Active limited-angle tomographic phase microscope

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.

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.

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.

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.

Optofluidic devices offer flexibility for a variety of tasks involving biological specimen. We propose a system for three-dimensional (3D) sensing and identification of biological micro-organisms. This system consists of a microfluidic device along with a digital holographic microscope and relevant statistical recognition algorithms. The microfluidic channel is used to house the micro-organisms, while the holographic microscope and a CCD camera record their digital holograms. The holograms can be computationally reconstructed in 3D using a variety of algorithms, such as the Fresnel transform. Statistical recognition algorithms are used to analyze and identify the micro-organisms from the reconstructed wavefront. Experimental results are presented. Because of computational reconstruction of wavefronts in holographic imaging, this technique offers unique advantages that allow one to image micro-organisms within a deep channel while removing the inherent microfluidic-induced aberration through interferometery.

Recent developments in ultra-high-definition (UHD) displays have a major impact on holographic displays as well as conventional display systems. A spatial light modulator (SLM) can reproduce hologram data through wavefront modulation of the incident light, and a holographic printer can record a high-quality hologram by sequentially recording a number of holograms reproduced by the SLM on a holographic material. As UHD (4K) resolution SLMs have been popularized, higher quality holograms can be reproduced compared to previous 2K resolution SLMs. When applied to the holographic printer, it is possible to manufacture holographic optical elements (HOEs) having a multifunctioning property and a wide field of view. In this paper, we introduce the holographic printer system using an amplitude UHD SLM and its applications. The holographic printer consists of optical systems to generate high-quality hologram from UHD SLM and mechanical systems to record the hologram on holographic material. The details of the total system are introduced. Furthermore, we introduce a holographic near-eye display system using UHD SLM and an image combiner HOE which is manufactured by the holographic printer.

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.

A method to improve the radial resolution using special structured light is proposed in the field of digital holographic microscopy (DHM). A specimen is illuminated with circular symmetrical structured light that makes the spectrum have radial movement, so that high frequency components of the specimen are moved into the passband of the receiver to overcome the diffraction limit. In the DHM imaging system, Computer Generated Hologram (CGH) technology is used to generate the required structured light grating. Then the grating is loaded into a spatial light modulator (SLM) to obtain specific structured illumination. After recording the hologram, digital reconstruction, for the microstructure of a binary optical element that needs to observe radial distribution, the radial resolution of the specimen is improved experimentally compare it with the result of one-dimensional sinusoidal structured light imaging. And a method of designing structured light is presented.

Liquid crystal on silicon (LCoS) spatial light modulators (SLMs) are versatile scientific tools relevant to an increasingly wide variety of research and technological applications including digital holography, wavefront correction, optical tweezing, and non-mechanical beam steering to name a few. Since SLMs are used in a multitude of different ways, some aspects of device performance (e.g., response time) are crucial to certain applications while being irrelevant to others. In this work we couple our standard SLMs with a thermo-electric cooler, allowing for tunability of the device operating temperature from 0° to 75 °C. We show that there is an inherent tradeoff between the liquid crystal response time and the phase stability of an SLM, and that the operating temperature offers a means of controlling this tradeoff. Furthermore, this paper aims to provide the reader with a brief but thorough explanation of SLM operating principles and device structure, defines the performance metrics of the SLM, and provides a methodology for measuring the specifications. By allowing control over the SLM operating temperature and detailing how temperature affects device functionality, SLM users are afforded greater experimental flexibility and will be better able to tailor the performance of their device for the given project or application at hand.

A microscope operating in Digital Holographic Microscopy (DHM) and classical widefield epi-fluorescence microscopy in a time sequential manner is developed to study morphological alterations of mouse myoblast cells under simulated microgravity in real time.

We report speckle-free digital holographic phase microscopy (DHPM) and tomography using partially spatially coherent monochromatic light with improved image sharpness, resolution and phase noise. Effects of high spatial and temporal coherence of laser light in DHPM, such as speckle effect, spurious fringe generation are discussed and these effects lead to poor phase reconstruction. We present our recent work on the speckle-free phase microscopy and spatial coherence gated tomography with improved phase reconstruction and resolution.

.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.

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.