Quantitative amplitude and phase contrast imaging in a scanning transmission X-ray microscope

Phase contrast in X-ray imaging provides lower radiation dose, and dramatically higher contrast at multi-keV photon energies when compared with absorption contrast. We describe here the use of a segmented detector in a scanning transmission X-ray microscope to collect partially coherent bright field images. We have adapted a Fourier filter reconstruction technique developed by McCallum, Landauer and Rodenburg to retrieve separate, quantitative maps of specimen phase shift and absorption. This is demonstrated in the imaging of a germanium test pattern using 525eV soft X-rays.     

硬X射线相衬成像及其生物应用

X射线相衬技术大大拓宽X射线的应用领域。本文综述X射线相衬成像的物理学原理、X射线相衬成像装置的一般结构和应用方法,并对各种不同相衬成像的方法、成像算法和生物应用逐一阐述。     

Some improvements in the phase contrast microscope

Some considerations on the improvement of the image quality of the phase contrast microscope are presented. The significance of glare and image fidelity discussed theoretically and the dependence of image fidelity and contrast on the phase plate characteristics are investigated on the basis of images calculated for different parameters of the annular phase plate. This paper also describes an anti-reflection technique for the phase plate and demonstrates its effectiveness in the elimination of glare.     

Linear phase imaging using differential interference contrast microscopy

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Accurate structure determination from image reconstruction in ADF STEM

Annular dark-field (ADF) imaging in a scanning transmission electron microscope results in direct structure images of the atomic configuration of the specimen. Since such images are almost perfectly incoherent they can be treated as a convolution between a point-spread function, which is simply the intensity of the illuminating electron probe, and a sharply peaked object function that represents the projected structure of the specimen. Knowledge of the object function for an image region of perfect crystal allows the point-spread function to be directly determined for that image. We examine how the object function for an image can then be reconstructed using a Wiener filter, the CLEAN algorithm and a maximum entropy reconstruction. Prior information is required to perform a reconstruction, and we discuss what nature of prior information is suitable for ADF imaging.     

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

Using the optical methods described, phase specimens can be observed with a modified light microscope in enhanced clarity, purified from typical artifacts which are apparent in standard phase contrast illumination. In particular, haloing and shade‐off are absent, lateral and vertical resolution are maximized and the image quality remains constant even in problematic preparations which cannot be well examined in normal phase contrast, such as specimens beyond a critical thickness or covered by obliquely situated cover slips. The background brightness and thus the range of contrast can be continuously modulated and specimens can be illuminated in concentric‐peripheral, axial or paraxial light. Additional contrast effects can be achieved by spectral color separation. Normal glass or mirror lenses can be used; they do not need to be fitted with a phase plate or a phase ring. The methods described should be of general interest for all disciplines using phase microscopy. Microsc. Res. Tech., 76:1050–1056, 2013. © 2013 Wiley Periodicals, Inc.     

Images of paraffin monolayer crystals with perfect contrast: minimization of beam-induced specimen motion

Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem in monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots, each of a diameter of around 7 nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, where the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support; however great care is needed in practice and the problem remains of how to best image ice-embedded biological structures in the absence of a strong, conductive support film.     

High-angle triple-axis specimen holder for three-dimensional diffraction contrast imaging in transmission electron microscopy

Electron tomography requires a wide angular range of specimen-tilt for a reliable three-dimensional (3D) reconstruction. Although specimen holders are commercially available for tomography, they have several limitations, including tilting capability in only one or two axes at most, e.g. tilt-rotate. For amorphous specimens, the image contrast depends on mass and thickness only and the single-tilt holder is adequate for most tomographic image acquisitions. On the other hand, for crystalline materials where image contrast is strongly dependent on diffraction conditions, current commercially available tomography holders are inadequate, because they lack tilt capability in all three orthogonal axes needed to maintain a constant diffraction condition over the whole tilt range. We have developed a high-angle triple-axis (HATA) tomography specimen holder capable of high-angle tilting for the primary horizontal axis with tilting capability in the other (orthogonal) horizontal and vertical axes. This allows the user to trim the specimen tilt to obtain the desired diffraction condition over the whole tilt range of the tomography series. To demonstrate its capabilities, we have used this triple-axis tomography holder with a dual-axis tilt series (the specimen was rotated by 90° ex-situ between series) to obtain tomographic reconstructions of dislocation arrangements in plastically deformed austenitic steel foils.     

A phase congruency‐based green fluorescent protein and phase contrast image fusion method in nonsubsampled shearlet transform domain

Image fusion technique is an effective way to merge the information contained in different imaging modalities by generating a more informative composite image. Fusion of green fluorescent protein (GFP) and phase contrast images is of great significance to the subcellular localization, the functional analysis of protein, and the expression of gene. In this article, a phase congruency (PC)‐based GFP and phase contrast image fusion method in nonsubsampled shearlet transform (NSST) domain is presented. The input images are decomposed by the NSST to acquire the multiscale and multidirection representations. The high‐frequency coefficients are fused with a strategy based on PC and parameter‐adaptive pulse coupled neural network (PA‐PCNN), while the low‐frequency coefficients are integrated through a local energy (LE)‐based rule. Finally, the fused image is generated by conducting the inverse NSST on the merged high‐ and low‐frequency coefficients. Experimental results illustrate that the presented method outperforms several state‐of‐the‐art GFP and phase contrast image fusion algorithms on both qualitative and quantitative assessments.     

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0·3–1·0 μm). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than ～10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the ‘restored’ images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.     

Micro‐CT versus synchrotron radiation phase contrast imaging of human cochlea

High‐resolution images of the cochlea are used to develop atlases to extract anatomical features from low‐resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption‐based micro‐CT to synchrotron radiation phase contrast imaging (SR‐PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR‐PCI and micro‐CT. Images were visually compared and contrast‐to‐noise ratios (CNRs) were computed from n = 27 regions‐of‐interest (enclosing soft tissue) for quantitative comparisons. Three‐dimensional (3D) models of cochlear internal structures were constructed from SR‐PCI images using a semiautomatic segmentation method. SR‐PCI images provided superior visualization of soft tissue microstructures over conventional micro‐CT images. CNR improved from 7.5 ± 2.5 in micro‐CT images to 18.0 ± 4.3 in SR‐PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR‐PCI images are very promising for developing atlas‐based segmentation methods for postoperative evaluation of cochlear implant surgery.     

Quantitative analysis of backscattered-electron contrast in scanning electron microscopy

Backscattered-electron scanning electron microscopy (BSE-SEM) imaging is a valuable technique for materials characterisation because it provides information about the homogeneity of the material in the analysed specimen and is therefore an important technique in modern electron microscopy. However, the information contained in BSE-SEM images is up to now rarely quantitatively evaluated. The main challenge of quantitative BSE-SEM imaging is to relate the measured BSE intensity to the backscattering coefficient η and the (average) atomic number Z to derive chemical information from the BSE-SEM image. We propose a quantitative BSE-SEM method, which is based on the comparison of Monte–Carlo (MC) simulated and measured BSE intensities acquired from wedge-shaped electron-transparent specimens with known thickness profile. The new method also includes measures to improve and validate the agreement of the MC simulations with experimental data. Two different challenging samples (ZnS/Zn(O_xS_1–x)/ZnO/Si-multilayer and PTB7/PC₇₁BM-multilayer systems) are quantitatively analysed, which demonstrates the validity of the proposed method and emphasises the importance of realistic MC simulations for quantitative BSE-SEM analysis. Moreover, MC simulations can be used to optimise the imaging parameters (electron energy, detection-angle range) in advance to avoid tedious experimental trial and error optimisation. Under optimised imaging conditions pre-determined by MC simulations, the BSE-SEM technique is capable of distinguishing materials with small composition differences.     

An inexpensive and highly efficient device for observing a transmitted electron image in SEM

An inexpensive, efficient device that supplies a transmission mode to the conventional SEM has been developed. The transmitted electrons strike a metal plate, and these generate secondary electrons that are proportional to the quantity of the transmitted electrons. The generated electrons are collected by the secondary electron detector. Hence, the performance of this device is influenced by the number of secondary electrons generated in the metal plate. In order to construct a device that can attain the best transmitted electron image, the signal-to-noise ratio of images, obtained from various trial devices, were measured by a newly-developed digital image processing program. When the material and shape of the device are selected, to produce high-secondary emission, the efficiency of the device compares with that of a relatively expensive standard detector system (scintillator detector).     

针灸疗效评估中激光散斑衬比图像稳定性研究

激光散斑衬比成像技术可以为中医理疗提供无创、高效的疗效评估手段,但目前该评估方法的稳定性有待提高。基于能产生稳定散斑衬比的近红外激光光源,对针灸疗效进行散斑成像实验并获得原始实验数据,分析对比Roll算法与基于此算法加入时间抽样形成新的衬比处理RT(Roll with time-sampling)算法的数据处理结果。从衬比随时间分布的规律可以看出,RT算法处理所得的衬比分布更加均匀,消除了部分噪声干扰,因此RT算法能够提升激光散斑衬比分布的稳定性。     

基于静态小波变换的变透明度法融合GFP荧光与相衬图像

绿色荧光蛋白荧光图像与相衬图像的融合对蛋白质功能的研究和亚细胞结构的定位有重要价值。针对成功用于遥感图像融合的ARSIS概念下多尺度融合算法融合荧光图像与相衬图像时易产生伪像的缺点,提出变透明度的概念,根据直观视觉效果设计一组函数,为融合图像的每一像素分配尺度透明度,并基于静态小波变换的分解结果对源图像进行融合。先通过30组图像的融合实验,估计出所设计函数的相应参数,然后用于对另外117组图像的融合测试,分别计算融合结果的荧光区和非荧光区与荧光图像、相衬图像的质量指数Q和高频相关系数HPCC这两个表征融合效果的特征参数。结果表明,相比常用的融合算法--透明度法、棋盘格法和静态小波替换法,本方法在保持交互可变透明度的同时,提高了融合结果细节的清晰程度,降低了荧光图像背景对融合结果的影响。     

Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals

The contrast in high resolution electron micrographs of three different thin crystals has been compared quantitatively with that predicted theoretically from separate measurements of thier electron diffraction patterns. The crystals were vermiculite, a mineral which is not greatly affected by the electron beam, and two organic specimens, n-paraffin and purple membrane, which are both destroyed by doses of about 1 electron/Ų. The results, all at 4.0 to 4.5 Å resolution, show that the absolute contrast in images of vermiculite is roughly 1/5th of that expected for a theoretically perfect microscope, whereas images of paraffin and purple membrane seldom reach more than 1/25th of theoretical contrast. Much of this loss of contrast can be explained on the basis of known microscope parameters in the case of the non-beam-sensitive specimens. However, for the images of paraffin and purple membrane, it is necessary to postulate that beam-induced specimen movement results in further substantial blurring of the image. The tendency for such movement to occur may be unavoidable since the molecular structure is being destroyed during the exposure. The magnitude of this movement must be reduced before the image contrast will be able to approach the theoretical limit.     

A novel method of Z-contrast imaging in STEM applied to double-labelling

A novel method of Z-contrast imaging in the scanning transmission electron microscope (STEM) is presented. The technique relies on the element dependence of the angular distribution of the scattered electrons, and is realized with a detector consisting of a set of concentric rings. It is possible to discriminate 9-nm colloidal gold and silver specifically distributed on thin sections. In addition to this practical work, numerical evaluations are used to assess the method. With two smaller markers, this approach will be useful in discriminating closely spaced antigenic sites when steric hindrance occurs with double-labelling using probes of different sizes.     

Quantitative analysis of ultrathin doping layers in semiconductors using high-angle annular dark field images

It is well known that the high-angle annular dark field (HAADF) technique in scanning transmission electron microscopy is an incoherent imaging process in the lateral ( xy ) plane. However, as a consequence of the existence of partial coherence in the z direction, accurate quantitative interpretation of image intensity is difficult. The effects of coherence in the z direction can be reduced by increasing the inner collector angle of the annular detector so that the scattering from atoms in the z direction is essentially incoherent. We thus show that it is feasible to quantify the total As concentration of ultrathin InAs _x P_{1− x} layers in InP in a simple but accurate way using a thickness integrated Bloch wave calculation including phonon scattering with a large inner collector angle of the annular detector of around 150 mrad. We compare the As composition derived from this approach with that from the Fresnel method and high resolution imaging. We also show that the non-linear variation of the HAADF intensity with thickness is consistent with our simpler simulations for such conditions. Therefore, this approach enables us easily and quickly to quantify compositions using HAADF images. The tetragonal distortion due to lattice mismatch is also shown to influence the contrast and has been included in the calculations.