On-chip differential interference contrast microscopy using lensless digital holography.
Ontology highlight
ABSTRACT:
SUBMITTER: Oh C
PROVIDER: S-EPMC2859902 | biostudies-other | 2010 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Differential interference contrast (DIC) microscopy is widely used for observing unstained biological samples that are otherwise optically transparent. Combining this optical technique with machine vision could enable the automation of many life science experiments; however, identifying relevant features under DIC is challenging. In particular, precise tracking of cell boundaries in a thick ( ) slice of tissue has not previously been accomplished. We present a novel deconvolution algorithm that achieves the state-of-the-art performance at identifying and tracking these membrane locations. Our proposed algorithm is formulated as a regularized least squares optimization that incorporates a filtering mechanism to handle organic tissue interference and a robust edge-sparsity regularizer that integrates dynamic edge tracking capabilities. As a secondary contribution, this paper also describes new community infrastructure in the form of a MATLAB toolbox for accurately simulating DIC microscopy images of in vitro brain slices. Building on existing DIC optics modeling, our simulation framework additionally contributes an accurate representation of interference from organic tissue, neuronal cell-shapes, and tissue motion due to the action of the pipette. This simulator allows us to better understand the image statistics (to improve algorithms), as well as quantitatively test cell segmentation and tracking algorithms in scenarios, where ground truth data is fully known.
| S-EPMC5839128 | biostudies-literature
Project description:Differential image contrast (DIC), through the numerical managing and manipulation of complex wavefronts obtained by digital holography (DH), is investigated. We name the approach Dynamical Differential Holographic Image Contrast (DDHIC). DDHIC dispenses from special optics and/or complex setup configurations with moveable components, as usually occurs in classical DIC, that is not well-suited for investigating objects experiencing dynamic evolution during the measurement. In fact, the technique presented here, is useful for floating samples since it allows, from a single recording, to set a posteriori the best conditions for DIC imaging in conjunction with the numerical focusing feature of DH. By DDHIC, the movies can be easily built-up to offering dynamic representation of phase-contrast along all directions, thus improving the visualization. Furthermore, the dynamic representation is useful for making the proper choice of other key parameters of DIC such as the amount of shear and the bias, with the aim to optimize the visualized phase-contrast imaging as favorite representation for bio-scientists. Investigation is performed on various biological samples.
| S-EPMC3038448 | biostudies-other
Project description:High-resolution wide field-of-view (FOV) microscopic imaging plays an essential role in various fields of biomedicine, engineering, and physical sciences. As an alternative to conventional lens-based scanning techniques, lensfree holography provides a new way to effectively bypass the intrinsical trade-off between the spatial resolution and FOV of conventional microscopes. Unfortunately, due to the limited sensor pixel-size, unpredictable disturbance during image acquisition, and sub-optimum solution to the phase retrieval problem, typical lensfree microscopes only produce compromised imaging quality in terms of lateral resolution and signal-to-noise ratio (SNR). Here, we propose an adaptive pixel-super-resolved lensfree imaging (APLI) method which can solve, or at least partially alleviate these limitations. Our approach addresses the pixel aliasing problem by Z-scanning only, without resorting to subpixel shifting or beam-angle manipulation. Automatic positional error correction algorithm and adaptive relaxation strategy are introduced to enhance the robustness and SNR of reconstruction significantly. Based on APLI, we perform full-FOV reconstruction of a USAF resolution target (~29.85?mm2) and achieve half-pitch lateral resolution of 770?nm, surpassing 2.17 times of the theoretical Nyquist-Shannon sampling resolution limit imposed by the sensor pixel-size (1.67µm). Full-FOV imaging result of a typical dicot root is also provided to demonstrate its promising potential applications in biologic imaging.
| S-EPMC5603528 | biostudies-literature
Project description:Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue. Our aim is to improve the DIC microscope's phase measurement to remove the phase bias that occurs in the presence of strong scattering. A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution. We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish. Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.
| S-EPMC7734411 | biostudies-literature
Project description:Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.
| S-EPMC3785893 | biostudies-literature
Project description:UnlabelledThe Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca(2+) and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease - Tlag) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow Trelease, in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences.ImportanceSpores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination.
| S-EPMC4524193 | biostudies-literature
Project description:A multi-angle lensfree holographic imaging platform that can accurately characterize both the axial and lateral positions of cells located within multi-layered micro-channels is introduced. In this platform, lensfree digital holograms of the micro-objects on the chip are recorded at different illumination angles using partially coherent illumination. These digital holograms start to shift laterally on the sensor plane as the illumination angle of the source is tilted. Since the exact amount of this lateral shift of each object hologram can be calculated with an accuracy that beats the diffraction limit of light, the height of each cell from the substrate can be determined over a large field of view without the use of any lenses. We demonstrate the proof of concept of this multi-angle lensless imaging platform by using light emitting diodes to characterize various sized microparticles located on a chip with sub-micron axial and lateral localization over approximately 60 mm(2) field of view. Furthermore, we successfully apply this lensless imaging approach to simultaneously characterize blood samples located at multi-layered micro-channels in terms of the counts, individual thicknesses and the volumes of the cells at each layer. Because this platform does not require any lenses, lasers or other bulky optical/mechanical components, it provides a compact and high-throughput alternative to conventional approaches for cytometry and diagnostics applications involving lab on a chip systems.
| S-EPMC2898907 | biostudies-literature
Project description:Left-right asymmetry is a fundamental feature of body plans, but its formation mechanisms and roles in functional lateralization remain unclear. Accumulating evidence suggests that left-right asymmetry originates in the cellular chirality. However, cell chirality has not yet been quantitatively investigated, mainly due to the absence of appropriate methods. Here we combine 3D Riesz transform-differential interference contrast (RT-DIC) microscopy and computational kinematic analysis to characterize chiral cellular morphology and motility. We reveal that filopodia of neuronal growth cones exhibit 3D left-helical motion with retraction and right-screw rotation. We next apply the methods to amoeba Dictyostelium discoideum and discover right-handed clockwise cell migration on a 2D substrate and right-screw rotation of subcellular protrusions along the radial axis in a 3D substrate. Thus, RT-DIC microscopy and the computational kinematic analysis are useful and versatile tools to reveal the mechanisms of left-right asymmetry formation and the emergence of lateralized functions.
| S-EPMC5736583 | biostudies-literature
Project description:Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.
| S-EPMC2753545 | biostudies-literature
Project description:We demonstrate digital plasmonic holography for direct in-plane imaging with propagating surface-plasmon waves. Imaging with surface plasmons suffers from the lack of simple in-plane lenses and mirrors. Lens-less digital holography techniques, however, rely on digitally decoding an interference pattern between a reference wave and an object wave. With far-field diffractive optics, this decoding scheme provides a full recording, i.e., a hologram, of the amplitude and phase of the object wave, giving three-dimensional information from a two-dimensional recording. For plasmonics, only a one-dimensional recording is needed, and both the phase and amplitude of the propagating plasmons can be extracted for high-resolution in-plane imaging. Here, we demonstrate lens-less, point-source digital plasmonic holography using two methods to record the plasmonic holograms: a dual-probe near-field scanning optical microscope and lithographically defined circular fluorescent screens. The point-source geometry gives in-plane magnification, allowing for high-resolution imaging with relatively lower-resolution microscope objectives. These results pave the way for a new form of in-plane plasmonic imaging, gathering the full complex wave, without the need for plasmonic mirrors or lenses.
| S-EPMC6107013 | biostudies-literature