Zernike Phase Contrast Imaging Research Articles

Temporal phase-contrast ghost imaging

We introduce a phase-contrast ghost-imaging scheme for the characterization of temporal phase objects in terms of intensity correlations at two photodetectors. The technique is analogous to Zernike's phase-contrast imaging method and is based on utilizing a suitable filter function which renders the small-amplitude phase variations visible in the intensity correlation function. The approach is insensitive to temporal distortions and offers a promising method to analyze the phases of optical pulses.

Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-microscopy

The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.

Non-iterative phase retrieval by phase modulation through a single parameter

We report on a novel non-iterative phase retrieval method with which the complex-valued transmission function of an object can be retrieved with a non-iterative computation, with a limited number of intensity measurements. The measurements are taken in either real space or Fourier space, and for each measurement the phase in its dual space is modulated according to a single optical parameter. The requirement found for the phase modulation function is a general one, which therefore allows for plenty of customization in this method. It is shown that quantitative Zernike phase contrast imaging is one special case of this general method. With simulations we investigate the sampling requirements for a microscopy setup and for a Coherent Diffraction Imaging (CDI) setup.

Elemental x-ray imaging using Zernike phase contrast**Project supported by the National Basic Research Program of China (Grant No. 2012CB825801) and the National Natural Science Foundation of China (Grant Nos. 11505188, and 11305173).

We develop an element-specific x-ray microscopy method by using Zernike phase contrast imaging near absorption edges, where a real part of refractive index changes abruptly. In this method two phase contrast images are subtracted to obtain the target element: one is at the absorption edge of the target element and the other is near the absorption edge. The x-ray exposure required by this method is expected to be significantly lower than that of conventional absorption-based x-ray elemental imaging methods. Numerical calculations confirm the advantages of this highly efficient imaging method.

The use of contrast enhancement techniques in X-ray imaging of lithium–ion battery electrodes

Understanding the microstructural morphology of Li–ion battery electrodes is crucial to improving the electrochemical performance of current Li–ion battery systems and in developing next-generation power systems. The use of 3D X-ray imaging techniques, which are continuously evolving, provides a non-invasive platform to study the relationship between electrode microstructure and performance at various time and length scales. In addition to characterizing a weakly (X-ray) absorbing graphite electrode at multiple length scales, we implement an approach for obtaining improved nano-scale image contrast on a laboratory X-ray microscope by combining information obtained from both absorption–contrast and Zernike phase-contrast X-ray images.

Zernike x-ray ptychography.

We present an imaging technique combining Zernike phase-contrast imaging and ptychography. The contrast formation is explained by following the theory of Zernike phase-contrast imaging. The method is demonstrated with x-rays at a photon energy of 6.2keV, showing how ptychographic reconstruction of a phase sample leads to a Zernike phase-contrast image appearing in the amplitude reconstruction. In addition, the results presented in this Letter indicate an improvement of the resolution of the reconstructed object in the case of Zernike ptychography compared with the conventional one.

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.

Towards tender X-rays with Zernike phase-contrast imaging of biological samples at 50 nm resolution.

X-ray microscopy is a commonly used method especially in material science application, where the large penetration depth of X-rays is necessary for three-dimensional structural studies of thick specimens with high-Z elements. In this paper it is shown that full-field X-ray microscopy at 6.2 keV can be utilized for imaging of biological specimens with high resolution. A full-field Zernike phase-contrast microscope based on diffractive optics is used to study lipid droplet formation in hepatoma cells. It is shown that the contrast of the images is comparable with that of electron microscopy, and even better contrast at tender X-ray energies between 2.5 keV and 4 keV is expected.

Zernike Phase-Contrast Electron Tomography of Microtubule-Related Complexes in Axonemes

In cryo-electron microscopy of vitreously frozen biological specimens, phase-plate imaging can greatly increase the signal-to-noise ratio of the micrographs. When routinely used, this imaging technique may be the method of choice for cryo-electron tomography, in which each image in the tilt-series data set suffers from a high noise background due to the low-electron-dose exposure. Utilizing the Zernike phase-contrast imaging technique, we have carried out an electron tomographic study of the axonemal microtubule doublets and their associated radial spokes. The reconstructed tomogram displays a strong contrast. The quality of the resultant density maps, obtained by sub-volume averaging of a single tomogram in this study, is comparable with that from the averaging of many cryo-tomograms without using the phase-plate.

Zernike phase contrast cryo-electron tomography of whole bacterial cells

Cryo-electron tomography (cryo-ET) provides three-dimensional (3D) structural information of bacteria preserved in a native, frozen-hydrated state. The typical low contrast of tilt-series images, a result of both the need for a low electron dose and the use of conventional defocus phase-contrast imaging, is a challenge for high-quality tomograms. We show that Zernike phase-contrast imaging allows the electron dose to be reduced. This limits movement of gold fiducials during the tilt series, which leads to better alignment and a higher-resolution reconstruction. Contrast is also enhanced, improving visibility of weak features. The reduced electron dose also means that more images at more tilt angles could be recorded, further increasing resolution.

3D analysis of a LiCoO2–Li(Ni1/3Mn1/3Co1/3)O2 Li-ion battery positive electrode using x-ray nano-tomography

A full-field high-resolution x-ray nano-tomography technique, transmission x-ray microscopy (TXM), was used to reveal the 3D morphology of the lithium ion battery composite positive electrode: LiCoO2 (LCO)–Li(Ni1/3Mn1/3Co1/3)O2 (NMC). The TXM method allowed the unambiguous chemical identification of oxide particles by tuning the x-ray energy relative to the transition-metal absorption edges. The NMC particles have a much rougher surface compared to the LCO particles. Cracks due to processing exist in both LCO and NMC particles but the NMC particles exhibit more severe cracking and also tend to have internal pores in addition to radial cracks. Further, the carbon-based phases including the binder and the conductive carbon were identified using Zernike phase contrast imaging.

X-ray mosaic nanotomography of large microorganisms

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.

Resolving the Three‐Dimensional Microstructure of Polymer Electrolyte Fuel Cell Electrodes using Nanometer‐Scale X‐ray Computed Tomography

AbstractThe electrodes of a polymer electrolyte fuel cell (PEFC) are composite porous layers consisting of carbon and platinum nanoparticles and a polymer electrolyte binder. The proper composition and arrangement of these materials for fast reactant transport and high electrochemical activity is crucial to achieving high performance, long lifetimes, and low costs. Here, the microstructure of a PEFC electrode using nanometer‐scale X‐ray computed tomography (nano‐CT) with a resolution of 50 nm is investigated. The nano‐CT instrument obtains this resolution for the low‐atomic‐number catalyst support and binder using a combination of a Fresnel zone plate objective and Zernike phase contrast imaging. High‐resolution, non‐destructive imaging of the three‐dimensional (3D) microstructures provides important new information on the size and form of the catalyst particle agglomerates and pore spaces. Transmission electron microscopy (TEM) and mercury intrusion porosimetry (MIP) is applied to evaluate the limits of the resolution and to verify the 3D reconstructions. The computational reconstructions and size distributions obtained with nano‐CT can be used for evaluating electrode preparation, performing pore‐scale simulations, and extracting effective morphological parameters for large‐scale computational models.

Quantitative 3D imaging of yeast by hard X‐ray tomography

Full-field hard X-ray tomography could be used to obtain three-dimensional (3D) nanoscale structures of biological samples. The image of the fission yeast, Schizosaccharomyces pombe, was clearly visualized based on Zernike phase contrast imaging technique and heavy metal staining method at a spatial resolution better than 50 nm at the energy of 8 keV. The distributions and shapes of the organelles during the cell cycle were clearly visualized and two types of organelle were distinguished. The results for cells during various phases were compared and the ratios of organelle volume to cell volume can be analyzed quantitatively. It showed that the ratios remained constant between growth and division phase and increased strongly in stationary phase, following the shape and size of two types of organelles changes. Our results demonstrated that hard X-ray microscopy was a complementary method for imaging and revealing structural information for biological samples.

Novel Measurement Method of Small Phase with Interference Phase Contrast

A novel method to measure small phase in micro-holography is discussed. It originated from Zernike phase contrast imaging technique, which has been a successful linear phase-to-intensity mapping approach toward phase object. Instead of phase-changing plate, an interference phase contrast technique that is based on in-line holography is utilized to obtain a hologram in the proposed method. The intensity distribution of hologram presents a monotone function with respect to phase distribution of phase object. Thus, the phase can be directly detected through hologram without phase unwrapping. In the work, the contrast of hologram is analyzed toward various phase difference between object wave and reference wave. An optimal solution to measure small phase of phase object is developed, which extends the measuring range of phase. Simulation on a computer is performed to validate theoretically the proposed method.

Nanoscale X-Ray Microscopic Imaging of Mammalian Mineralized Tissue

A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Lightsource operating from 5 to 15 keV X-ray energy with 14 to 30 microm2 field of view has been used for high-resolution (30-40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 microm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30-40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51-54% of pure crystal density and plate-like areas had 44-53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies.

A 3.5-nm Structure of Rat TRPV4 Cation Channel Revealed by Zernike Phase-contrast Cryoelectron Microscopy

The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV.

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.

Practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy

A number of practical issues must be addressed when using thin carbon films as quarter-wave plates for Zernike phase-contrast electron microscopy. We describe, for example, how we meet the more stringent requirements that must be satisfied for beam alignment in this imaging mode. In addition we address the concern that one might have regarding the loss of some of the scattered electrons as they pass through such a phase plate. We show that two easily measured parameters, (1) the low-resolution image contrast produced in cryo-EM images of tobacco mosaic virus particles and (2) the fall-off of the envelope function at high resolution, can be used to quantitatively compare the data quality for Zernike phase-contrast images and for defocused bright-field images. We describe how we prepare carbon-film phase plates that are initially free of charging or other effects that degrade image quality. We emphasize, however, that even though the buildup of hydrocarbon contamination can be avoided by heating the phase plates during use, their performance nevertheless deteriorates over the time scale of days to weeks, thus requiring their frequent replacement in order to maintain optimal performance.

New Frontiers in Cryo-electron Tomography with Zernike Phase Contrast Imaging for Transmission Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008