Conventional Phase Contrast Research Articles

Detection of Zn Atoms on Ferritin by Annular Dark-Field cryo-STEM

Metal ions play essential roles in many aspects of biological chemistry, including oxygen transport, enzyme catalysis, and maintenance of biopolymer integrity. Cryo-electron microscopy is in principle sensitive to metal ions due to the stronger Coulomb potential relative to surrounding light elements, but conventional defocus phase contrast may not be the ideal approach to visualization. In tomography even small gold nanoparticles (<5 nm) are difficult to identify.We explore the alternative modality of scanning transmission EM (STEM), recently applied to cryo-microscopy and tomography. Annular dark-field STEM provides a quantitative image contrast based on atomic scattering, and has been exploited most effectively for mass measurements of macromolecules. Angular resolution of the scattering can provide further information to distinguish relatively heavy and light elements; in materials science this is known as Z contrast. We ask whether cryo-STEM can detect the presence and location of metal ions in protein complexes.Ferritin proteins forms a nearly spherical, highly symmetrical 3D shell structure by self-assembly of 24 polypeptide subunits. Ferritins bind iron as the primary physiological substrate, but also other metals. Zn binds to ferritin at precise ferroxidase sites within the structure. Taking advantage of these properties, we used cryo-STEM to identify Zn ions within the protein structure. Images were processed by single-particle alignment and averaging. Even in very small datasets, Zn stands out clearly at the predicted binding sites. The data provide an experimental measure of the signal contribution from single Zn atoms in cryo-STEM. This is key to understanding atomic detection of metals in 3D macromolecular and cellular contexts, as well as to use of synthetic metal tags as specific molecular labels.

석면섬유 자동계수를 위한 고효율 현미경법의 영상처리 알고리즘 개선

We developed a high-throughput microscopy (HTM) method which enabled us to replace a conventional phase contrast microscopy (PCM) method that has been used as a standard analytical method for airborne asbestos. We could obtain the concentration of airborne asbestos fibers under detection limit by automated image processing and analysis using HTM method. Here we propose an improved image processing algorithm with variable parameters to enhance the accuracy of the HTM analysis. Since the variable parameters that compensate the difference of the brightness are applied to the individual images in our new image processing method, it is possible to enhance the accuracy of the automatic image analysis method for sample slides with low asbestos concentration that caused errors in binary image processing. We demonstrated that enumeration of fibers by improved image processing algorithm remarkably enhanced the accuracy of HTM analysis in comparison with PCM. The improved HTM method can be a potential alternative to conventional PCM.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Bicuspid Valve-Related Aortic Disease: Flow Assessment with Conventional Phase-Contrast MRI

Abnormal blood flow with bicuspid aortic valve (BAV) has been characterized with four-dimensional flow magnetic resonance imaging (MRI), but this approach is time consuming and requires technical expertise. We assess the relationship between different leaflets fusion patterns with BAV, eccentric systolic flow, and dilation patterns of the ascending aorta using two-dimensional (2D) phase-contrast (PC) MRI. Fifty-nine patients with BAV who underwent cardiac MRI were identified; 47 had right-left (RL) aortic leaflet fusion and 12 had right-noncoronary (RN) fusion. Flow displacement was calculated, and patients with abnormal displacement (>0.1) were classified as either rightward or leftward. Patterns of aortopathy were determined (0-3), and correlation between leaflet fusion, flow direction, aortopathy type, and other clinical parameters was performed with Pearson correlation, the Fisher exact test and chi-square analysis. Normal systolic flow was seen in 24% of cases and was significantly correlated with normal aortas (P = .011). Abnormal flow displacement with RL fusion was strongly associated with rightward deviation (36 of 37 cases), whereas RN fusion skewed leftward (seven of eight cases; P < .01). In patients with aortopathy, RL fusion was strongly associated with type 2 aortopathy and RN with type 3 aortopathy (P < .01). Conventional PC MRI can identify abnormal systolic flow and differences in jet orientation with BAV. RL leaflet fusion is associated with rightward flow deviation and type 2 aortopathy, whereas RN fusion is linked to leftward deviation and type 3 aortopathy. The presence and direction of eccentric flow jets may help risk stratify these patients for valve-related aortic disease.

Condenser-free contrast methods for transmitted-light microscopy.

Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared.

White-light diffraction tomography of unlabelled live cells

We present a technique called white-light diffraction tomography (WDT) for imaging microscopic transparent objects such as live unlabelled cells. The approach extends diffraction tomography to white-light illumination and imaging rather than scattering plane measurements. Our experiments were performed using a conventional phase contrast microscope upgraded with a module to measure quantitative phase images. The axial dimension of the object was reconstructed by scanning the focus through the object and acquiring a stack of phase-resolved images. We reconstructed the three-dimensional structures of live, unlabelled, red blood cells and compared the results with confocal and scanning electron microscopy images. The 350 nm transverse and 900 nm axial resolution achieved reveals subcellular structures at high resolution in Escherichia coli cells. The results establish WDT as a means for measuring three-dimensional subcellular structures in a non-invasive and label-free manner. The three-dimensional structures of transparent objects, such as living cells, are captured by an imaging technique that uses white-light illumination and diffraction tomography to collect a stack of phase-based images.

Accelerated radial Fourier-velocity encoding using compressed sensing

PurposePhase Contrast Magnetic Resonance Imaging (MRI) is a tool for non-invasive determination of flow velocities inside blood vessels. Because Phase Contrast MRI only measures a single mean velocity per voxel, it is only applicable to vessels significantly larger than the voxel size. In contrast, Fourier Velocity Encoding measures the entire velocity distribution inside a voxel, but requires a much longer acquisition time. For accurate diagnosis of stenosis in vessels on the scale of spatial resolution, it is important to know the velocity distribution of a voxel. Our aim was to determine velocity distributions with accelerated Fourier Velocity Encoding in an acquisition time required for a conventional Phase Contrast image. Materials and MethodsWe imaged the femoral artery of healthy volunteers with ECG – triggered, radial CINE acquisition. Data acquisition was accelerated by undersampling, while missing data were reconstructed by Compressed Sensing. Velocity spectra of the vessel were evaluated by high resolution Phase Contrast images and compared to spectra from fully sampled and undersampled Fourier Velocity Encoding. By means of undersampling, it was possible to reduce the scan time for Fourier Velocity Encoding to the duration required for a conventional Phase Contrast image. ResultsAcquisition time for a fully sampled data set with 12 different Velocity Encodings was 40min. By applying a 12.6–fold retrospective undersampling, a data set was generated equal to 3:10min acquisition time, which is similar to a conventional Phase Contrast measurement. Velocity spectra from fully sampled and undersampled Fourier Velocity Encoded images are in good agreement and show the same maximum velocities as compared to velocity maps from Phase Contrast measurements. ConclusionCompressed Sensing proved to reliably reconstruct Fourier Velocity Encoded data. Our results indicate that Fourier Velocity Encoding allows an accurate determination of the velocity distribution in vessels in the order of the voxel size. Thus, compared to normal Phase Contrast measurements delivering only mean velocities, no additional scan time is necessary to retrieve meaningful velocity spectra in small vessels.

Fundamental relationship between the noise properties of grating-based differential phase contrast CT and absorption CT: Theoretical framework using a cascaded system model and experimental validation

Using a grating interferometer, a conventional x-ray cone beam computed tomography (CT) data acquisition system can be used to simultaneously generate both conventional absorption CT (ACT) and differential phase contrast CT (DPC-CT) images from a single data acquisition. Since the two CT images were extracted from the same set of x-ray projections, it is expected that intrinsic relationships exist between the noise properties of the two contrast mechanisms. The purpose of this paper is to investigate these relationships. First, a theoretical framework was developed using a cascaded system model analysis to investigate the relationship between the noise power spectra (NPS) of DPC-CT and ACT. Based on the derived analytical expressions of the NPS, the relationship between the spatial-frequency-dependent noise equivalent quanta (NEQ) of DPC-CT and ACT was derived. From these fundamental relationships, the NPS and NEQ of the DPC-CT system can be derived from the corresponding ACT system or vice versa. To validate these theoretical relationships, a benchtop cone beam DPC-CT/ACT system was used to experimentally measure the modulation transfer function (MTF) and NPS of both DPC-CT and ACT. The measured three-dimensional (3D) MTF and NPS were then combined to generate the corresponding 3D NEQ. Two fundamental relationships have been theoretically derived and experimentally validated for the NPS and NEQ of DPC-CT and ACT: (1) the 3D NPS of DPC-CT is quantitatively related to the corresponding 3D NPS of ACT by an inplane-only spatial-frequency-dependent factor 1∕f (2), the ratio of window functions applied to DPC-CT and ACT, and a numerical factor C(g) determined by the geometry and efficiency of the grating interferometer. Note that the frequency-dependent factor is independent of the frequency component f(z) perpendicular to the axial plane. (2) The 3D NEQ of DPC-CT is related to the corresponding 3D NEQ of ACT by an f (2) scaling factor and numerical factors that depend on both the attenuation and refraction properties of the image object, as well as C(g) and the MTF of the grating interferometer. The performance of a DPC-CT system is intrinsically related to the corresponding ACT system. As long as the NPS and NEQ of an ACT system is known, the corresponding NPS and NEQ of the DPC-CT system can be readily estimated using additional characteristics of the grating interferometer.

Oziella sibirica (Acari: Eriophyoidea: Phytoptidae), a new eriophyoid mite species described using confocal microscopy, COI barcoding and 3D surface reconstruction

Oziella sibirica sp. nov., collected from sedges (Cyperaceae: Carex macroura) in Siberia, Russia, is herein describedbased on the external morphology of all active instars using primarily conventional phase contrast microscopy, andon the female internal genitalia and prodorsal shield design using confocal laser scanning microscopy (CLSM)imaging and a 3D modelling technique. A partial mitochondrial COI gene sequence of O. sibirica sp. nov. is alsoprovided, through GenBank, and this represents the first published record of any gene sequence data for the familyPhytoptidae. We present remarks on the phylogenetic significance of the position of setae 3a in immature instars oferiophyoids and on the ontogenic variability of the empodium morphology of O. sibirica sp. nov. Using this speciesas a model, we propose a method for describing the internal genitalia of eriophyoids based on CLSM. We advocatethe use of CLSM imaging as a new, relatively simple technique for observing and describing the internal genitalia oferiophyoids, as these largely unexplored genitalic structures may provide phylogenetically meaningful informationfor improving the classification of this poorly understood group of mites. In addition, CLSM may complementconventional light microscopy techniques in facilitating the interpretation of external structures such as body ornamentation or chaetotaxy.

High-resolution, low-dose phase contrast X-ray tomography for 3D diagnosis of human breast cancers

Mammography is the primary imaging tool for screening and diagnosis of human breast cancers, but ~10-20% of palpable tumors are not detectable on mammograms and only about 40% of biopsied lesions are malignant. Here we report a high-resolution, low-dose phase contrast X-ray tomographic method for 3D diagnosis of human breast cancers. By combining phase contrast X-ray imaging with an image reconstruction method known as equally sloped tomography, we imaged a human breast in three dimensions and identified a malignant cancer with a pixel size of 92 μm and a radiation dose less than that of dual-view mammography. According to a blind evaluation by five independent radiologists, our method can reduce the radiation dose and acquisition time by ~74% relative to conventional phase contrast X-ray tomography, while maintaining high image resolution and image contrast. These results demonstrate that high-resolution 3D diagnostic imaging of human breast cancers can, in principle, be performed at clinical compatible doses.

Optimum aberration coefficients for recording high-resolution off-axis holograms in a Cs-corrected TEM

Amongst the impressive improvements in high-resolution electron microscopy, the Cs-corrector also has significantly enhanced the capabilities of off-axis electron holography. Recently, it has been shown that the signal above noise in the reconstructable phase can be significantly improved by combining holography and hardware aberration correction. Additionally, with a spherical aberration close to zero, the traditional optimum focus for recording high-resolution holograms ("Lichte's defocus") has become less stringent and both, defocus and spherical aberration, can be selected freely within a certain range. This new degree of freedom can be used to improve the signal resolution in the holographically reconstructed object wave locally, e.g. at the atomic positions. A brute force simulation study for an aberration corrected 200 kV TEM is performed to determine optimum values for defocus and spherical aberration for best possible signal to noise in the reconstructed atomic phase signals. Compared to the optimum aberrations for conventional phase contrast imaging (NCSI), which produce "bright atoms" in the image intensity, the resulting optimum values of defocus and spherical aberration for off-axis holography enable "black atom contrast" in the hologram. However, they can significantly enhance the local signal resolution at the atomic positions. At the same time, the benefits of hardware aberration correction for high-resolution off-axis holography are preserved. It turns out that the optimum is depending on the object and its thickness and therefore not universal.

4D phase contrast flow imaging for in-stent flow visualization and assessment of stent patency in peripheral vascular stents – A phantom study

Purpose4D phase contrast flow imaging is increasingly used to study the hemodynamics in various vascular territories and pathologies. The aim of this study was to assess the feasibility and validity of MRI based 4D phase contrast flow imaging for the evaluation of in-stent blood flow in 17 commonly used peripheral stents. Materials and methods17 different peripheral stents were implanted into a MR compatible flow phantom. In-stent visibility, maximal velocity and flow visualization were assessed and estimates of in-stent patency obtained from 4D phase contrast flow data sets were compared to a conventional 3D contrast-enhanced magnetic resonance angiography (CE-MRA) as well as 2D PC flow measurements. ResultsIn all but 3 of the tested stents time-resolved 3D particle traces could be visualized inside the stent lumen. Quality of 4D flow visualization and CE-MRA images depended on stent type and stent orientation relative to the magnetic field. Compared to the visible lumen area determined by 3D CE-MRA, estimates of lumen patency derived from 4D flow measurements were significantly higher and less dependent on stent type. A higher number of stents could be assessed for in-stent patency by 4D phase contrast flow imaging (n=14) than by 2D phase contrast flow imaging (n=10). Conclusions4D phase contrast flow imaging in peripheral vascular stents is feasible and appears advantageous over conventional 3D contrast-enhanced MR angiography and 2D phase contrast flow imaging. It allows for in-stent flow visualization and flow quantification with varying quality depending on stent type.

Response

We greatly appreciate the interest in our Technical Note on the measurement of eccentric blood flow in the ascending aorta (1), and applaud Dr.Della Corte et al (2) on their recent publication regarding restricted aortic cusp motion with bicuspid aortic valve (BAV). We believe that this type of correlation between aortic valve imaging features and concrete endpoints like interval aortic growth is exactly the direction this field needs to head. Regarding our group's recent article on wall shear stress (WSS) in the context of BAV, the data demonstrate that flow eccentricity is associated with marked skewing of systolic WSS profiles and focally increased WSS at the aortic convexity (3). The Technical Note in question, however, does not attempt to make a direct correlation with WSS in patients, but instead suggests that a straightforward parameter like flow displacement may prove to be a useful surrogate for the more complex and difficult to measure parameter of WSS. Second, we apologize if our equation regarding “center of velocity” was unclear. Our data are volumetric with three-directional velocity information. From this larger dataset, we exported a plane at a standardized level for the analysis we describe. Ultimately, this type of evaluation could be performed with conventional 2D phase contrast MRI, but we consider this a strength rather than a weakness of the approach. We agree that the cause versus consequence of aortic dilation issue is problematic, and caution against using aortic diameter for “in vivo validation” in the table reviewing imaging parameters of flow eccentricity. As pointed out, a proper evaluation of these parameters would require a systematic, prospective study. The goal of our Technical Note was more modest: to show that the flow displacement parameter we propose is simple, reliable, and reproducible. (We would strongly encourage adding a column for reproducibility in the aforementioned table.) More recently, we have started to investigate the relationship between flow displacement and key clinical endpoints, and are soon to publish our preliminary results (4). Michael D. Hope MD*, Monica Sigovan PhD*, Petter Dyverfeldt PhD*, David Saloner PhD*, * Department of Radiology, University of California, San Francisco, San Francisco, California.

X-ray spatial frequency heterodyne imaging

We derive an expression for the electric field at the image plane from the Kirchhoff-Fresnel integral for a grating in contact with an object in an x-ray, in-line imaging configuration that shows the grating to give a heterodyning effect. We also show how image contrast in a harmonic of the grating is enhanced depending on the first space derivative of the phase function. Experiments are reported where a Fourier component of the image intensity gives contrast at the edges of objects significantly larger than that found with conventional in-line phase contrast imaging.

Investigation of stenotic jets using 3D-PC-UTE

Background Conventional phase contrast (PC) velocity measurements fail in situation of high velocities and complex flow, as after a stenosis. Specifically, volume flow and peak velocities are underestimated, and velocity-to-noise ratio (VNR) is decreased due to flow voids. These effects are much dependent on the echo time (TE). Reducing TE will drastically improve the results, both concerning VNR and accuracy. The method of ultra-short TE (UTE) allows for submillisecond TE, and has recently been implemented for 2D through-plane PC(2D PC-UTE). This work aims to investigate peak velocities and visualize stenotic jets in a 3D volume using PC-UTE.

Evaluation of Sensitivity of Fluorescence-Based Asbestos Detection by Correlative Microscopy

Fluorescence microscopy (FM) has recently been applied to the detection of airborne asbestos fibers that can cause asbestosis, mesothelioma and lung cancer. In our previous studies, we discovered that the E. coli protein DksA specifically binds to the most commonly used type of asbestos, chrysotile. We also demonstrated that fluorescent-labeled DksA enabled far more specific and sensitive detection of airborne asbestos fibers than conventional phase contrast microscopy (PCM). However, the actual diameter of the thinnest asbestos fibers visualized under the FM platform was unclear, as their dimensions were below the resolution of optical microscopy. Here, we used correlative microscopy (scanning electron microscopy [SEM] in combination with FM) to measure the actual diameters of asbestos fibers visualized under the FM platform with fluorescent-labeled DksA as a probe. Our analysis revealed that FM offers sufficient sensitivity to detect chrysotile fibrils as thin as 30-35nm. We therefore conclude that as an analytical method, FM has the potential to detect all countable asbestos fibers in air samples, thus approaching the sensitivity of SEM. By visualizing thin asbestos fibers at approximately tenfold lower magnifications, FM enables markedly more rapid counting of fibers than SEM. Thus, fluorescence microscopy represents an advanced analytical tool for asbestos detection and monitoring.

Magnetic resonance 4D flow characteristics of cerebrospinal fluid at the craniocervical junction and the cervical spinal canal

To evaluate the applicability of 4D phase contrast (4D PC) MR imaging in the assessment of cerebrospinal fluid dynamics in healthy volunteers and patients with lesions at the craniocervical junction or the cervical spinal canal. Ten healthy volunteers and four patients with lesions including Chiari I malformation and cervical canal stenoses were examined by a cardiac-gated 4D PC imaging sequence on 1.5T MRI. Phase contrast images were postprocessed allowing for flow quantification and flow pathline visualisation. Velocity data were compared with conventional axial 2D phase contrast images. The 4D PC sequence allowed for flow quantification and visualisation in all individuals. Bland-Altman analysis showed good agreement of 2D and 4D PC velocity data. In healthy volunteers, CSF flow was homogeneously distributed in the anterior and anterolateral subarachnoid space with the flow directed caudally during systole and cranially during diastole. Flow velocities were closely related to the width of the subarachnoid space. Patients showed grossly altered CSF flow patterns with formation of flow jets with increased flow velocities. 4D PC MR imaging allows for a detailed assessment of CSF flow dynamics helping to distinguish physiological from complex pathological flow patterns at the craniocervical junction and the cervical spine.

Phase shifting interferometry of cold atoms

We propose a scheme to engage phase shifting interferometry on cold atomic samples and present the simulation results under several experimentally achievable conditions nowadays. This method allows far-detuning, low power probing, and is intrinsically nondestructive. This novel detection means yields image quality superior to the conventional phase contrast imaging at certain conditions and could be experimentally realized. Furthermore, the longitudinal resolution of imaging by this manner is mainly set by optical interference and can be better than the diffraction limit. This scheme also provides special advantages to diagnose the surface-trapped clouds, with which phase imaging on the fabricated wires and atoms altogether is possible as well.

Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from Vibrio alginolyticus

Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm.

Ultra-high Contrast Amplification in Bright-field Images

In this paper, a new way for visualization of unstained transparent specimens is described, which is based on bright-field imaging and promises an improved resolution and contrast. The final results can be compared with conventional phase contrast images, and the new method may lead to superior results in most specimens.