laser as the source and a Fresnel biprism for splitting and recombining the beams simultaneously. In the overlapping area, linear interference fringes with high contrast are produced. The frequency of the interference pattern could be easily adjusted by displacement of the biprism along the optical axis without a decrease in fringe contrast. To evaluate the validity of the method, the spatial noise and temporal stability of the setup are compared with the common off-axis DH microscope based on a Mach-Zehnder interferometer. It is shown that the proposed technique has low mechanical noise as well as superb temporal stability with sub-nanometer precision without any external vibration isolation. The higher temporal stability improves the capabilities of the microscope for studying micro-object fluctuations, particularly in the case of biological specimens. Experimental results are presented using red blood cells and silica microspheres to demonstrate the system performance.

Introducing a microscope objective in an interferometric setup induces a phase curvature on the resulting wavefront. In digital holography, the compensation of this curvature is often done by introducing an identical curvature in the reference arm and the hologram is then processed using a plane wave in the reconstruction. This physical compensation can be avoided, and several numerical methods exist to retrieve phase contrast images in which the microscope curvature is compensated. Usually, a digital array of complex numbers is introduced in the reconstruction process to perform this curvature correction. Different corrections are discussed in terms of their influence on the reconstructed image size and location in space. The results are presented according to two different expressions of the Fresnel transform, the single Fourier transform and convolution approaches, used to propagate the reconstructed wavefront from the hologram plane to the final image plane.

The concept of numerical parametric lenses (NPL) is introduced to achieve wavefront reconstruction in digital holography. It is shown that operations usually performed by optical components and described in ray geometrical optics, such as image shifting, magnification, and especially complete aberration compensation (phase aberrations and image distortion), can be mimicked by numerical computation of a NPL. Furthermore, we demonstrate that automatic one-dimensional or two-dimensional fitting procedures allow adjustment of the NPL parameters as expressed in terms of standard or Zernike polynomial coefficients. These coefficients can provide a quantitative evaluation of the aberrations generated by the specimen. Demonstration is given of the reconstruction of the topology of a microlens.