Differential Phase Contrast Microscopy Research Articles

Neural space-time model for dynamic multi-shot imaging.

Computational imaging reconstructions from multiple measurements that are captured sequentially often suffer from motion artifacts if the scene is dynamic. We propose a neural space-time model (NSTM) that jointly estimates the scene and its motion dynamics, without data priors or pre-training. Hence, we can both remove motion artifacts and resolve sample dynamics from the same set of raw measurements used for the conventional reconstruction. We demonstrate NSTM in three computational imaging systems: differential phase-contrast microscopy, three-dimensional structured illumination microscopy and rolling-shutter DiffuserCam. We show that NSTM can recover subcellular motion dynamics and thus reduce the misinterpretation of living systems caused by motion artifacts.

Single-shot differential phase contrast microscopy using ring-shaped polarisation multiplexing illumination.

We propose a differential phase contrast microscopy that enables single-shot phase imaging for unstained biological samples. The proposed approach employs a ring-shaped LED array for polarisation multiplexing illumination and a polarisation camera for image acquisition. As such, multiple images of different polarisation angles can be simultaneously captured with a single shot. Through polarisation demultiplexing, the sample phase can therefore be recovered from the single-shot measurement. Both simulations and experiments demonstrate the effectiveness of the approach. We also demonstrate that ring-shaped illumination enables higher contrast and lower-distortion imaging results than disk-shaped illumination does. The proposed single-shot approach potentially enables phase contrast imaging for live cell samples in vitro. Lay Description: We propose a microscopy that enables imaging of transparent samples, unstained cells, etc. We demonstrate that the proposed method enables higher contrast and lower-distortion imaging results than conventional methods, and significantly improves imaging efficiency. The proposed method potentially enables dynamic imaging for live cell samples in vitro.

Comparison of differential phase Contrast, Lorentz and non-Lorentz transmission electron microscopy for microstructural and magnetic imaging of alnico magnet

In this work, non-Lorentz, Lorentz transmission electron microscopy (LTEM) and differential phase contrast (DPC) methods were used to observe the phase and magnetic structure of the alnico magnet. All observations were performed on alnico-5 in transverse orientation. The results showed the magnetic contrast of the domain walls in the conventional TEM images despite the effects of the lens magnetic field, which was verified and explained using DPC. Experimental methodology and sample results are summarized with advice for further research.

Untrained deep learning-based differential phase-contrast microscopy.

Quantitative differential phase-contrast (DPC) microscopy produces phase images of transparent objects based on a number of intensity images. To reconstruct the phase, in DPC microscopy, a linearized model for weakly scattering objects is considered; this limits the range of objects to be imaged, and requires additional measurements and complicated algorithms to correct for system aberrations. Here, we present a self-calibrated DPC microscope using an untrained neural network (UNN), which incorporates the nonlinear image formation model. Our method alleviates the restrictions on the object to be imaged and simultaneously reconstructs the complex object information and aberrations, without any training dataset. We demonstrate the viability of UNN-DPC microscopy through both numerical simulations and LED microscope-based experiments.

Polarization Switching and Correlated Phase Transitions in Fluorite-Structure ZrO2 Nanocrystals.

Unconventional ferroelectricity in fluorite-structure oxides enables tremendous opportunities in nanoelectronics owing to their superior scalability and silicon compatibility. However, their polarization order and switching process remain elusive due to the challenges of visualizing oxygen ions in nanocrystalline films. In this work, the oxygen shifting during polarization switching and correlated polar-nonpolar phase transitions are directly captured among multiple metastable phases in freestanding ZrO2 thin films by low-dose integrated differential phase-contrast scanning transmission electron microscopy (iDPC-STEM). Bidirectional transitions between antiferroelectric and ferroelectric orders and interfacial polarization relaxation are clarified at unit-cell scale. Meanwhile, polarization switching is strongly correlated with Zr-O displacement in reversible martensitic transformation between monoclinic and orthorhombic phases and two-step tetrahedral-to-orthorhombic phase transition. These findings provide atomic insights into the transition pathways between metastable polymorphs and unravel the evolution of polarization orders in (anti)ferroelectric fluorite oxides.

Dislocation-twin interaction in medium entropy alloy containing a high density of oxygen interstitials

Dislocation-twin interactions play important roles in tuning the plastic deformation of materials. Here, we report the great influence of interstitial oxygen atoms on dislocation-twin interactions in NiCoCrO medium entropy alloy through in-situ transmission electron microscopy mechanical testing, atomic resolution energy-dispersive X-ray spectroscopy characterizations, and integrated differential phase-contrast scanning transmission electron microscopy imaging. The spreading lattice distortion in medium-entropy alloy was found to enable large oxygen interstitial solubility. Oxygen atoms occupied both the tetrahedral and octahedral interstitial sites, and tended to be enriched at defects such as twin boundaries and stacking faults. Oxygen-enriched regions near the twin boundary hindered dislocations from direct transmitting or cross-slip into another twin plane during plastic deformation, resulting in significant dislocation entanglement, partial dissociation of dislocations, and the deviation of slip directions. Our results give insight into the strengthening mechanisms associated with the synergetic effects of twin boundary and interstitial atoms.

Single-shot color-coded LED microscopy for quantitative differential phase contrast imaging

Common quantitative differential phase contrast microscopy imaging usually uses multistep circular or annular illuminations and color multiplexed illuminations, bringing about low capture efficiency and poor phase reconstruction, respectively. We present a new single-shot quantitative differential phase contrast microscopy imaging method, which uses R/B-G annular LED multiplexed illumination to achieve better phase reconstruction by capturing 1 colored image. By deriving the phase transfer function of the illumination and simulation verification, the superiority of it is confirmed. Through phase reconstruction simulations for circular step and gradual samples, we verify that the R/B-G annular multiplexed illumination has better axial phase reconstruction accuracy and weaker lateral anisotropy than other color multiplexed illuminations. As a demonstration, quantitative phase imaging is performed on Quantitative Phase Target, validating the axial accuracy, lateral resolution, and lateral anisotropy of our method. Imaging and phase reconstruction were also performed on mouse kidney tissue slice and motional silica microspheres to visually express the utility and practicability of this method. Our research provides a better color multiplexed avenue for the real-time quantitative phase imaging in dispersion-free, weak-phase samples, especially living biological tissues.

Quantitative differential phase contrast phase reconstruction for sparse samples

Quantitative differential phase contrast (qDPC) microscopy can achieve phase imaging of unlabeled cell samples with high spatial resolution. However, qDPC imaging is easily affected by the noise generated in the experiments. In this manuscript, the L0-norm regularization was introduced into qDPC phase reconstruction for sparse samples (L0-qDPC), including the quantitative phase target and cell samples. After the detailed comparison of phase fidelity, imaging contrast, resolution, and convergence rate, L0-qDPC method can provide a stable qDPC phase imaging without parameter adjustment for sparse samples due to strong constraint and good robustness of L0-norm based on sparse prior, compared with the L2-norm and total variation regularization.

Creating two-dimensional solid helium via diamond lattice confinement

The universe abounds with solid helium in polymorphic forms. Therefore, exploring the allotropes of helium remains vital to our understanding of nature. However, it is challenging to produce, observe and utilize solid helium on the earth because high-pressure techniques are required to solidify helium. Here we report the discovery of room-temperature two-dimensional solid helium through the diamond lattice confinement effect. Controllable ion implantation enables the self-assembly of monolayer helium atoms between {100} diamond lattice planes. Using state-of-the-art integrated differential phase contrast microscopy, we decipher the buckled tetragonal arrangement of solid helium monolayers with an anisotropic nature compressed by the robust diamond lattice. These distinctive helium monolayers, in turn, produce substantial compressive strains to the surrounded diamond lattice, resulting in a large-scale bandgap narrowing up to ~2.2 electron volts. This approach opens up new avenues for steerable manipulation of solid helium for achieving intrinsic strain doping with profound applications.

Effects of an inhomogeneous electron density distribution on the hydrogen distribution in TiZrTaNbAl multi-principal element alloys

A body-centered cubic equiatomic TiZrTaNbAl multi-principal element alloy (MPEA) with elemental fluctuations was investigated to further understand the relationship between the microstructure and hydrogen distribution. In this study, a composition dependence of the hydrogen distribution was observed in the TiZrTaNbAl MPEA. An inhomogeneous electron density distribution of the MPEA was revealed by advanced differential phase-contrast scanning electron microscopy (DPC-STEM) for the first time. The results showed that the electron density has a significant effect on the hydrogen distribution in TiZrTaNbAl MPEAs. This work provides new insight into the design of materials with high hydrogen storage capacity and high hydrogen embrittlement resistance.

Unblocking Ion-occluded Pore Channels in Poly(triazine imide) Framework for Proton Conduction.

Poly(triazine imide) or PTI is an ordered graphitic carbon nitride hosting Å-scale pores attractive for selective molecular transport. AA'-stacked PTI layers are synthesized by ionothermal route during which ions occupy the framework and occlude the pores. Synthesis of ion-free PTI hosting AB-stacked layers has been reported, however, pores in this configuration are blocked by the neighboring layer. The unavailability of open pore limits application of PTI in molecular transport. Herein, we demonstrate acid treatment for ion depletion which maintains AA' stacking and results in open pore structure. We provide first direct evidence of ion-depleted open pores by imaging with the atomic resolution using integrated differential phase-contrast scanning transmission electron microscopy. Depending on the extent of ion-exchange, AA' stacking with open channels and AB stacking with closed channels are obtained and imaged for the first time. The accessibility of open channels is demonstrated by enhanced proton transport through ion depleted PTI.

Dual-modality quantitative phase-contrast microscopy based on pupil phase modulation (DQPCM)

Phase-contrast optical microscopy that converts phase modulation of transparent samples into intensity modulation plays an indispensable role in biomedical research. In this study, we propose a dual-modality quantitative phase contrast microscopic (DQPCM) platform that integrates quantitative differential interference phase-contrast microscopy (qDIC-PCM) and quantitative Zernike phase-contrast microscopy (qZ-PCM) in the same microscopy. Both imaging modalities are realized through spatial light modulator (SLM) based pupil phase modulation, and switching between the two imaging modes is performed by rotating two polarizers. Both imaging modalities have the merits of high stability and low coherent noise due to their common path interference structure and partially coherent illumination. The experimental results indicate that qDIC-PCM is superior to qZ-PCM in quantitative phase imaging of samples with certain scattering, while qZ-PCM has higher spatial resolution than qDIC-PCM. The proposed technique will be very helpful for imaging the dynamic processes of biological samples with high spatiotemporal resolution.

Time-lapse imaging using dual-color coded quantitative differential phase contrast microscopy.

.Significance: Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity measurements. However, for live cell imaging, motion artifacts and image acquisition time are important issues. For live cell imaging, a large number of intensity measurements can limit the imaging quality and speed. The minimum number of intensity measurements in qDPC can greatly enhance performance for live imaging.Aim: To obtain high-contrast, isotropic qDPC images with two intensity measurements and perform time-lapse imaging of biological samples.Approach: Based on the color-coded design, a dual-color linear-gradient pupil is proposed to achieve isotropic phase contrast response with two intensity measurements. In our method, the purpose of designing a dual-color coded pupil is twofold: first, to obtain a linear amplitude gradient for asymmetric illumination, which is required to get a circular symmetry of transfer function, and second, to reduce the required number of frames for phase retrieval.Results: To demonstrate the imaging performance of our system, standard microlens arrays were used as samples. We performed time-lapse quantitative phase imaging of rat astrocytes under a low-oxygen environment. Detailed morphology and dynamic changes such as the apoptosis process and migration of cells were observed.Conclusions: It is shown that dual-color linear-gradient pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only two-axis measurements. The reduced number of frames helps in achieving faster imaging speed as compared to the typical qDPC system. The imaging performance of our system is evaluated by time-lapse imaging of rat astrocytes. Different morphological changes in cells during their life cycle were observed in terms of quantitative phase change values.

Automated stain-free histomorphometry of peripheral nerve by contrast-enhancing techniques and artificial intelligence

BackgroundTraditional histopathologic evaluation of peripheral nerve using brightfield microscopy is resource-intensive, necessitating complex sample preparation. Label-free imaging techniques paired with artificial intelligence-based image reconstruction and segmentation may facilitate peripheral nerve histomorphometry. New methodHerein, the utility of label-free phase contrast techniques paired with artificial intelligence-based image processing for imaging of mammalian peripheral nerve is demonstrated. ResultsFresh frozen murine sciatic nerve sections were imaged in transmission modalities using differential interference and phase contrast microscopy and in epifluorescent modality following staining with myelin-specific dye. Deep learning was employed to predict epifluorescent images from transmitted phase contrast images, and machine learning employed for automated segmentation of myelinated axons for reporting of axons counts and g-ratios. Comparison with existing methodsConventional peripheral nerve histomorphometry employs resource intensive resin embedding, ultra-microtome sectioning, and staining steps. Herein we demonstrate feasibility of high-throughput nerve histomorphometry via label-free phase contrast imaging of frozen sections. ConclusionsClinical applications of label-free phase contrast microscopy paired with deep learning algorithms are discussed.

Atomic-resolved structural and electric field analysis of the passivation interface of MIS-HEMTs

The passivation interfaces of GaN-based MIS-HEMTs with Si3N4 and ZrO2/Si3N4 bilayers were investigated through atomic resolution scanning transmission electron microscope–energy dispersive spectroscopy–differential phase contrast microscopy methods. It is found that the Si3N4/GaN interface exhibits atomic disorder fluctuation, and the GaN surface is discontinuous at the depth of 1–2 atomic layers. An oxide layer of ∼2 nm is formed at the ZrO2/GaN interface, and the GaN surface is atomically flat. Furthermore, the local minimum of the potential is located at the Si3N4/GaN interface, while it is distributed in the GaN side at the ZrO2/GaN interface. The electric field or potential distribution is affected by the crystal orientation of the polycrystalline ZrO2 layer. Finally, the difference in passivation mechanism is discussed.

Morphology analysis of unlabeled red blood cells based on quantitative differential phase contrast microscopy.

The current classical blood smear technique to observe the morphology of single red blood cells (RBCs) for classification is a laborious and error-prone process. To objectively evaluate the morphology of blood cells, we established a method of computational imaging based on a programmable light emitting diode array. By using quantitative differential phase contrast (qDPC), we characterized the morphology of unlabeled RBCs as well as blood smears. By focusing on comparing the difference of imaging between unlabeled RBCs and stained RBCs under multimode microscopic imaging technology, we demonstrated that qDPC could clearly differentiate discocytes and spherocytes in both unlabeled RBCs and blood smears. The phase map provided by quantitative phase imaging further enhanced the classification accuracy. According to statistical analysis from morphological indexes, the qDPC imaging has a significantly improvement in non-circularity, texture inhomogeneity and equivalent diameters of cells. Thus, this method has a significant superiority in the capability to analyze the morphology of RBCs and could be applied to clinical assays for determining morphological, functional, and structural deterioration of RBCs.

Self-calibrated 3D differential phase contrast microscopy with optimized illumination.

3D phase imaging recovers an object's volumetric refractive index from intensity and/or holographic measurements. Partially coherent methods, such as illumination-based differential phase contrast (DPC), are particularly simple to implement in a commercial brightfield microscope. 3D DPC acquires images at multiple focus positions and with different illumination source patterns in order to reconstruct 3D refractive index. Here, we present a practical extension of the 3D DPC method that does not require a precise motion stage for scanning the focus and uses optimized illumination patterns for improved performance. The user scans the focus by hand, using the microscope's focus knob, and the algorithm self-calibrates the axial position to solve for the 3D refractive index of the sample through a computational inverse problem. We further show that the illumination patterns can be optimized by an end-to-end learning procedure. Combining these two, we demonstrate improved 3D DPC with a commercial microscope whose only hardware modification is LED array illumination.

Low-coherence quantitative differential phase-contrast microscopy using Talbot interferometry.

This paper presents a simple, cost-efficient, and highly stable quantitative differential phase-contrast (PC) microscopy based on Talbot interferometry. The proposed system is composed of an optical microscope coupled with a pair of Ronchi amplitude gratings that utilizes a light-emitting diode as a low temporal coherence light source. The quantitative differential PC images of the microscopic transparent samples are reconstructed by analyzing the deformation of moiré patterns using a phase-shifting procedure. Low temporal coherence leads to eliminating speckle noise and undesirable interferences to obtain high-quality images. The spatial phase stability of the system is investigated and compared to two other common-path interferometers. Additionally, the performance of the method is verified by the experimental results of a standard resolution test target and phase biological samples.

An Effective Deep Learning Framework for Cell Segmentation in Microscopy Images.

Cell segmentation is a common step in cell behavior analysis. Reliably and automatically segmenting cells in microscopy images remains challenging, especially in differential inference contrast microscopy images and phase-contrast microscopy images. In this paper, we propose a deep learning solution combining a Mask RCNN architecture with Shape-Aware Loss to produce cell instance segmentation. Our approach outperforms prior works in cell segmentation, achieving an IOU of 91.91% on the DIC-C2DH-HeLa dataset and an IOU of 94.93 % on the PhC-C2DH-U373 dataset. Our framework can calculate cell instance segmentation masks from both types of microscopy images without any additional post-processing.Clinical Relevance - The proposed approach produces accurate instance segmentation in Differential Inference Contrast and Phase-Contrast microscopy images. The segmentation results can be reliably used in cell behavior analysis and cell tracking.

High-precision 3D drift correction with differential phase contrast images.

Single molecule localization microscopy (SMLM) usually requires long image acquisition time at the order of minutes and thus suffers from sample drift, which deteriorates image quality. A drift estimation method with high precision is typically used in SMLM, which can be further combined with a drift compensation device to enable active microscope stabilization. Among all the reported methods, the drift estimation method based on bright-field image correlation requires no extra sample preparation or complicated modification to the imaging setup. However, the performance of this method is limited by the contrast of bright-field images, especially for the structures without sufficient features. In this paper, we proposed to use differential phase contrast (DPC) microscopy to enhance the image contrast and presented a 3D drift correction method with higher precision and robustness. This DPC-based drift correction method is suitable even for biological samples without clear morphological features. We demonstrated that this method can achieve a correction precision of < 6 nm in both the lateral direction and axial direction. Using SMLM imaging of microtubules, we verified that this method provides a comparable drift estimation performance as redundant cross-correlation.