Common-path multimodal three-dimensional fluorescence and phase imaging system

Digital holographic microscope is an ideal tool for quantitative phase contrast imaging of living cells. It yields the thickness distribution of the object under investigation from a single hologram. From a series of holograms the dynamics of the cell under investigation can be obtained. But two-beam digital holographic microscopes has low temporal stability due to uncorrelated phase changes occurring in the reference and object arms. One way to overcome is to use common path techniques, in which, the reference beam is derived from the object beam itself. Both the beams travel along the same path, increasing the temporal stability of the setup. In self-referencing techniques a portion of the object beam is converted into reference beam. It could be achieved by example, using a glass plate to create two laterally sheared versions of the object beam at the sensor, which interfere to produce the holograms/interferograms. This created a common path setup, leading to high temporal stability (~0.6nm). This technique could be used to map cell membrane fluctuations with high temporal stability. Here we provide an overview of our work on the development of temporally stable quantitative phase contrast techniques for dynamic imaging of micro-objects and biological specimen including red blood cells.

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.

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.

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.

Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast, to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA microscope objectives is numerically extended. For example, two different biological cells, floating at different depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full- field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase images of biological cells have been demonstrated.

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

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 microscopy (DHM) is a powerful tool for observation of biological samples. The main advantage of DHM imaging is the possibility to reconstruct the intensity image and the phase image of an observed sample in real-time. The background of phase images is deformed and this deformation is changing during a long-term experiment. We describe here a novel procedure of deformation compensation for sequence of phase images. This novel procedure is based on deformation compensation procedure for a single phase image which uses the least square method. The novel procedure is applicable to compensate deformation during observation in real-time. The proposed procedure is applied to phase images of cells obtained by a transmitted-light digital holographic microscope.

Compact in-line lensfree digital holographic microscope.

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.

Fluorescent images that are blurred by large focus displacements are corrected by phase compensation. This is possible when the defocusing distance is obtained from an external measurement. A multi-modal digital holography that enables both quantitative phase imaging and 2D fluorescent imaging provides complimentary information from the same measuring object, such as living cells with fluorescent labels. In this paper, we adapted Zernike polynomials to correct the defocused fluorescent images, and used coefficients that are linear to the defocusing distances. Experimental results from bead images are presented.

Digital holographic microscopy (DHM), which provides quantitative phase imaging (QPI), has been widely applied in material and biological applications. The performance of DHM technologies relies heavily on computational reconstruction methods to provide accurate phase measurements. For example, non-telecentric DHM systems should compensate for the spherical wavefront associated with a non-telecentric configuration. The size of the ±1 diffraction orders in the hologram spectrum depends inversely on the radius of the curvature of the spherical wavefront introduced by the non-telecentric DHM system. Therefore, one can estimate the radius of curvature of the spherical wavefront by analyzing the hologram spectrum. Here, we outline the steps for the automatic reconstruction of phase images without distortions and with minimum user input from a hologram recorded in a non-telecentric DHM system. The proposed reconstruction approach can be divided into six main steps. The first step automatically selects the +1 diffraction order in the hologram spectrum. Secondly, the spherical wavefront parameters and the interference angle are estimated by analyzing the size and position of the selected +1 order. The third and fourth steps are the spatial filtering of the +1 order and the compensation of the interference angle, respectively. The next step involves the estimation of the center of the spherical wavefront. Finally, there is a fine-tuning step to optimize the estimated parameters and provide a phase image with minimum phase distortions. We have identified the relevant metrics in each step, compared multiple approaches, and selected the one with the higher performance for all our experimental holograms.

In this study, we present a novel single-shot polarization-resolved digital holographic microscope (PDHM) that integrates both double field-of-view (DFoV) and polarization imaging for real-time, live-cell imaging. This innovative system overcomes the limitations of conventional techniques by simultaneously capturing two distinct regions of the sample, each with polarization information. Using a carefully configured Sagnac setup, the reference beam is split into orthogonally polarized components, each modulated with a distinct spatial carrier frequency. This allows the independent reconstruction of polarization-resolved complex fields. At the same time, a beam splitter placed in the object path spatially folds two separate regions of the sample onto the same sensor area, thereby achieving angular multiplexing without the need for additional optics. This configuration allows both spatially and polarization-resolved information in a single acquisition. The system is experimentally validated on live MCF-7 breast cancer cells, where it successfully reconstructs phase anisotropy and internal cellular features in real time. Its ability to capture wide-field, label-free, and polarization-sensitive information in a compact format makes it a promising tool for biomedical imaging and dynamic cell analysis.

We present a digital holographic microscope that has a wide field of view. Off-axis holograms are recorded with a magnified image of microscopic objects and numerically reconstructed by calculation of scalar diffraction in the Fresnel approximation. Holograms are recorded by CCD. The distance between neighbouring pixels of a CCD is only of the order of 5 micrometer. The corresponding maximum resolvable frequency is of the order of 100 linepairs /mm. The maximum angle between the reference and object wave is therefore limited to a few degrees. The higher magnification by an objective lens with the higher power makes the wider angle object beam. Off-axis holograms with the high power objective lens has the limitation of magnification and a field of view. We present a new type of imaging system that overcomes the limitation of magnification and a field of view. It is consisted of an objective lens and additional lens array. It makes the nearly same angle between object beams and a reference beam. The overlapped angles are also less than the maximum limited angle due to CCD pixel size. It also has a maximum field of view which is decided inherently by an objective lens. It therefore overcomes the limitation of the size of CCD.

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.