Imaging of soft and hard materials using a Boersch phase plate in a transmission electron microscope

Using two levels of electron beam lithography, vapor phase deposition techniques, and FIB etching, we have fabricated an electrostatic Boersch phase plate for contrast enhancement of weak phase objects in a transmission electron microscope. The phase plate has suitable dimensions for the imaging of small biological samples without compromising the high-resolution capabilities of the microscope. A micro-structured electrode allows for phase tuning of the unscattered electron beam, which enables the recording of contrast enhanced in-focus images and in-line holograms. We have demonstrated experimentally that our phase plate improves the contrast of carbon nanotubes while maintaining high-resolution imaging performance, which is demonstrated for the case of an AlGaAs heterostructure. The development opens a new way to study interfaces between soft and hard materials.     

Hard X-ray microscopy with Zernike phase contrast

Zernike phase contrast has been added to a full‐field X‐ray microscope with Fresnel zone plates that was in operation at 6.95 keV. The spatial resolution has also been improved by increasing the magnification of the microscope objective looking at the CsI(Tl) scintillation crystal. Cu no. 2000 meshes and a zone plate have been imaged to see the contrast as well as the spatial resolution. A Halo effect coming from the Zernike phase contrast was clearly visible on the images of meshes.     

Effect of the phase shift due to dynamical scattering on the contrast of crystal structure images

The contrast of through-focus images of a niobium tungsten bronze, 2Nb₂O₅·7WO₃, taken by a 1 MV high resolution electron microscope, is discussed in terms of the transfer function, in which, in addition to the conventional phase aberration, the phase shift due to the dynamical scattering is taken into account. It is found that the phase shift may be advantagous in the formation of structure images, as long as the crystal very thin, i.e., a few nanometers in thickness. It is also made clear why the best defocus for optimum structure images differs from the Scherzer defocus, depending on the crystal thickness. An almost exact contrast reversal occurs at overfocus only in the images of extremely thin crystals, because the positive range of the transfer function is made narrower due to the above phase shift. The discussion is supported by the computer simulation of the image contrast.     

Upgrading a microscope with a spiral phase plate

We present the implementation of a spiral phase plate in a standard bright-field microscope to enhance the contrast of phase and amplitude samples. The method can be employed in all types of microscopy where standard phase contrast methods are applicable, for example, in bright-field transmission or reflection microscopy using an illumination source with partial spatial coherence. The spiral phase filter is placed into an accessible Fourier plane of the imaging path of the microscope. It is shown that this produces not only a strong contrast enhancement but in theory also improves the spatial resolution of the microscope for white light. A series of different set-ups for transmissive or reflective samples, including epi-illumination, are presented to demonstrate the practical range of applications of this contrasting method. A minute shift of the spiral phase plate out of the centre results in relief-like images that are similar to those obtained by differential interference contrast microscopy. A series of such relief-like images can be numerically processed to obtain quantitative phase and amplitude information of the sample.     

The tuning of a Zernike phase plate with defocus and variable spherical aberration and its use in HRTEM imaging

With the advent of the double-hexapole aberration corrector in transmission electron microscopy the spherical aberration of the imaging system has become a tunable imaging parameter like the objective lens defocus. Now Zernike phase plates, altering the phase of the diffracted electron wave, can be approximated more perfectly than with the lens defocus alone, and the amount of phase change can be adjusted within wide limits. The tuning of the phase change allows an optimum contrast transfer in high-resolution imaging even for thick crystalline objects, thus surpassing the limits of the well-known Scherzer lamda/4 phase plate to the imaging of thin objects. The optimum values for the spherical aberration and the lens defocus are derived, and the limits and imperfections of the approximation explored. A mathematical link to the channelling approximation of high-energy electron diffraction shows how the image contrast of atomic columns can be improved systematically within wide thickness limits. Depending on the specimen thickness different combinations of spherical aberration and defocus are favourable: positive spherical aberration with an underfocus, zero spherical aberration with zero defocus, as well as negative spherical aberration with an overfocus.     

Theoretical aspects of image formation in the aberration-corrected electron microscope

The theoretical aspects of image formation in the transmission electron microscope (TEM) are outlined and revisited in detail by taking into account the elastic and inelastic scattering. In particular, the connection between the exit wave and the scattering amplitude is formulated for non-isoplanatic conditions. Different imaging modes are investigated by utilizing the scattering amplitude and employing the generalized optical theorem. A novel obstruction-free anamorphotic phase shifter is proposed which enables one to shift the phase of the scattered wave by an arbitrary amount over a large range of spatial frequencies. In the optimum case, the phase of the scattered wave and the introduced phase shift add up to −π/2 giving negative contrast. We obtain these optimum imaging conditions by employing an aberration-corrected electron microscope operating at voltages below the knock-on threshold for atom displacement and by shifting optimally the phase of the scattered electron wave. The optimum phase shift is achieved by adjusting appropriately the constant phase shift of the phase plate and the phase shift resulting from the defocus and the spherical aberration of the corrected objective lens. The realization of this imaging mode is the aim of the SALVE project (Sub-Å Low-Voltage Electron microscope).     

Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate

Imaging of weak amplitude and phase objects, such as unstained vitrified biological samples, by conventional transmission electron microscopy (TEM) suffers from poor object contrast since the amplitude and phase of the scattered electron wave change only very little. In phase contrast light microscopy the imaging of weak phase objects is greatly enhanced by the use of a quarter-wave phase plate, which produces high signal contrast by shifting the phase of the scattered light. An analogous quarter-wave plate for the electron microscope, designed as an electrostatic einzel lens, was proposed by Boersch in 1947 but the small dimensions of the device have impeded its realization up to now. We here present the first fabrication and application of a miniaturized electrostatic einzel lens driven as TEM quarter-wave phase plate. Phase modulation is generated by the electrostatic field confined to the inside of a microstructured ring electrode. This field affects the phase velocity of the unscattered part of the electron wave. By varying its strength the phase shift of the primary beam can be adjusted to pi/2, producing strong phase contrast independent of spatial frequency. The phase plate proves to be mechanically stable and does not impair image quality, in particular it does not reduce the high-resolution signal. The expected residual lens effect of the einzel lens is minimal. Our microlens is supported by conducting rods arranged in a threefold symmetry. This particular geometry provides optimized single-sideband signal transfer for spatial frequencies otherwise obstructed by the supporting rods.     

A study of the human hair structure with a Zernike phase contrast X-ray microscope

We have observed the internal structure of human hair shafts with a transmission Zernike phase contrast hard X‐ray microscope. Due to the high spatial resolution and the high contrast of the microscope, we could image scales, macrofibrils, medulla and melanin without staining. The structure of a black hair shaft is compared with that of a white one.     

Contrast in confocal scanning microscopy with a finite detector

The optical properties of a general scanning microscope are determined within the framework of Fourier imaging theory. For a simple model optical system, with Gaussian lens and detector apertures, the contrast transfer function can be expressed in terms of elementary functions. The theory predicts that spatial resolution and depth discrimination vary continuously with detector aperture and that defocus phase contrast is present in transmission images obtained with a symmetric objective, collector lens confocal microscope.     

Effect of a physical phase plate on contrast transfer in an aberration-corrected transmission electron microscope

In this theoretical study we analyze contrast transfer of weak-phase objects in a transmission electron microscope, which is equipped with an aberration corrector (C(s)-corrector) in the imaging lens system and a physical phase plate in the back focal plane of the objective lens. For a phase shift of pi/2 between scattered and unscattered electrons induced by a physical phase plate, the sine-type phase contrast transfer function is converted into a cosine-type function. Optimal imaging conditions could theoretically be achieved if the phase shifts caused by the objective lens defocus and lens aberrations would be equal to zero. In reality this situation is difficult to realize because of residual aberrations and varying, non-zero local defocus values, which in general result from an uneven sample surface topography. We explore the conditions--i.e. range of C(s)-values and defocus--for most favourable contrast transfer as a function of the information limit, which is only limited by the effect of partial coherence of the electron wave in C(s)-corrected transmission electron microscopes. Under high-resolution operation conditions we find that a physical phase plate improves strongly low- and medium-resolution object contrast, while improving tolerance to defocus and C(s)-variations, compared to a microscope without a phase plate.     

High-resolution electron microscopy of human enamel crystals

The structure of enamel crystals obtained from four human premolars has been studied by high-resolution electron microscopy (HREM) in the [0001], [***2110], [***1540], [***0110] and [***1213] crystallographic directions at various microscope defocus and crystal thickness values. The resolution obtained has not previously been reported for human enamel crystals. In all cases, it was possible to match the experimental images to images calculated using the atomic positions of mineral hydroxyapatite. However, a deviation from hexagonal symmetry characterized by marked (***1010) planes of intensity different from the one of the (***3030) and {***1010}-type planes was observed. In this work, we present an improvement of Scherzer resolution of 0.25-0.20 nm over previous work on biological enamel crystals. This improvement of resolution has permitted the incorporation of crystallographic reflections of higher spatial frequencies into the imaging process of the microscope and has led to a more precise structure determination of the crystals studied.     

Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.     

In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C_s corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.     

3D visualization of subcellular structures of Schizosaccharomyces pombe by hard X-ray tomography

Cellular structures of the fission yeast, Schizosaccharomyces pombe, were examined by using hard X-ray tomography. Since cells are nearly transparent to hard X-rays, Zernike phase contrast and heavy metal staining were introduced to improve image contrast. Through using such methods, images taken at 8 keV displayed sufficient contrast for observing cellular structures. The cell wall, the intracellular organelles and the entire structural organization of the whole cells were visualized in three-dimensional at a resolution better than 100 nm. Comparison between phase contrast and absorption contrast was also made, indicating the obvious advantage of phase contrast for cellular imaging at this energy. Our results demonstrate that hard X-ray tomography with Zernike phase contrast is suitable for cellular imaging. Its unique abilities make it have potential to become a useful tool for revealing structural information from cells, especially thick eukaryotic cells.     

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.     

Design of a microfabricated, two-electrode phase-contrast element suitable for electron microscopy

A miniature electrostatic element has been designed to selectively apply a 90 degrees phase shift to the unscattered beam in the back focal plane of the objective lens, in order to realize Zernike-type, in-focus phase contrast in an electron microscope. The design involves a cylindrically shaped, biased-voltage electrode, which is surrounded by a concentric grounded electrode. Electrostatic calculations have been used to determine that the fringing fields in the region of the scattered electron beams will cause a negligible phase shift as long as the ratio of electrode length to the transverse feature size is greater than 5:1. Unlike the planar, three-electrode einzel lens originally proposed by Boersch for the same purpose, this new design does not require insulating layers to separate the biased and grounded electrodes, and it can thus be produced by a very simple microfabrication process. Scanning electron microscope images confirm that mechanically robust devices with feature sizes of approximately 1 microm can be easily fabricated. Preliminary experimental images demonstrate that these devices do apply a 90 degrees phase shift between the scattered and unscattered electrons, as expected.     

Energy filtering transmission electron microscopy using the new JEM-2010FEF

The new JEM-2010FEF electron microscope provides useful techniques based on energy filtering as an omega-type energy filter is integrated into a thermal field-emission 200 kV transmission electron microscope. For example, the zero-loss imaging improves the contrast of high resolution lattice images as well as images of precipitates or lattice defects in alloys. The acquisition time for elemental mapping with core-loss electrons is one order in magnitude shorter than with energy-dispersive X-ray spectroscopy. The removal of inelastically scattered electrons enables us to observe weak lines in convergent-beam electron diffraction patterns from a thicker specimen with a probe size 1–2 nm in diameter. A combination of the field emission gun and sensitive recording media such as an imaging plate and a slow-scan CCD camera makes the energy filtering more powerful.     

Restoration of weak phase-contrast images recorded with a high degree of defocus: the "twin image" problem associated with CTF correction

Relatively large values of objective-lens defocus must normally be used to produce detectable levels of image contrast for unstained biological specimens, which are generally weak phase objects. As a result, a subsequent restoration operation must be used to correct for oscillations in the contrast transfer function (CTF) at higher resolution. Currently used methods of CTF correction assume the ideal case in which Friedel mates in the scattered wave have contributed pairs of Fourier components that overlap with one another in the image plane. This "ideal" situation may be only poorly satisfied, or not satisfied at all, as the particle size gets smaller, the defocus value gets larger, and the resolution gets higher. We have therefore investigated whether currently used methods of CTF correction are also effective in restoring the single-sideband image information that becomes displaced (delocalized) by half (or more) the diameter of a particle of finite size. Computer simulations are used to show that restoration either by "phase flipping" or by multiplying by the CTF recovers only about half of the delocalized information. The other half of the delocalized information goes into a doubly defocused "twin" image of the type produced during optical reconstruction of an in-line hologram. Restoration with a Wiener filter is effective in recovering the delocalized information only when the signal-to-noise ratio (S/N) is orders of magnitude higher than that which exists in low-dose images of biological specimens, in which case the Wiener filter approaches division by the CTF (i.e. the formal inverse). For realistic values of the S/N, however, the "twin image" problem seen with a Wiener filter is very similar to that seen when either phase flipping or multiplying by the CTF is used for restoration. The results of these simulations suggest that CTF correction is a poor alternative to using a Zernike-type phase plate when imaging biological specimens, in which case the images can be recorded in a close-to-focus condition, and delocalization of high-resolution information is thus minimized.     

基于波前编码技术的眼底成像系统

提出了一种混合光学-数字眼底成像系统,该系统不需要探测和补偿不同人眼带来的像差的动态变化,即可获取高分辨率的视网膜图像。该系统在出瞳位置放置一块三次相位掩膜板对眼底图像进行编码,然后采用维纳滤波算法复原获取清晰图像。利用基于光学成像的二维卷积模型对该系统进行了仿真实验,结果表明,针对不同个体的人眼像差,三次相位板可以使系统的点扩散函数在一定范围内保持一致,从而校正包含离焦、彗差、像散、三叶草在内的人眼像差,获得清晰的眼底图像。该系统的设计简化了传统眼底相机的复杂光学结构,是一种便携式结构。     

Aberration-corrected microscopy for structural biology applications

This study explores the potential of a C _s-corrected transmission electron microscope for structural studies of biological samples, in particular isolated macromolecular complexes. A 300-kV transmission electron microscope, equipped with a C _s corrector was employed to record sets of images at different defocus and C _s settings. The experiments were designed to determine whether imaging with large defocus benefits from C _s correction. Defocus contrast in biological imaging has a stronger influence on image resolution than any other parameter. We find the results are in good agreement with theoretical framework, verifying that the typical imaging conditions required for biological investigations are not affected by C _s correction.