Wavefront error analysis and compensation in a digital holographic microscope

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.

Process engineering and failure analysis of MEMS and MOEMS require static and dynamical characterization of both their in-plane and out of plane response to an excitation. A remarkable characteristic of Digital Holography Microscopes (DHM) is the extremely short acquisition time required to grab the whole information necessary to provide 3D optical topography of the sample: a unique frame grab, without any vertical or lateral scan provides the information over the full field of view. First, it ensures DHM measurements to be insensitive to vibrations. Second, it opens the door to fast dynamical characterization of micro-systems. For periodic movement analysis, DHM can operate in stroboscopic mode with standard cameras. It enables precise characterization up to excitation frequencies of 100 kHz with recovery cycle of 10% simply by triggering properly the camera. Pulsed sources can be used for investigation of higher excitation frequencies. For non periodic movement analysis fast acquisition cameras and postponed treatment are used. DHM are therefore unique and very efficient tool for dynamical characterization of in- plane and out-of-plane response. In this paper we show the basics of the technology and illustrate process engineering and failure analysis using DHM with an example of in and out of plane characterization of movements of a variable capacitor using the stroboscopic mode of acquisition.

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.

Digital holographic microscopy (DHM) is an important technique that may be used for quantitative phase imaging of unstained biological cell samples. Since the DHM technology is not commonly used in clinics or bioscience research labs, at present there is no well-accepted focusing criterion for unstained samples that users can follow while recording image plane digital holograms of cells. The usual sharpness metrics that are useful for auto-focusing of stained cells do not work well for unstained cells as there is no amplitude contrast. In this work, we report a practical method for estimating the best focus plane for unstained cells in the digital hologram domain. The method is based on an interesting observation that for the best focus plane the fringe pattern associated with individual unstained cells predominantly shows phase modulation effect in the form of bending of fringes and minimal amplitude modulation. This criterion when applied to unstained red blood cells shows that the central dip in the doughnut-like phase profile of cells is maximal in this plane. The proposed methodology is helpful for standardizing the usage of DHM technology across different users and application developmentefforts. LAY DESCRIPTION: Digital holographic microscopy (DHM) is slowly but steadily becoming an important microscopy modality and gaining acceptability for basic bio-science research as well as clinical usage. One of the important features of DHM is that it allows users to perform quantitative imaging of unstained transparent cells. Instead of using dyes or fluorescent labelling, DHM systems use quantitative phase as a contrast mechanism which depends on the natural refractive index variation within the cell samples. Since minimal wet lab processing is required in order to image cell samples with a DHM, cells can be imaged in their natural state. While DHM is gaining popularity among users, the imaging protocols across the labs or users need to be standardized in order to make sure that the same quantitative phase parameters are used for tasks such as quantitative phased based cellclassification. One of the important operational tasks for any microscopy work is to focus the sample under study. While focusing comes naturally to users of brightfield microscopes based on image contrast, the focusing is not straightforward when samples are unstained so that they do not offer any amplitude contrast. When performing quantitative phase imaging, defocus can actually change the phase profile of the cell due to near-zone (Fresnel) diffraction effects. So unless a standardized focusing methodology is used, it will be difficult for multiple DHM users (potentially at different sites) to agree on quantitative results out of their phase images. DHM literature has prior works which perform numerical focusing of recovered complex wave-field in the hologram plane to find the best focus plane. However such methods are not user friendly and do not allow user the same focusing experience as in a brightfield microscope. The numerical focusing is therefore a reasonably good method for an optics researcher but not necessarily so for a microscopy technician looking at cell samples with a DHM system in a clinicalsetting. The present work provides a simple focusing criterion for unstained samples that works directly in the hologram domain. The technique is based on an interesting observation that the when an unstained cell sample is in the best-focus plane, its corresponding hologram (or fringe pattern) predominantly shows phase modulation manifested by bending of fringes at the location of the cell. This criterion can be converted into a simple numerical method as we have used to find the best-focus plane using a stack of through focus holograms. We believe that the technique can be used manually by visually observing the holograms or can be converted to an auto-focus algorithm for a motorized DHMsystem.

Super-resolved Mirau digital holography by structured illumination

Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images. Self-referencing geometry is the simplest of the common-path techniques, in which a portion of the object beam itself acts as the reference, leading to compact setups using fewer optical elements. However, it has reduced field of view, and the reference may contain object information. Here, we describe the development of a common-path digital holographic microscope, employing a shearing plate and converting one of the beams into a separate reference by employing a pin-hole. The setup is as compact as self-referencing geometry, while providing field of view as wide as that of a two-beam microscope. The microscope is tested by imaging and quantifying the morphology and dynamics of human erythrocytes.

In this paper we present a newly developed digital transmission holographic microscope. The microscope enables using arbitrarily low coherent illumination (both spatially and temporally) in conjunction with the off-axis holography. The setup of the microscope, its function and the object wave reconstruction procedure are described. The optical sectioning effect, similar to a confocal microscope, resulting from the use of low spatially coherent light source is demonstrated. The microscope has been tailored for studies of living cell dynamics. Time-lapse phase reconstruction series of live cells activities were carried out. The different behavior related to changes in the cell cycle is demonstrated.

Inflammatory bowel diseases (IBD) are inflammatory disorders of the gastrointestinal tract characterized by a chronic relapsing disease course. As uncontrolled intestinal inflammation can result in severe disease complications, recent treatment targets of IBD evolved toward seeking the absence of mucosal and histological inflammation. However, this approach requires adequate histological evaluation of IBD disease activity. The diagnostic challenge of histological examination of intestinal inflammation is documented by the multitude of proposed histological scoring systems. In this context, we review quantitative phase imaging (QPI) techniques such as digital holographic microscopy (DHM) for characterizing intestinal inflammation. DHM determines optical path-length delays in a stain-free manner, thereby providing the tissue refractive index as a biophysical marker that directly correlates to tissue density. Recently, DHM has been successfully applied in cell biology, cancer cell research and infectious-induced cellular alterations. We summarized the capabilities of DHM and related QPI techniques to assess the severity of intestinal inflammation in experimental colitis as well as in colonic samples from human IBD patients. Moreover, we illustrate major advantages of DHM facilitated multimodal evaluation of epithelial wound healing processes as assessed by physical parameters like cell volume, density, thickness and dry mass in vitro. Furthermore, potential limitations of DHM and future utilities of QPI are discussed. In conclusion, DHM represents a promising, easy-to-use quantitative tool to provide accurate and objective assessment of intestinal inflammation and may pave the way towards automated label-free digital pathology and related in vitro cell culture analysis in future.

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.

In this paper, we present a modified transmission digital holographic microscope that can be used to image the state of polarization. The resulting device, called polarization digital holographic microscope (Pol-DHM), records in off-axis geometry the interference between two orthogonally polarized reference waves and the object wave transmitted by a microscopic sample and magnified by a microscope objective. A CCD camera records the resulting hologram. Using a single hologram, we reconstruct separately the amplitude and phase of two wave fronts, which are used to represent the object wave's state of polarization, represented by the azimuth and the phase difference associated to the polarization ellipse. ne proposed method is illustrated with two applications. The first application is to use a 10-times magnification microscope objective to measure the bireftingence induced by internal stresses in transparent materials such as a bended optical fiber. The second application is to use a 20-times microscope objective to image the state of polarization of a thin concrete sample and reveals birefringent properties of the different aggregates found in the concrete.

In this paper we present a modified transmission digital holographic microscope that can be used to image the state of polarization of biological tissue. The resulting device, called polarization-sensitive phase-shifting digital holographic microscope (PS-DHM), records in on-axis geometry the interference between the reference and object beams with the same polarization, but were acquired at two orthogonally polarizations. The object wave transmitted by the biological tissue and magnified by a microscope objective. CCD camera records the two resulting holograms at vertical and horizontal polarizations. The PS-DHM system was upgraded with the liquid crystal variable retarder to perform phase shifts in the reference beam. The polarization-dependent phase-shifted holograms are recorded by rotating the half-wave plates. Using a single hologram, we reconstruct separately the phase map at each polarization, which are used then to represent the phase difference at two orthogonal states of polarization. This phase difference reflects the polarization-dependent refractive index associated to the anisotropy of biological tissue under study. The reconstruction and least-square unwrapping algorithms are used to extract phase information of biological tissues at different polarization states. The birefringence of tissue is obtained from the above-phase distributions. The proposed method is illustrated with applications to three samples: the cancer cells hematoblast, the pollen cells and the heart tissue. The results show that polarization sensitivity exists in the cancer cells hematoblast tissue, the pollen cells itself are not the anisotropic, but their walls are birefringent, and any significant anisotropy in the heart tissue was not watched. These results will provide reference for clinic diagnoses and pathological research.KeywordsDigital holographic microscopyAnisotropyRefractive indexPolarization sensitive imaging

.A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach–Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss Physcomitrella patens. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

For the first time, transmission digital holography microscopy is applied to observe coal palynofacies, which are organic fossil microcomponents contained in the coal grains. The recorded holograms were produced by using microscope lenses with 20x and 40x of lateral magnification respectively, and He-Ne laser of wavelength 594.5 nm. The results show that reflection digital holography microscopy is required for observing relative opaque particles, because the phase recovery is strong diminished by light transmission in those cases. On the other hand, the phase distribution is related to the relief of the particles and the variations of their refraction index. Therefore, a priori information should be necessary to properly relate the phase information to physical features of the particles. Numerical unwrapping procedures are also crucial. Procedures with special requirements can be needed for analysing fast varying phase distributions. However, digital holography microscopy becomes a high performance tool for 3D modelling of fossil particles if the above requirements are enough fulfilled.

We present a method for fast geometrical inspection of micro deep drawing parts. It is based on single-shot two-wavelength contouring digital holographic microscopy (DHM). Within the capturing process, spatial multiplexing is utilized in order to record the two required holograms in a single-shot. For fast evaluation, determining the locations where the object is in focus and stitching all focus object's areas together is achieved digitally without the need for any external intervention using an autofocus algorithm. Thus, the limited depth of focus of the microscope objective is improved. The autofocus algorithm is based on minimizing the total variation (TV) of phase difference residuals of the two-wavelength measurements. In contrast to standard DHM, an object side telecentric microscope objective is used for overcoming the image scaling distortions caused by a conventional microscope objective. The method is used to reconstruct the 3D geometrical shape of a cold drawing micro cup. Experimental results verify the improvement of DHM's depth of focus.

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.