High-resolution Phase Contrast Imaging Research Articles

Single Electron Self-coherence and Its Wave/Particle Duality in the Electron Microscope.

Intensities in high-resolution phase-contrast images from electron microscopes build up discretely in time by detecting single electrons. A wave description of pulse-like coherent-inelastic interaction of an electron with matter implies a time-dependent coexistence of coherent partial waves. Their superposition forms a wave package by phase decoherence of 0.5 - 1 radian with Heisenbergs energy uncertainty ΔEH = ħ/2 Δt-1 matching the energy loss ΔE of a coherent-inelastic interaction and sets the interaction time Δt. In these circumstances, the product of Planck's constant and the speed of light hc is given by the product of the expression for temporal coherence λ2/Δλ and the energy loss ΔE. Experimentally, the self-coherence length was measured by detecting the energy-dependent localization of scattered, plane matter waves in surface proximity exploiting the Goos-Hänchen shift. Chromatic-aberration Cc-corrected electron microscopy on boron nitride (BN) proves that the coherent crystal illumination and phase contrast are lost if the self-coherence length shrinks below the size of the crystal unit cell at ΔE > 200 eV. In perspective, the interaction time of any matter wave compares with the lifetime of a virtual particle of any elemental interaction, suggesting the present concept of coherent-inelastic interactions of matter waves might be generalizable.

Deep learning-based segmentation of subcellular organelles in high-resolution phase-contrast images.

Although quantitative analysis of biological images demands precise extraction of specific organelles or cells, it remains challenging in broad-field grayscale images, where traditional thresholding methods have been hampered due to complex image features. Nevertheless, rapidly growing artificial intelligence technology is overcoming obstacles. We previously reported the fine-tuned apodized phase-contrast microscopy system to capture high-resolution, label-free images of organelle dynamics in unstained living cells (Shimasaki, K. et al. (2024). Cell Struct. Funct., 49: 21-29). We here showed machine learning-based segmentation models for subcellular targeted objects in phase-contrast images using fluorescent markers as origins of ground truth masks. This method enables accurate segmentation of organelles in high-resolution phase-contrast images, providing a practical framework for studying cellular dynamics in unstained living cells.Key words: label-free imaging, organelle dynamics, apodized phase contrast, deep learning-based segmentation.

Immuno-surveillance and protection of the human cochlea.

Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

Automated human induced pluripotent stem cell colony segmentation for use in cell culture automation applications

Human induced pluripotent stem cells (hiPSCs) have demonstrated great promise for a variety of applications that include cell therapy and regenerative medicine. Production of clinical grade hiPSCs requires reproducible manufacturing methods with stringent quality-controls such as those provided by image-controlled robotic processing systems. In this paper we present an automated image analysis method for identifying and picking hiPSC colonies for clonal expansion using the CellXTM robotic cell processing system. This method couples a light weight deep learning segmentation approach based on the U-Net architecture to automatically segment the hiPSC colonies in full field of view (FOV) high resolution phase contrast images with a standardized approach for suggesting pick locations. The utility of this method is demonstrated using images and data obtained from the CellXTM system where clinical grade hiPSCs were reprogrammed, clonally expanded, and differentiated into retinal organoids for use in treatment of patients with inherited retinal degenerative blindness.

High-resolution phase-contrast X-ray imaging for biological soft tissue

Minimizing the distance between sample and detector achieves high spatial resolution while maintaining high contrast of biological tissue.

X-ray volumetric quantitative phase imaging by Foucault differential filtering with linear scanning

Non-interferometric X-ray quantitative phase imaging (XQPI), much simpler than the interferometric scheme, has provided high-resolution and reliable phase-contrast images. We report on implementing the volumetric XQPI images using concurrent-bidirectional scanning of the orthogonal plane on the optical axis of the Foucault differential filter; we then extracted data in conjunction with the transport-intensity equation. The volumetric image of the laminate microstructure of the gills of a fish was successfully reconstructed to demonstrate our XQPI method. The method can perform 3D rendering without any rotational motion for laterally extended objects by manipulating incoherent X-rays using the pinhole array.

Reflectional quantitative phase-contrast microscopy (RQPCM) with annular epi-illumination.

Quantitative phase microscopy (QPM) is a label-free microscopic technique that exploits the phase of a wave passing through a sample; hence, it has been applied to many fields, including biomedical research and industrial inspection. However, the high spatiotemporal resolution imaging of reflective samples still challenges conventional transmission QPM. In this paper, we propose reflectional quantitative phase-contrast microscopy based on annular epi-illumination of light-emitting diodes. The unscattered wave from the sample is successively phase-retarded by 0, π/2, π, and 3π/2 through a spatial light modulator, and high-resolution phase-contrast images are obtained, revealing the finer structure or three-dimensional tomography of reflective samples. With this system, we have quantitatively obtained the contour of tissue slices and silicon semiconductor wafers. We believe that the proposed system will be very helpful for the high-resolution imaging of industrial devices and biomedical dynamics.

Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call “specimen induced decoherence”, is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.

High-Resolution X-ray Phase-Contrast Imaging and Sensory and Rheometer Tests in Cooked Edamame.

Although several reports exist on the use of X-ray analysis in vegetables and fruits to examine internal disorders, cavities, and porosity, information on X-ray analysis of qualities, such as texture, is lacking as well as information on X-ray analysis of legumes. Therefore, this study aimed to perform X-ray analysis with sensory and rheometer tests in cooked vegetable soybean (edamame). Edamame is popular worldwide due to its deliciousness and nutritional value. Vascular structures and cracks around them were clearly visualized using X-ray phase-contrast computed tomography (CT) imaging. In addition, we observed the fine structure of the seed coat, which could be important for seed development, germination, and processing. The density in the edamame beans declined as the boiling time increased, promoting a reduction in hardness described in sensory and rheometer tests. The reduction in density proceeded from the gap between cotyledons, the opposite side of the hypocotyl, and the crack. Collectively, the findings show that the high-resolution X-ray phase-contrast CT imaging conducted in a nondestructive manner may help in effectively evaluating the quality of vegetables and in observing the internal structures related to plant development.

High-resolution and sensitivity bi-directional x-ray phase contrast imaging using 2D Talbot array illuminators.

Two-dimensional (2D) Talbot array illuminators (TAIs) were designed, fabricated, and evaluated for high-resolution high-contrast x-ray phase imaging of soft tissue at 10-20 keV. The TAIs create intensity modulations with a high compression ratio on the micrometer scale at short propagation distances. Their performance was compared with various other wavefront markers in terms of period, visibility, flux efficiency, and flexibility to be adapted for limited beam coherence and detector resolution. Differential x-ray phase contrast and dark-field imaging were demonstrated with a one-dimensional, linear phase stepping approach yielding 2D phase sensitivity using unified modulated pattern analysis (UMPA) for phase retrieval. The method was employed for x-ray phase computed tomography reaching a resolution of 3 µm on an unstained murine artery. It opens new possibilities for three-dimensional, non-destructive, and quantitative imaging of soft matter such as virtual histology. The phase modulators can also be used for various other x-ray applications such as dynamic phase imaging, super-resolution structured illumination microscopy, or wavefront sensing.

Micro- and Nano-Scales Three-Dimensional Characterisation of Softwood.

Understanding the mechanical response of cellular biological materials to environmental stimuli is of fundamental importance from an engineering perspective in composites. To provide a deep understanding of their behaviour, an exhaustive analytical and experimental protocol is required. Attention is focused on softwood but the approach can be applied to a range of cellular materials. This work presents a new non-invasive multi-scale approach for the investigation of the hygro-mechanical behaviour of softwood. At the TOMCAT beamline of the Paul Scherrer Institute, in Switzerland, the swelling behaviour of softwood was probed at the cellular and sub-cellular scales by means of 3D high-resolution phase-contrast X-ray imaging. At the cellular scale, new findings in the anisotropic and reversible swelling behaviour of softwood and in the origin of swelling hysteresis of porous materials are explained from a mechanical perspective. However, the mechanical and moisture properties of wood highly depend on sub-cellular features of the wood cell wall, such as bordered pits, yielding local deformations during a full hygroscopic loading protocol.

Single-shot x-ray phase-contrast and dark-field imaging based on coded binary phase mask

We introduce a coded-mask-based multi-contrast imaging method for high-resolution phase-contrast and dark-field imaging. The method uses a binary phase mask designed to provide an ultra-high-contrast pattern and reference-free single-shot measurement and an algorithm based on maximum-likelihood optimization and automatic differentiation to perform simultaneous reconstruction of absorption, phase, and dark-field object images. Further, we demonstrate that the method has great potential for real-time quantitative phase imaging and wavefront sensing when combined with deep learning.

X-Ray Ptychography with a Laboratory Source.

X-ray ptychography has revolutionized nanoscale phase contrast imaging at large-scale synchrotron sources in recent years. We present here the first successful demonstration of the technique in a small-scale laboratory setting. An experiment was conducted with a liquid metal-jet x-ray source and a single photon-counting detector with a high spectral resolution. The experiment used a spot size of 5 μm to produce a ptychographic phase image of a Siemens star test pattern with a submicron spatial resolution. The result and methodology presented show how high-resolution phase contrast imaging can now be performed at small-scale laboratory sources worldwide.

Pilot imaging of ex vivo meniscus using phase-contrast enhanced synchrotron micro-tomography

Purpose: We aimed to investigate the feasibility of performing synchrotron radiation-based phase-contrast micro-tomography of ex vivo human meniscus. An advantage with the use of synchrotron light is that the high radiation and coherence enables fast imaging with high resolution. Additionally, the use of phase contrast has the benefit of achieving high contrast in soft tissues. Thus, in this pilot study, for the first time we obtained and analyzed high-resolution phase-contrast images with the purpose to visualize and describe collagen structures of the healthy meniscus.

Ab initio description of bonding for transmission electron microscopy

The simulation of transmission electron microscopy (TEM) images or diffraction patterns is often required to interpret their contrast and extract specimen features. This is especially true for high-resolution phase-contrast imaging of materials, but electron scattering simulations based on atomistic models are widely used in materials science and structural biology. Since electron scattering is dominated by the nuclear cores, the scattering potential is typically described by the widely applied independent atom model. This approximation is fast and fairly accurate, especially for scanning TEM (STEM) annular dark-field contrast, but it completely neglects valence bonding and its effect on the transmitting electrons. However, an emerging trend in electron microscopy is to use new instrumentation and methods to extract the maximum amount of information from each electron. This is evident in the increasing popularity of techniques such as 4D-STEM combined with ptychography in materials science, and cryogenic microcrystal electron diffraction in structural biology, where subtle differences in the scattering potential may be both measurable and contain additional insights. Thus, there is increasing interest in electron scattering simulations based on electrostatic potentials obtained from first principles, mainly via density functional theory, which was previously mainly required for holography. In this Review, we discuss the motivation and basis for these developments, survey the pioneering work that has been published thus far, and give our outlook for the future. We argue that a physically better justified ab initio description of the scattering potential is both useful and viable for an increasing number of systems, and we expect such simulations to steadily gain in popularity and importance.

Intraocular Pressure Decrease Does Not Affect Blood Flow Rate of Ophthalmic Artery in Ocular Hypertension.

PurposeTo investigate if decrease of IOP affects the volumetric blood flow rate in the ophthalmic artery (OA) in patients with previously untreated ocular hypertension.MethodsSubjects with untreated ocular hypertension (n = 30; mean age 67 ± 8 years; 14 females) underwent ophthalmologic examination and a 3-Tesla magnetic resonance imaging investigation. The magnetic resonance imaging included three-dimensional high-resolution phase-contrast magnetic resonance imaging to measure the OA blood flow rate. The subjects received latanoprost once daily in the eye with higher pressure, the untreated eye served as control. The same measurements were repeated approximately 1 week later.ResultsThe mean OA blood flow rate before and after treatment was 12.4 ± 4.4 and 12.4 ± 4.6 mL/min in the treated eye (mean ± SD; P = 0.92) and 13.5 ± 5.2 and 13.4 ± 4.1 mL/min in the control eye (P = 0.92). There was no significant difference between the treated and control eye regarding blood flow rate before (P = 0.13) or after treatment (P = 0.18), or change in blood flow rate after treatment (0.1 ± 3.1 vs. −0.1 ± 4.0 mL/min, P = 0.84). Latanoprost decreased the IOP by 7.2 ± 3.1 mm Hg in the treated eye (P < 0.01).ConclusionsThe results indicate that a significant lowering of IOP does not affect the blood flow rate of the OA in ocular hypertension subjects. The ability to maintain blood supply to the eye independent of the IOP could be a protective mechanism in preserving vision in subjects with ocular hypertension.

Deep learning for high-resolution and high-sensitivity interferometric phase contrast imaging

In Talbot-Lau interferometry, the sample position yielding the highest phase sensitivity suffers from strong geometric blur. This trade-off between phase-sensitivity and spatial resolution is a fundamental challenge in such interferometric imaging applications with either neutron or conventional x-ray sources due to their relatively large beam-defining apertures or focal spots. In this study, a deep learning method is introduced to estimate a high phase-sensitive and high spatial resolution image from a trained neural network to attempt to avoid the trade-off for both high phase-sensitivity and high resolution. To realize this, the training data sets of the differential phase contrast images at a pair of sample positions, one of which is close to the phase grating and the other close to the detector, are numerically generated and are used as the inputs for the training data set of a generative adversarial network. The trained network has been applied to the real experimental data sets from a neutron grating interferometer and we have obtained improved images both in phase-sensitivity and spatial resolution.

Growth of endotaxial Ge nanocrystals in Si(100) matrix via low-energy ion implantation

Embedded structures in a crystalline substrate, endotaxial structures, play a major role in thermoelectric and optoelectronic applications. Here, we have fabricated Ge nanostructures inside Si(100) matrix via low-energy Ge+ ion implantation. Thermally grown SiO2 layer over the Si substrate has been used as a protective coating to avoid low-energy sputtering of the Si surface. 300 keV Ge ions are implanted into Si(100) matrix at two different fluences, 1 × 1015 and 5 × 1015 ions/cm2. After annealing the as-implanted specimens at 800 °C under the inert atmosphere for 1 h, the growth of Ge nanoclusters has been studied by Raman spectroscopy. Endotaxial nature of the Ge nanocrystals has been studied using cross-sectional high-resolution TEM. The compatibility between Ge and Si at the nanocrystal/matrix interface has been discussed in detail using high-resolution phase-contrast imaging.

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.

The application of harmonic techniques to enhance resolution in mesh-based x-ray phase imaging

X-ray phase-contrast imaging produces significantly higher contrast than the conventional attenuation-based imaging. However, phase contrast typically requires small low-power sources or precisely machined and aligned gratings to generate the required spatial coherence. To overcome this limitation, we use a simple wire mesh to produce a periodic intensity pattern in the illumination. Distortions in this pattern upon propagation through an object can be used to produce phase contrast images. Unlike Talbot-grating-based techniques, this directly provides a measure of the phase gradient. Due to periodicity, fast Fourier transforms enable rapid retrieval of the phase contrast image. The high contrast of the mesh pattern allows a significant relaxation of the coherence requirement. However, the resolution of the phase contrast image is normally limited by the mesh period. We demonstrate that employing wider windowing functions during the Fourier processing substantially improves the spatial resolution at the cost of introducing artifacts in the reconstructed images. In order to remove these artifacts, a method is presented to combine images acquired while spatially shifting the mesh. Experimental results demonstrate the successful removal of these artifacts. This offers a method for significant resolution improvement in mesh-based phase-contrast imaging, enabling high resolution phase contrast images with a significant relaxation of source coherence requirements.